C-1-01
A random-forest based method that can predict detailed
enzyme functions and also identify specificity determining
residues
Chioko Nagao 1

Nozomi Nagano 2chio@nibio.go.jp

n.nagano@aist.go.jp

Kenji Mizuguchi 1
kenji@nibio.go.jp

1

2

National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka
567-0085, Japan
Computational Biology Research Center, AIST, 2-4-7 Aomi, Koto-ku, Tokyo
135-0064, Japan

Keywords: Enzyme function, EC number, Random forests, Specificity determining residues
Determining enzyme functions is essential for understanding chemical reactions occurring inliving cells. Although many prediction methods have been developed, it remains a significant
challenge to predict enzyme functions at the fourth-digit level of the Enzyme Commission (EC)numbers [1]. A small number of mutations can often drastically change functional specificity ofenzymes. Therefore, information about these specificity determining residues (SDRs) canpotentially help discriminate detailed functions. However, because these residues must be
identified by mutagenesis experiments, the available information is limited, and the lack ofexperimentally verified SDRs has hindered the development of detailed function prediction
methods and computational identification of SDRs.
In this study, we developed EFPrf, a novel method for predicting enzyme functions at the
fourth-digit level of EC numbers and identified a set of putative SDRs (rf-SDRs) by using amachine-learning technique known as random forests [2]. For each enzyme in each CATH
homologous superfamily [3], binary predictors were constructed by random forests with full-length
sequence similarities and the residue similarities for active sites, ligand binding sites andconserved sites as input attributes. From the most highly contributing attributes, we obtained the
rf-SDRs. In a cross-validated benchmark assessment, EFPrf showed a prediction performancecomparable to that of a related method currently available (precision=0.98, recall=0.89). The
rf-SDRs included many residues, whose importance for specificity had been validated
experimentally. The analysis of the rf-SDRs revealed both a general tendency that functionallydiverged superfamilies tend to include more active site residues in their rf-SDRs than in lessdiverged suprefamilies, and more complicated relationships that the rf-SDRs strongly depend on
the mechanisms of functional diversification in each superfamily.
[1] Webb E. C., NC-IUBMB, Enzyme Nomenclature 1992, Recommendations of the Nomenclature
Committee of the International Union of Biochemistry and Molecular Biology on theNomenclature and Classification of Enzymes. Academic Press, 1992.
[2] Breiman L., Random Forests, Machine Learning Journal: 5-32, 2001.
[3] Orengo C. A., Michie A. D., Jones S., Jones D. T., Swindells M. B., Thornton J. M., CATH -a
hierarchic classification of protein domain structures-, Structure 5: 1093-1108, 1997.

C-1-02
Large-Scale Fragment Molecular Orbital Calculations
toward Evaluating the Drug Effects of AIDS Agents

1

2

Yoichiro Yagi 1,2

Yoshinobu Naoshima 1,2

yagi@sp.ous.ac.jp

naoshima@sp.ous.ac.jp

Institute of Natural Sciences, Okayama University of Science, 1-1 Ridai-cho,
Kita-ku, Okayama 700-0005, Japan
Graduate School of Informatics, Okayama University of Science, 1-1 Ridai-cho,
Kita-ku, Okayama 700-0005, Japan

Keywords: HIV-1 protease, AIDS agent, drug effects, FMO calculation, quantum biology
In our continuous efforts to evaluate and predict the drug effects of currently approved HIV-1
protease inhibitors, we have carried out a large-scale fragment molecular orbital (FMO)
calculation at FMO-MP2/6-31G level on the complexes of HIV-1 protease with five different
peptidomimetic HIV-1 protease inhibitors such as Ritonavir (RTV), Indinavir (IDV), Nelfinavir(NFV), Darunavir (DRV), and Saquinavir (SQV) using ABINIT-MP/BioStation system [1-3]. In
addition, we performed the analogous FMO calculations toward four different complexes of renin
and pepsin with RTV and SQV to explore a relationship between the computed energies and theunwanted side effects of HIV-1 protease inhibitors. Renin and pepsin are aspartic proteases andexist in human kidney and stomach, respectively. It has been reported that RTV gives acute renal
failure and severe gastrointestinal injury, while no renal toxicity is attributed to SQV.
FMO computations of five different inhibitor-bound HIV-1 protease complexes indicated thatthe calculated binding energy for the HIV-1 protease complexes can be correlated to the clinicallymeasured pharmacokinetic parameters such as maximum drug concentration (Cmax) and area
under the drug concentration-time curve (AUC) of the AIDS agents. We found that HIV-1 proteaseinhibitors having large values of Cmax and AUC such as RTV can strongly interact with HIV-1
protease, and that HIV-1 protease inhibitors with relatively large binding and interaction energies
can be highly effective as an AIDS agent [1-3]. When a drug was administered, it reaches its peaklevel in the blood. A drug travels through the blood to tissues in the body, is then metabolized andremoved from the blood. Cmax exhibits the highest drug level in the blood. AUC is universally
regarded as a measure of the extent of drug absorption. Although the reasons for the correlation of
the computed energy with Cmax and AUC are not clear, these two pharmacokinetic parameters
may be useful as indications for drug effects of AIDS agents [3]. For the human proteasecomplexes with RTV and SQV, the binding energy of RTV is larger than that of SQV. Furthermore,
it was found that the interaction energy of RTV with the active site aspartic acid in both humanproteases is large, while that of SQV with the active site is very small. We can safely say that thelarge interactions between the human proteases and RTV are implicated as a possible cause ofrenal dysfunction and severe gastrointestinal injury due to RTV inhibitor. Similar FMO4-MP2
calculations are under way to discuss the protease-inhibitor interactions in more detail.
[1] Yagi, Y., Iwasa, A., and Naoshima, Y., Large-Scale Quantum Chemical Calculation on the
Complexes of HIV-1 Protease and Inhibitors: A Relationship of Calculation Energies with Drug
Effects of Inhibitors, Proceedings of International Conference on Modeling and Simulation
Technology, JSST2011, JSST, 51-57, 2011.
[2] Yagi, Y. and Naoshima, Y., Large-Scale Quantum Chemical Calculations for the Drug Effects of
AIDS Agents, Abstracts of Joint Conference on Informatics in Biology, Medicine and Pharmacology
2012, C66_50, 2012[3] Yagi, Y., Hattori, Y., and Naoshima, Y., Large-Scale Quantum Chemical Calculation on the
Complexes of HIV-1 Protease and Inhibitors: A Relationship of Calculation Energies with Drug
Effects of Inhibitors, Trans. Japan Soc. for Simulation Tech., 4(2):41-50, 2012.

C-1-03
FMO calculations for nano-biotechnology
Yuji Mochizuki 1,2 Yoshio Okiyama 2 Kaori Fukuzawa 2,3
fullmoon@rikkyo.ac.jp okiyama@iis.u-tokyo.ac.jp kaori.fukuzawa@mizuho-ir.co.jp

Chiduru Watanabe 2 Kouichiro Kato 3 Takayuki Tsukamoto 3
chiduru@iis.u-tokyo.ac.jp kouichiro.kato@mizuho-ir.co.jp takayuki.tsukamoto@mizuho-ir.co.jp

Tatsuya Nakano 4 Shigenori Tanaka 5nakano@nihs.go.jp tanaka2@kobe-u.ac.jp
1

2

3

4

5

Department of Chemistry & Research Center for Smart Molecules, Faculty of Science,
Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo
153-8505, Japan
Mizuho Information & Research Institute Inc., 2-3 Kanda-nishiki-cho, Chiyoda-ku,
Tokyo 101-8443, Japan
Division of Medicinal Safety Science, National Institute of Health, 1-18-1 Kamiyoga,
Setagaya-ku, Tokyo 158-8501, Japan
Graduate School of System Informatics, Department of Computational Science, Kobe
University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan

Keywords: four-body corrected fragment molecular orbital (FMO4) method, solid, peptide
The fragment molecular orbital (FMO) method [1] has now been popular in the field ofcomputational biochemistry and pharmachemistry, where proteins with a few hundred amino acidresidues can be routinely computed within Kitaura’s original two-body expansion (FMO2) of fragments.
In Ref. [2], we developed the four-body extension (FMO4) in which the contribution from up to
fragment tetramers are included. FMO4 provides better accuracy than does FMO2, and furthermore it
makes the treatment of 3-dimensional cluster models of solids (with proper band-gap) tractable. The
applicability of FMO4 to solids is promising to attack various important problems in the
nano-biotechnology as an emerging field (e.g. fabrication of safer implants or protein-based chemicalsensors). In this context, we [3] recently reported the FMO4 calculations to investigate theinteraction between a designed peptide, with sequence of Arg1-Lys2-Leu3-Pro4-Asp5-Ala6[4], and the silica surface modeled by a large cluster model including 257 silicon atoms(under an explicitly hydrated condition). The electron correlation effect was taken into
account at the second-order perturbation (MP2) level with the Cholesky decomposition
technique for acceleration. The importance of three charged residues (Arg1, Lys2 and Asp4)
in the peptide-silica interaction was found, where the contributions from not only theelectrostatic attraction but also the charge-transfer were addressed. The poster will
resume the details of calculations and results in Ref. [3].Additionally, some preliminary
data about ionic solids such as apatite and alkali halide will be presented as well.

[1] The Fragment Molecular Orbital Method: Practical Applications to Large Molecular Systems,
edited by D. G. Fedorov, K. Kitaura (2009, Boca Raton, CRC press).
[2] "Development of the four-body corrected fragment molecular orbital (FMO4) method", T
Nakano, Y. Mochizuki, K. Yamashita, C. Watanabe, K. Fukuzawa, K. Segawa, Y. Okiyama, T.
Tsukamoto, S. Tanaka, Chem. Phys. Lett., 523 (2012) 128-133.
[3] "Modeling of peptide - silica interaction based on four-body corrected fragment molecularorbital (FMO4) calculations", Y. Okiyama, T. Tsukamoto, C. Watanabe, K. Fukuzawa, S. Tanaka,
Y. Mochizuki, Chem. Phys. Lett., 566 (2013) 25-31.
[4] "A Hexapeptide Motif that Electrostatically Binds to the Surface of Titanium", K. Sano, K.
Shiba, J. Amer. Chem. Soc. 125 (2003) 14234-24235.

C-1-04
A practical procedure to calculate intermolecularinteractions including statistical information between aprotein and a large ligand
Hiroyuki Sato 1

Kentaro Takai 2

shryk@jp.fujitsu.com

takai.kentaro@jp.fujitsu.com

Hajime Sugiyama 2

Yoshiaki Tanida 1

sugiyama.hajime@jp.fujitsu.com

tanida.yoshiaki@jp.fujitsu.com

Azuma Matsuura 1

Shunji Matsumoto 2

matsuura.azuma@jp.fujitsu.com

shun@jp.fujitsu.com

Hiroaki Suga 3
hsuga@chem.s.u-tokyo.ac.jp

1

2

3

Design Engineering Lab., Hardware Technologies Laboratories, Fujitsu Laboratories Ltd.,
10-1 Morinosato-Wakamiya, Atsugi 243-0197, Japan
Bio IT Development, Business Planning & Promotion Div., Healthcare & Educational Systems
Unit, Fujitsu Ltd., 1-9-3 Nakase, Mihama, Chiba 261-8588, Japan
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo,
7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

Keywords: Intermolecular interactions, Average structure, Water molecule, FMO, MD
We investigated the method to compute intermolecular interactions around the binding site in thesystem composed of the histone deacetylase SIRT2[1], a nonstandard cyclic peptide (S2iL5[2]) andsolvent, especially in view of the influence of water molecules.
First, we performed 1 s molecular dynamics (MD) simulation in the system and obtained anequilibrium time region corresponding to local fluctuations of SIRT2 from the MD trajectory. In
this region, we selected the water molecules whose RMSF values were as small as those of SIRT2.
These water molecules were considered to be less fluctuated and thus stable. In order to include
the statistical information in one typical structure, we averaged the structures of the complex withthe stable water molecules in the region and then optimized the obtained structure by the molecularmechanics method. Next, we investigated the conformation of the binding sites and calculated theintermolecular interaction energy by the fragment molecular orbital (FMO[3]) method. We found thatthe optimized structure reproduced the binding site that had been frequently caused in the
trajectory although the structure without the stable water molecules did not. We also found that theinteraction energy between SIRT2 and each residue of S2iL5 with the stable water molecules weredifferent from that without the stable water molecules by about 20 kcal/mol in the maximum. This
indicates that it is important for the intermolecular interaction by the quantum mechanical method
to determine the structure of the system including the stable water molecules.
A series of techniques presented here is considered to be a practical procedure to include statisticalinformation in the intermolecular interactions by quantum mechanical method, relating the quantum
mechanical method and the classical MD simulation.
[1] Finnin, M.S., et al., Nature Structual Biology, vol.8, num.7:621-625, 2001.
[2] Morimoto, J., Hayashi, Y., and Suga, H., Angew. Chem. Int. Ed., 51:3423-3427, 2012.
[3] Kitaura, K., et al., M., Chem., Phys., Lett., 313:701-706, 1999.

C-1-05
Analysis of antibody-antigen interactions andprediction of their complex structures

1

Yuko Tsuchiya 1

Kenji Mizuguchi 1

tsuchiya@nibio.go.jp

kenji@nibio.go.jp

National institute of biomedical innovation, 7-6-8 Saito-Asagi, Ibaraki-city,
Osaka 567-0085, Japan

Keywords: Antibody-antigen interaction, Complex structure prediction, Drug design
A deeper understanding of antibody-antigen interactions is essential for the design of antibody
drugs. To design an antibody drug having a high affinity with the target antigen, it is desirable toobtain an antibody structure in complex with its target antigen. Because it is generally not easy todetermine protein complex structures by experimental methods, such as X-ray crystallography and
NMR, model structures of protein complexes predicted by bioinformatics techniques are
considered to be important for drug discovery.
Previous studies have found canonical conformations of antibody proteins, in particular,
Complementarity-Determining Regions, based on the analyses of their sequences and structures[1,2]. In terms of antibody-antigen interactions, an antigen binding site on an antibody, paratope,
and an antibody binding site on an antigen, epitope, have been analyzed and several epitope
prediction methods have been developed [3].
In this study, we performed a careful analysis of antibody-antigen interactions from multiple
perspectives, and developed a method for predicting paratopes and epitopes separately, towards
accurate prediction of antibody-antigen complexes suitable for drug design. We are now
developing a method for predicting antibody-antigen complex structures, where a probablecombination of the predicted paratope and epitope sites is selected based on a scoring function
constructed from our new observations, combined with the knowledge of antibody-antigen
interactions elucidated previously.
[1] Chothia, C., and Lesk, A.M., Canonical Structures for the Hypervariable Regions of
Immunoglobulins, J Mol Biol, 196:901-917, 1987.
[2] Shirai, H., Kidera, A., and Nakamura, H., H3-rules: identification of CDR-H3 structures inantibodies, FEBS Letters, 455:188-197, 1999.
[3] Andersen, P.H., Nielsen, M., and Lund, O., Prediction of residues in discontinuous B-cell
epitopes using protein 3D structures, Protein Science, 15:2558-2567, 2006.

C-1-06
Screening and Design of Small Molecules that Reversibly
Bind to Streptavidin
Moe Kohno 1
2470493421@mail.ecc.u-tokyo.ac.jp

Takayoshi Okabe 3
tokabe@mol.f.u-tokyo.ac.jp

1
2
3

Takuya Terai 1

Shigeru Sugiyama 2t-terai@mol.f.u-tokyo.ac.jp

sugiyama@chem.eng.osaka-u.ac.jp

Tetsuo Nagano 3

Yasuteru Urano 1

tlong@mol.f.u-tokyo.ac.jp

uranokun@m.u-tokyo.ac.jp

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7 -3-1, Hongo,
Bunkyo-ku, Tokyo 113-8654, Japan
Graduate School of Science, Osaka University, 1-8, Yamadaoka, Suita-shi,Osaka 565-0871,
Japan
Open Innovation Center for Drug Discovery, The University of Tokyo, 7 -3-1, Hongo,
Bunkyo-ku, Tokyo 113-8654, Japan

Keywords: Ligand docking, Protein-small molecule interaction
The interaction between streptavidin (from Streptmyces avidinii) and biotin is known as thestrongest non-covalent interaction in nature (K d = 10 -13 M) 1 . Streptavidin-biotin system is used formany applications in biotechnology, such as affin ity column chromatography (isolation), SPR
sensor chip (immobilization) or fluorescence imaging (detection). However, for some applications,a few disadvantages of this system have been pointed out. One of the major limitations of thissystem is the almost irreversible binding under physiological conditions. To disrupt a
streptavidin-biotin complex, it is necessary to denature the streptavidin using harsh condition.
In order to make the binding of biotin with streptavidin more easily reversible under mild
conditions, much work has been done to modify streptavidin by genetic mutations. In contrast,
another approach to develop biotin analogs with reduced affinity to streptavidin has been hardly
reported, and the reported biotin analogs provide only partial reversibility. In this research, we
screen a large chemical library to search for novel chemical scaffolds that bind to streptavidin, andaim to optimize the hit compounds based on information from X-ray crystal structure.
First, we performed the screening for avidin (from hen egg) which has identical tertiary structurewith streptavidin. 166,480 compounds were screened by using fluorescence polarization, and weselected 773 compounds as primary hits. These compounds were subsequently screened for
streptavidin, dose-dependently assayed, and analyzed by SPR (Surface Plasmon Resonance). Manybiotin analogs were detected as hits, but we excluded these compounds. Finally, 6 compounds wereselected as novel ligand scaffolds for streptavidin which exhibited fully reversible binding to theprotein (K d = 10 -4 ~10 -6 M). We characterized the binding mode of compound 1 which showed thestrongest affinity (Figure). Based on information from X-ray crystal structure and derivativesynthesis, the amide moiety fixed in cis form was most
likely to be essential for the binding. We synthe size
further derivatives of compound 1 and optimizing theaffinity to streptavidin. Then we will demonstrate theutility of the new reversible scaffold by applying it for
affinity column chromatography.
Blank Subtracted Sensorgrams

RU

35
30
25

Response

20
15
10

5
0

-5

-10

-50

0

50

100

150

[1] N. M. Green, Adv. Protein Chem., 29:85-133, 1975.

RUmax

Tim e

Kd = 3.90×10-6 M
0

10

20
[Compound] (μM)

30

40

200

250
s

C-1-07
Standard binding free energy calculation fortheophylline-RNA aptamer system: alchemicaltransformation and metadynamics
Yoshiaki Tanida 1

Takashi Mitsui 2

tanida.yoshiaki@jp.fujitsu.com

tmitsui@jp.fujitsu.com

Azuma Matsuura 1
matsuura.azuma@jp.fujitsu.com

1
2

Fujitsu Laboratories Ltd., Atsugi, Kanagawa 243 -0197, Japan
Fujitsu Ltd., Mihama, Chiba 261-8588, Japan

Keywords: Standard free energy, Alchemical transformation, Metadynamics
Binding affinity of theophylline to RNA apatamer (PDB id: 1EHT) is calculated using alchemicaltransformation, in which the interactions of the ligand with its surroundings are decreased to zero[1]. The parmbsc0 force field is used for describing potential functions of RNA to improve theaccuracy of simulation [2]. A quadratic potential restraining the translation of the ligand is added
to keep the relation to the standard state condition. The calculated binding free energy is -8.9
kcal/mol, which agrees well with the experimental value of -8.9 kcal/mol. This result indicates thatexperimental binding energy is well reproduced by simulations for alchemical route.
In order to realize the computer-aided drug design (CADD), understanding of the kinetics ofassociation/dissociation phenomena is also necessary. Then, we perform metadynamics simulation
to explore the free energy surface (FES) starting from the equilibrated binding geometry [3]. The
distance between U23(C1’) and TEP(C8) and the angle of G26(C6)-TEP(C2)-TEP(C8) are chosen
as a set of collective variables (CVs) used here as shown (Figure 1). Metadynamics is a dynamics
in the space of the CVs. In Figure 2, the FES shows a physical path for the ligand from the bindingsite to the external solution. A
barrier from the binding site to theunbound state is estimated to be
U23(C1’)13.5 kcal/mol while taking theexperimental binding affinity into
TEP(C8)account. This value gives us
G26(C6)
information for recognizing thedissociation process of the ligandfrom the binding site and for
TEP(C2)
ligand optimization.
It is shown that alchemical
Figure 2. Free energy surface
transformation and metadynamics
reconstructed using metadynamics
Figure 1. Theophylline-RNA
simulation are powerful methods
as a function of the angle
binding geometry after
G26(C6)-TEP(C2)-TEP(C8) and offor exploring the properties of
equilibrium procedure.
the distance U23(C1’)-TEP(C8).
Particular
atoms
used
are
ligand in rational drug design.
also shown.

[1] S. Boresch, F. Tettinger, F. Leitgeb, and M. Karplus, J. Phys. Chem. B, 107: 9535-9551, 2003.
[2] A. Perez, I. Marchan, D. Svozil, J. Sponer, T. E. Cheatham III, C. A. Laughton, and M. Orozco,
Biophys. J., 92: 3817-3829, 2007.
[3] F. L. Gervasio, LA. Laio, and M. Parrinello, J. Am. Chem. Soc., 127: 2600-2607, 2005.

C-1-08
Molecular Dynamics Simulation of Shiga Toxin
Kazumi Omata 1

Hisashi Okumura 2,3

komata@hosp.ncgm.go.jp

hokumura@ims.ac.jp

Yoshiharu Mori 2

Kiyotaka Nishikawa 4

ymori@ims.ac.jp

1
2
3
4

knishika@mail.doshisha.ac.jp

Medical Sociology Section, National Center for Global Health and medicine , 1-21-1 Toyama,
Shinjuku, Tokyo 162-0052, Japan
Institute for Molecular Science, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, JapanThe Graduate University for Advanced Studies, 38 Nishigonaka, Myodaiji, Okazaki, Aichi
444-8585, Japan
Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakotani, Tatara, Kyotanabe,
Kyoto 610-0394, Japan

Keywords: Shiga toxin, Ligand docking, Molecular dynamics, O157
To develop a drug against infection by Shiga toxin (Stx) -producing Escherichia coli O157:H7 [1],this study investigates docking of Stx2 B subunit (Stx2 B) an d a peptide neutralizer, using
molecular dynamics simulations.
The used software is Generalized-Ensemble Molecular Biophysics (GEMB) [2]. The PDB code
1R4P is employed for the initial structure of Stx2 B, and the amino acid sequence of the neutralizer
is MAPPPRRRRA. The number of residues of Stx2 B is 350, so that the required number of watermolecules is estimated to be about 20,000. The force field is AMBER99SB for Stx2 B and TIP3P
for water molecules.
Several simulations have shown that Arg's included in the neutralizer are attracted to Asp's andGlu's located at the binding sites of Stx2 B. In a representative result, among the amino acids in thesequence R9-R8-R7-R6-P5-P4-P3 of the neutralizer, R9 and R8 have electrostatic interactions with
E15 and D16 of Stx2 B, respectively; R7 and P4 are thought to have small effects on the binding
because they are directed towards the outside of Stx2 B from its inside; although R6 interacts with
W33 through its alkyl part, this interaction is not electrostatic; P5 is located at the foot of R6 andnot participating the binding; it is possible that P3 interacts with alkyl of R32, but the distance
between them may be large. It can be suggested that two effects are important in simulations
considering water molecules: screening of electrostatic interactions betw een basic and acidic
residues; existence of hydrophobic residues in the neutralizer.
[1] Nishikawa, K., Watanabe, M., Kita, E., Igai, K., Omata, K., Yaffe, M.B., and Natori, Y., A
multivalent peptide library approach identifies a novel Shiga toxin inhibitor that inducesaberrant cellular transport of the toxin, The FASEB Journal, 20:2597-9, 2006.
[2] Okumura, H., Temperature and pressure denaturation of chignolin: Folding andunfolding simulation by multibaric-multithermal molecular dynamics method , Proteins,
80: 2397-2416, 2012.

C-1-09
Random Matrix Theory Analysis of Molecular Dynamics
Simulation
Masanori Yamanaka 1
yamanaka@phys.cst.nihon-u.ac.jp

1

Department of Physics, College of Science and Technology, Nihon University, 1-8
Kanada-Surugai, Chiyoda-ku, Tokyo 101-8308, Japan

Keywords: Molecular dynamics, All atom MD with solvent, Random matrix theory, Principal
component analysis, Protein-ligand interaction, Protein-protein interaction, Domain decomposition,
Correlation matrix, Variance-covariance matrix
We apply the random matrix theory [1,2,3] to analyze the time series data of motion of atoms ofproteins which is produced by the molecular dynamics simulation [4]. We perform all-atom
molecular dynamics simulation with solvent with the maximum duration of 1000ns. We study adata set with the different sampling-time intervals, 0.01fs, 0.1fs, 1fs, 10fs, 100fs, 1ps, and 10ps toobserve the characteristic motion at each time scale. The variance-covariance matrices areconstructed from the time series data. We calculate the probability density of thevariance-covariance matrix, eigenvalue distribution, unfolded eigenvalue distribution of thenearest-neighbor and the next nearest-neighbor level spacings, inverse participation ratio, etc.
They are the fundamental quantities which charact erize the universality class in the random matrix
theory. The results of the nearest-neighbor and the next nearest-neighbor level spacings agree wellwith the Gaussian orthogonal and Gaussian symplectic ensembles, respectively. On the other hand,
the raw eigenvalue distribution has a crossover behavior between the universal and non -universal
classes as a function of the sampling-time interval. Following the random matrix theory, weclassify the dynamically-correlated sectors of the protein domain by analyzing the eigenvalues
outside of the bulk and the inverse participation ratio, and decompose them into subsectors. Our
method is an attempt to improve and refine the principal compo nent analysis of protein domain [5].
As an example, we demonstrate our method in the bovine eye lens protein gamma-B(gamma-II)-crystallin, PDBID:4GCR to study the domain decomposition, and ubiquitin carboxy
terminal hydrolase L1 bound to ubiquitin vinylmethylester, PDBID:3KW5 to study theprotein-ligand and protein-protein dynamical interactions.
[1] E.P. Wigner, Proc. Cambridge Philos. Soc. 47, 790, 1951; F.J. Dyson, J. Math. Phys. 3, 140,
1962; F.J. Dyson and M.L. Mehta, J. Math. Phys. 4, 701, 1963; M.L. Mehta and F.J. Dyson, J.
Math. Phys. 4, 713, 1963.
[2] L. Laloux, P. Cizeau, J.-P. Bouchaud, and M. Potters, Phys. Rev. Lett. 83, 1467, 1999; V. Plerou,
P. Gopikrishnan, B. Rosenow, L.A.N. Amaral, and H.E. Stanley, Phys. Rev. Lett. 83 , 1471, 1999;
V. Plerou, P. Gopikrishnan, B. Rosenow, L.A.N. Amaral, T. Guhr and H.E. Stanley, Phy s. Rev.
E65, 066126, 2002.
[3] The first attempt to the protein is reported by R. Potestio, F. Caccioli, and P. Vivo, Phy. Rev.
Lett. 103, 268101, 2009 by the solvable elastic network models. Here, we perform all-atom
molecular dynamics simulation with water.
[4] M. Yamanaka, Journal of the Physical Society of Japan 82, 083801, 2013.
[5] For a review, see Akio Kitao, Normal Mode Analysis: Theory and Applications to Biologicaland Chemical Systems. Chapter 12: edited by Qiang Cui & Ivet Bahar, , Chapman & Hall/CRC,
Boca Raton, FL, USA, 233-251, 2006, and references therein.

C-1-10
Binding Mechanism of KNI-272 with HIV-1 Protease
Takahisa Hayashi*, Takuya Sugimoto, Shuhei Sakamoto, Akihiro Kawano, Tatsusada Yoshida, Hiroshi Chuman*c400803063@tokushima-u.ac.jp

Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78 Shomachi, Tokushima, 770-8505, Japan

Keywords: FMO, HIV-1 protease, neutron crystallography, structural water, X-ray crystallography
The complex of structure of HIV-1 protease (HIV-1 PR) with a transition-state analog inhibitor KNI-272 hasbeen determined recently by both neutron and high-resolution X-ray crystallographies. As shown in Figure 1,
the complex structure contains several structural water molecules, which can mediate hydrogen-bonding
interactions between HIV-1 PR and KNI-272. [1] In this study, MM/MD and ONIOM (QM/MM)(M06-2X/6-31G*: Amber) calculations were carried out to quantitatively examine possible roles of thesestructural water molecules. After the ONIOM optimization, we performed the FMO-Inter Fragment Interaction
Energy (FMO-IFIE： HF/6-31G*) analysis of KNI-272 with amino acid residues and structural water molecules, andexamined the energitic contribution of each structural water molecule in the binding. Catalytic Asp25/25’
residues have the largest contribution to the binding. Figure 2 shows FMO-IFIE together with the classicaldispersion ones (Edisp). As can be seen in Figure 2, W1 (water molecule 1 shown in Figure 1), intervening
between KNI-272 and Ile50/50’ in HIV-1 PR, makes the most potent hydrogen-bonding interaction. W2 andW3 (water molecules 2 and 3) are also important for the binding of KNI-272 with between HIV-1 PR
through hydrogen-bonding/electrostatic interactions. These results confirm that these structural watermolecules inside HIV-1 PR play a decisive role in the binding of KNI-272 with HIV-1 PR, and suggest that
it is very necessary to consider the “hydrogen-bonding/electrostatic network” among an inhibitor, HIV-1-PRand structural water molecules, when designing a new potent inhibitor of HIV-1 PR.

Figure 1. Complex of HIV-1 PR with KNI-272.
Atoms in shadow areas are treated as thequantum region in the ONIOM optimization.

Figure 2. FMO-IFIE and Edisp of KNI-272 with structural
waters in HIV-1 PR.

[1] M. Adachi et al., Proc. Natl. Acad. Sci.USA, 2009, 106, 4641–4646.

C-1-11
LERE-QSAR Analysis of Binding of -Lactum Hydroxamic Acid
Derivatives with Tumor Necrosis Factor-Alpha Converting Enzyme
Koh Nonoshita * , Takuya Sugimoto, Ayana Hamano, Akihiro Kawano, Shuhei Sakamoto,
Tatsusada Yoshida, and Hiroshi Chuman*c400903038@tokushima-u.ac.jp

Graduate School of Pharmaceutical Sciences, The University of Tokushima ,1-78 Shomachi, Tokushima 770-8505, Japan

Keywords: LERE, ONIOM, Tumor necrosis factor-alpha converting enzyme

ΔGobs = 0.129 [ΔEbind(ONIOM/HF/ME) + Edisp+ ΔGsolpol] + 16.1
n = 11, r = 0.912, s = 0.844 kcal/mol

Gly349

Leu348ON
H

Glu406

O
O
O

H
O

Me O

H
N

N

Leu401
H

N

Me

O HN
O

NH2
X Asn447
His405
Zn
Val440Ser441N
N
His409
N H O
H O
HN
H N
N
N
H
Ala439 H
O
His415OH
HN N

O

Figure 1. Schematic representation of a -lactum
hydroxamic acid derivative bound in the TACE
active site. Atoms in shadow areas are treated as thequantum region in the ONIOM calculation.
20
10

DGobs
DGcalcEdisp

DGsolpolDEbind(ONIOM/HF/ME)a. X = H, b. X = isobutyl, c. X = methoxy

Energy (kcal/mol)

Tumor necrosis factor-alpha converting enzyme(TACE) is a metal enzyme that contains a zinc atom,
and TACE converts TNF-alpha into an activatedform by hydrolysis. Rheumatism arthritis andCrohn's disease are caused by overproduction of theactivated form TNF-alpha. In this study, we
examined the atomic and electronic mechanism
underlying binding between TACE and hydroxamic
acid derivatives, which have a -lactum ring [1](Figure 1), using the ONIOM calculation and LERE
(Linear Expression by Representative Energy
terms)-QSAR procedure [2]. We constructedcomplex structures of TACE with each hydroxamic
acid derivative using the MM/MD and ONIOM
calculations. In the current study, we assumed thatthe observed overall free-energy change (ΔGobs) inthe complex formation can be expressed as a sum of
the intrinsic interaction energy (ΔEbindONIOM) andpolar contribution of the solvation free-energychange (ΔGsolpol), and dispersion interaction energychange(Edisp),calculated
with
the
ONIOM(HF/6-31G: Amber) mechanical embedding(ME) method, PB calculation, and MM calculation,
respectively. The sum of these three representative
energy terms is nicely linear with ΔGobs, resulting in
the following LERE-QSAR equation;

0-10
d. X = allyloxy, e. X = benzyloxy,
f. X = phenoxy, g. X = 4-nitrobenzyloxy,-20 h. X = 3, 5-di-Me-benzyloxy,
i. X = 3, 5-di-CF3-benzyloxy,
j. X = 2-methyl-4-quinolinylmethoxy (IK-682)-30

Figure 2 shows that there is a negative correlation
between ΔEbind(ONIOM/HF/ME) and ΔGsolpol, andthat ΔGobs parallels with Edisp. These results suggestthat the variation of ΔGobs among the derivatives is-40
governed by Edisp. We also discuss a detailed bindinga b c d e f g hij DPCmechanism by decomposing the binding energy
polobtained with the FMO calculation into individual Figure 2. Variation in ΔGobs, ΔGcalc,Edisp, ΔGsol ,and ΔEbind(ONIOM/HF/ME).
contributions of amino acid residues.
[1] J. J. W. Duan, et al., J. Med. Chem. 2002, 45, 4954–4957.
[2] T. Yoshida, S. Hitaoka, A. Mashima, T. Sugimoto, H. Matoba, and H. Chuman, J. Phys. Chem. B, 2012, 116,
10283–10289.

C-1-12
Spatial distribution of QM based interaction energy
between amino-acids and probe molecules
Hirofumi Watanabe, Tomohiro Sato, Daisuke Takaya, Hitomi Yuki, Noriaki Hashimoto,
Keiko Kumazawa and Teruki Honma
hirowatanabe@ssbc.riken.jp, Honma@gsc.riken.jp

RIKEN, Center for Life Science Technologies, 1-7-22 Suehiro-cho Tsurumi-ku Yokohama
Kanagawa 230-0045, Japan

Keywords: Quantum Chemical Calculation, Interaction energy, Molecular mechanics force field
Quantum chemical calculation and molecular dynamics simulation are used for prediction of theprotein-ligand binding affinity [1]. First one is used as QM based single point calculation, which
can deal with the molecular interactions accurately but have the problems in sampling. Second one
is performed with MM force field, which has the problem in accuracy of interactions. Thus wehave to use both QM based and MM based methods and select better one for adjusting the situation.
For this purpose, we have to know how different between QM and MM for each situation. Current
comparative studies between QM and MM, are limited on the potential minimum or along few
directions. Thus, in this study, we evaluated interaction energies between amino acid residues andprobe molecules with spatially comprehensive arrangement. We employed B97D/aug-cc-pVDZ
method [2] because we confirmed this method provides only small deviation from higher level
quantum chemical calculation (CCSD(T)/CBS) [3]. We compared QM based interaction energy andMM force field energy (MMFF94x) for each amino acid and different types of probe molecules.
Figure 2 represents example of calculation for interaction energy PHE side chain and cation probe(NH 4 + ). QM calculation provides spatially larger attractive region and deeper potential minimum.
Comparison between various types of interaction will be discussed.

Figure1. Evaluation scheme of spatial
distribution of QM based interation energy.

Figure2. Contour plot of interaction energy(Phe-NH 4 + )with
QM(B97D:red)andMM(MMFF94x:blue).

[1] H. Watanabe et al. CBI Journal 10 (2010) 32-45.
[2] S. Grimme, J. Comput. Chem. 27, (2006), 1787-1799.
[3] The Benchmark Energy & Geometry Database (http://www.begdb.com/)C-1-13
Estimation of Non-covalent Interactions witha New Efficient Dispersion Corrected HF Approach
Tatsusada Yoshida 1, *, Akira Mashima 1 , Katsunori Sasahara 1 ,Yuto Shibata 1 , Masahiro Eguchi 1 , Seiji Hitaoka 2 , and Hiroshi Chuman 1*tyoshida@tokushima-u.ac.jp
1

Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78-1 Shomachi, Tokushima
770-8505, Japan
2
Institute for Materials Chemistry and Engineering and International Research Center for Molecular System,
Kyushu University, Fukuoka 819-0395, Japan

Keywords: BSSE, Dispersion, Hartree–Fock, LERE

[1] T. Yoshida, Y. Munei, S. Hitaoka, H. Chuman. J. Chem. Inf. Model., 2010, 50, 850–860.

M06-2X/6-31+G(d)B3LYP-D2/6-31+G(d)B2PLYP-D/6-31+G(d)MP2/6-31+G(d)HF-D/6-31+G(d)HF/6-31+G(d)MAE (kcal/mol)

An accurate estimation of binding interaction energy associated with complex formation of aligand and protein is important for understanding the binding mechanism and rational drug design.
Although the dispersion is one of the most important interaction as well as hydrogen-bonding andelectrostatic ones, its evaluation based on molecular orbital methods is still not easy task. In thisstudy, we examined our newly proposed dispersion correction for the Hartree–Fock method (HF-D)
on several complexes of biologically relevant molecules.
We selected a set of eighteen small non-covalently bonded complexes: six dispersion-dominated(DISP), nine hydrogen-bonded (HB), and three other types of complexes (others). The geometry ofeach complex was fully optimized at the MP2/6-311++G(d,p) level. In our HF-D approach, the HF
energy was corrected by introducing damped empirical dispersion energy term (f(R)C 6 /R 6 ) using asigmoid-type function. We calculated interaction energies using HF-D and the counterpoisecorrections were employed to reduce the basis set superposition errors (BSSE). The performanceof HF-D were confirmed by comparing with post-HF methods (CCSD(T) and MP2) and several
DFT based methods (M06-2X and Grimme’s dispersion corrected B3LYP-D2 and B2PLYP-D).
Figure 1 shows the mean absolute error (MAE) of
2.5
interactionenergies
estimatedfromthe
DISP (n = 6)
CCSD(T)/aug-cc-pVTZ reference values. The overall
HB (n = 9)
2.0
performance of HF-D is excellent; as expected, HF-D
others (n = 3)
more accurately reproduces interaction energies foroverall (n = 18)
all three types of complexes than the conventional HF
1.5
method. It should be noted that MAE of HF-D shows
significantly smaller than that of more expensive MP2
1.0
in all complexes (MAE(overall) = 0.40 and 1.01
kcal/mol, respectively). Although M06-2X gives thebest performance (MAE(overall) = 0.30) among the
0.5
tested methods, HF-D is competitive with the M06-2X
and other dispersion corrected B3LYP-D2 and
0.0
B2PLYP-D. HF-D approach is probably effective andpractical, compared with time-consuming post-HF
and DFT methods to quantitatively evaluate theinteraction energy of large molecular systems such ascomplex of a ligand with protein. We will also discussthebindingmechanismofaseriesofbenzenesulfonamides with carbonic anhydrase using Figure 1. Comparison of the performance of
LERE-QSAR procedure [1] in which HF-D approach HF-D and other methods for non-covalently
is introduced for estimation of binding interaction bonded complexes.
energy.

C-1-14
Refinement of Crystal Structures Using Partial Geometry
Optimization and Electron Density Calculations Based on
the Fragment Molecular Orbital Method.
Kaori Fukuzawa 1,2
kaori.fukuzawa@mizuho-ir.co.jp

Chiduru Watanabe 2
chiduru@iis.u-tokyo.ac.jp

Shigenori Tanaka 3
tanaka2@kobe-u.ac.jp

1
2
3
4

Naoki Watanabe 1
naoki.watanabe@mizuho-ir.co.jp

Yoshio Okiyama 2okiyama@iis.u-tokyo.ac.jp

Yuji Mochizuki 2,4
fullmoon@rikkyo.ac.jp

Mizuho Information & Research Institute Inc., 2 -3 Kanda Nishiki-Cho, Chiyoda-ku, Tokyo
101-8443, Japan
Institute of Industrial Science, The University of Tokyo, 4 -6-1 Komaba, Meguro-ku, Tokyo
153-8505, Japan
Graduate School of System Informatics, Department of Computational Science, Kobe
University, 1-1, Rokkodai, Nada-ku, Kobe 657-8501, Japan
Department of Chemistry & Research Center for Smart Molecules, Faculty of Science, Rikkyo
University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan

Keywords: Fragment molecular orbital (FMO) method, Protein-ligand interaction, Geometry
optimization, Electron density
The ab initio fragment molecular orbital (FMO) method was applied to refine X-ray crystal
structures of protein–ligand complex using ABINIT-MP/BioStation program package [1]. Partial
geometry optimization surrounding a ligand and electron density calculations were performed at
the MP2/6-31G* level for complex between human estrogen recepror  and 17-estradiol (PDB
ID: 3ERE) [2]. Members of hydrogen bond network were optimized: ligand, Glu353, Arg394,
Phe404, His524 and a water molecule. In addition to the inter-fragment interaction energy (IFIE),
the configuration analysis for fragment inter action (CAFI) was performed to analyze chargetransfer interactions on the basis of optimal hydrogen bond network structures. Strong chargetransfer interaction between the ligand and Glu353 was observed through a “shared proton” withlong OH distance of ligand (1.07 Å) and short ligand - Glu353 hydrogen bond distance (1.42 Å).
Such findings are consistent with the existence of considerable experimental electron density [3]between them. Three protonation states of His524 (HIE, HID, HIP) were also considered for the
calculations. Based on the optimized structure, strength of interaction, and electron density map,
we concluded His524 is most likely deprotonated HIE conformation.

[1] ABINIT-MP/BioStation download site; http://www.ciss.iis.u-tokyo.ac.jp/dl/index.php.
[2] Fukuzawa, K., Watanabe, N., Watanabe, C., Okiyama, Y., Tanaka, S., and Mochizuki, Y., in
preparation.
[3] EDS electron density server (http://eds.bmc.uu.se/eds).
Acknowledgement:
A portion of this research was supported by the grant for “Strategic Programs for Innovative
Research” Field No. 4: Industrial Innovations from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT)’s “Development and Use of Advanced, High -Performance,
General-Purpose Supercomputers Project.”C-1-15

FMO calculations with ABINIT-MP on K-computer
Yoshio Okiyama1

Chiduru Watanabe1

Kaori Fukuzawa1,2

okiyama@iis.u-tokyo.ac.jp

chiduru@iis.u-tokyo.ac.jp

kaori.fukuzawa@mizuho-ir.co.jp

1
2
3
4

Shigenori Tanaka3

Yuji Mochizuki1,4

tanaka2@kobe-u.ac.jp

fullmoon@rikkyo.ac.jp

Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku,
Tokyo 153-8505, Japan
Mizuho Information & Research Institute Inc., 2-3 Kanda Nishiki-Cho, Chiyoda-ku,
Tokyo 101-8443, Japan
Graduate School of System informatics, Kobe University, 1-1 Rokkodai, Nada-ku,
Kobe 657-8501, Japan
Faculty of Science, Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo
171-8501, Japan

Keywords: fragment molecular orbital (FMO) method, many-body expansion, Cholesky decomposition, massively parallel computing, K-computer
The K-computer, one of the fastest peta-scale supercomputers in the world, processes huge amountsof tasks at once with its massive parallelism and have started being utilized for drug discovery primarily
based on the classical mechanical approaches.
On the other hand, fragment molecular orbital (FMO) method proposed by Kitaura et al. in 1999
has revealed the electronic properties of large biomolecular systems quantum-mechanically and has agreat expectation for application to drug discovery. Especially, the four-body corrected FMO method(FMO4) [1], which is implemented to an FMO processing program, ABINIT-MP, enables us to divide
a molecular system more finely with keeping good accuracy and analyze inter-fragment interactions
with higher resolution favorable to the structure-based drug design (SBDD) [2]. While the number oftetrameric combinations of fragments needed for four-body correction voluminously increases as thewhole system size, one can simultaneously complete all the tasks of processing the combinations in a
moment by using the K-computer.
The Cholesky decomposition (CD) technique for two-electron repulsion integral approximation [3]is essential to accelerate the second-order Moeller-Plesset perturbation processes in FMO calculations
by keeping the Cholesky basis on memory and executing the DGEMM operations. For saving memory
and further acceleration, we introduce the CD approximation with one-center Cholesky basis (1CCD) [4], which limits the atomic orbital pairs as the Cholesky basis to those on the same atoms. The
computational times in Hartree-Fock processes are also reduced successfully by applying the 1C-CD
approximation.
Details of our trials and applications with ABINIT-MP on the K-computer will be shown in theposter session.
Acknowledgment This research was supported by the grant for “Strategic Programs for Innovative
Research” Field No. 4: Industrial Innovations from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT)’s “Development and Use of Advanced, High-Performance, GeneralPurpose Supercomputers Project.”
References[1] Nakano, T., et al., Chemical Physics Letters, 523:128–133, 2012.
[2] Watanabe, C., et al., Journal of Molecular Graphics and Modelling, 41:31–42, 2013.
[3] Okiyama, Y., et al., Chemical Physics Letters, 490:41–45, 2010.
[4] Aquilante, F., et al., Journal of Chemical Physics, 127:114107, 2007.

C-1-16
Molecular Simulation Analysis of Glucuronidation of
UDP-glucuronosyltransferase 1A7 and 1A10
Kenji Miura 1

Mika Ohta 1080m812m@stu.kobe-u.ac.jp

ohtam@med.kobe-u.ac.jp

Aki Sugano 1
sugano@med.kobe-u.ac.jp

Eiichi Maeda 1

Toshiyuki Sakaeda 2

Yutaka Takaoka 1

emaeda@med.kobe-u.ac.jp

sakaedat@pharm.kyoto-u.ac.jp

ytakaoka@med.kobe-u.ac.jp

1

2

Division of Medical Informatics and Bioinformatics, Kobe University Hospital, 7-5-2
Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
Center for Development Integrative Education in Pharmacy and Pharmaceutical Science,
Graduate School of Pharmaceutical Science, Kyoto University, 46-29
Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

Keywords: UDP-glucuronosyltransferase, Ligand docking, conjugation capacity
UDP-glucuronosyltransferase 1A (UGT1A) constitute an endoplasmic reticulum(ER)
membrane-bound enzyme family whose members catalyze glucuronidation. We previously
analyzed the bilirubin glucuronidation by UGT1A isoform 1 (UGT1A1) by means of (1)calculation of 3-D structure of mutant UGT1A1, (2) docking simulation of UGT1A1 and its
coenzyme (UDPGA), (3) induced fit of UGT1A1-UDPGA complex, and (4) docking simulation of
UGT1A1 and bilirubin. We found that the ratio of the docking when the hydroxyl group ofbilirubin oriented toward UDPGA correlated with in vitro conjugation capacity report previously[1]. Based on this molecular mechanism that glucuronidation by UGT1A1 requires the hydroxyl
group of ligand to locate toward the coenzyme [2], we reported the method to predict the
conjugation capacity of UGT1A1 by our molecular simulation [3]. In this study we analyzed the
glucuronidation of other UGT1A isoforms (UGT1A7 and UGT1A10) by our molecular simulation.
As a result, we found that ratio of the binding of coenzyme to the enzyme in reactive orientationand the ratio of the ligand orientation whose hydroxyl group locate toward the coenzyme also
correlated with in vitro conjugation capacity [4,5]. This result suggests that the conjugation
capacity of other UGT1A isoforms is determined by the similar mechanism as UGT1A1.
[1] Yamamoto K. et al., Contribution of two missense mutations (G71R and Y486D) of thebilirubin UDP glycosyltransferase (UGT1A1) gene to phenotypes of Gilbert’s syndrome andCrigler-Najjar syndrome type II, Biochim Biophys Acta 1406:267-273, 1998.
[2]
Takaoka
Y. et al., Ligandorientation
governs
conjugation
capacityofUDP-glucuronosyltransferase 1A1, Journal of Biochemistry, 148(1):25-28, 2010.
[3] Takaoka Y. et al., Method for predicting enzyme activity by computer simulation,
PCT/JP2009/004286, 2009.
[4] Villeneuve L. et al., Novel Functional Polymorphisms in the UGT1A7 and UGT1A9
Glucuronidating Enzymes in Caucasian and African-American Subjects and Their Impact on the
Metabolism of 7-Ethyl-10-hydroxycamptothecin and Flavopiridol Anticancer Drugs, The
Journal of Pharmacology and experimental therapeutics, 307(1):117-128, 2003.
[5] Dellinger R.W. et al., Importance of UDP-glucuronosyltransferase 1A10 (UGT1A10) in thedetoxification of polycyclic aromatic hydrocarbons: decreased glucuronidative activity of theUGT1A10 139Lys isoform, Drug Metabolism and Disposition, 34(6):943-949, 2006.

C-1-17
FMO-based cluster analysis for drug design
by multi-dimensional scaling
Ryo Kurauchi1

Shigenori Tanaka1

kurauchi@eniac.scitec.kobe-u.ac.jp

tanaka2@kobe-u.ac.jp

Kaori Fukuzawa2
kaori.fukuzawa@mizuho-ir.co.jp

Yoshio Okiyama 3okiyama@iis.u-tokyo.ac.jp
1
2
3
4
5

Akifumi Kato 2

Chiduru Watanabe 3

akifumi.kato@mizuho-ir.co.jp

chiduru@iis.u-tokyo.ac.jp

Yuji Mochizuki 4

Tatsuya Nakano 5

fullmoon@rikkyo.ac.jp

nakano@nihs.go.jp

Graduate School of System Informatics, Kobe University, Nada, Kobe 657-8501
Mizuho Information & Research Institute Inc.
Institute of Industrial Science, The University of Tokyo
Rikkyo University
National Institute of Health Sciences

Keywords: Protein-ligand docking, Drug design, Fragment molecular orbital method
Recently, in silico drug design has attracted a great deal of general attention. On the basis of the fragment
molecular orbital (FMO) method, a clustering method called VISCANA to analyze the pattern of interactions
between receptor protein and ligand molecule was proposed [1], in which conventional, hierarchicalclusterings for the inter-fragment interaction energies (IFIEs) were performed. Although this method isuseful to understand the similarity of the interaction pattern, it does not tell us how and why the patterns aredifferent between two clusters. In addition, while it can sort out the existing ligands and explain theirexperimental results, it cannot predict or design a novel ligand with favorable affinity and other appropriate
properties. Here, we study how to cope with these difficulties by a multi-dimensional scaling (MDS) methodfor drug design. The MDS method, which is somewhat related to the self-organizing map (SOM) [2], is amethod to construct a low-dimensional map from the knowledge of the distance between elements [3]. Thismethod can help us understand why the difference takes place between the clusters of ligands, and canpredict the binding characteristics of unknown ligands. We estimated the usefulness of this method for someexamples.
[1] S. Amari, M. Aizawa, J. Zhang, K. Fukuzawa, Y. Mochizuki, Y. Iwasawa, K. Nakata, H. Chuman, T.
Nakano, VISCANA: Visualized Cluster Analysis of Protein-Ligand Interaction Based on the ab Initio
Fragment Molecular Orbital Method for Virtual Ligand Screening, J. Chem. Inf. Model. 46, 221-230(2006).
[2] T. Kohonen, Self-Organizing Maps, 3rd ed., Springer (2000).
[3] W.S. Torgerson, Theory and Methods of Scaling, Wiley (1958).

C-1-18
Statistical analysis of 3D distribution of ligand atoms inProtein Data Bank using kernel density estimation
Tomohiro Sato 1
tomohiro@gsc.riken.jp

Hirofumi Watabnabe 1
hirowatanabe@ssbc.riken.jp

Teruki Honma 1
Honma@gsc.riken.jp

1

Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama
City, Kanagawa, 230-0045, Japan

Keywords: Protein-ligand complex, Protein Data Bank, Fragment Molecular Orbital method,
Kernel density estimation
Scoring functions have been widely used to evaluate protein-ligand interaction in virtual
screening. With increasing protein-ligand information in Protein Data Bank (PDB), various
empirical and knowledge-based scoring functions are reported[1]. Previous studies of empiricalscoring function, such as PMF score[2], used the structural information in PDB to train
distance-dependent interaction energies between protein-ligand atom pairs. In this study, we
analyzed the PDB data to estimate the 3D probability density distributions of ligand atoms forevery grid points around amino acid residues, rather than to assess the interaction only considering
the atom pair distance (Figure 1). Using kernel density estimation (KDE)[3], the probability
distributions of ligand atoms around 20 amino acids were estimated for each of the atom types
defined by PATTY[4]. As the result, the p-values were calculated for every grid points, and thencompared to the score by force field calculation.
[1] Huang, S. Y., Grinter, S. Z., Zou, X., Scoring functions and their evaluation methods forprotein-ligand docking: recent advances and future directions, Phys. Chem. Chem. Phys.,
12:12899-908, 2010.
[2] Muegge, I., Martin, Y. C., A General and Fast Scoring Function for Protein−Ligand
Interactions: A Simplified Potential Approach, J. Med. Chem., 42:791-804, 1999.
[3] Parzen, E., On estimation of a probability density function and mode, Ann. Math. Stat.
33:1065-1076, 1962.
[4] Bush, L. B., Sheridan, R. P., PATTY: A programmable atom type and language for automaticclassification of atoms in molecular databases , J. Chem. Inf. Comput. Sci., 33:756-762, 1993.

Figure 1. Brief procedure of the estimation of 3D distribution of ligand atoms.

C-1-19
FMO-based electron density analysis
to protein structure refinement
Chiduru Watanabe 1
chiduru@iis.u-tokyo.ac.jp

Kaori Fukuzawa 1,2
kaori.fukuzawa@mizuho-ir.co.jp

Akifumi Kato 2
akifumi.kato@mizuho-ir.co.jp

Shigenori Tanaka 3
tanaka2@kobe-u.ac.jp
1
2
3
4

Yoshio Okiyama 1
okiyama@iis.u-tokyo.ac.jp

Takayuki Tsukamoto 2
takayuki.tsukamoto@mizuho-ir.co.jp

Yuji Mochizuki 1,4
fullmoon@rikkyo.ac.jp

Institute of Industrial Science, The University of Tokyo, 4 -6-1 Komaba, Meguro-ku, Tokyo
153-8505, Japan
Mizuho Information & Research Institute Inc., 2 -3 Kanda Nishiki-Cho, Chiyoda-ku, Tokyo
101-8443, Japan
Graduate School of System Informatics, Department of Computational Science, Kobe
University, 1-1, Rokkodai, Nada-ku, Kobe 657-8501, Japan
Department of Chemistry & Research Center for Smart Molecules, Faculty of Science, Rikkyo
University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan

Keywords: Electron density, Refinement, Optimization, Fragment molecular orbital (FMO)
method, ABINIT-MP, BioStation
Electron densities obtained by X-ray diffraction analyses for biomolecules determine the nuclear
coordinates with some ambiguities in position of hydrogen atoms, protonation, conformation ofamino acid residues, and orientation of ligand binding, and so on. Optimization using the classicalmolecular mechanics (MM) generally refines the coordinates to obtain the final structures, which
are registered to PDB entry and used to understand functions of biomolecul es. However, the MM
optimization often fails to reproduce the distance of hydrogen bonding and such optimized
structure has inadequate coordinates in the framework of quantum mechanics for example. For
drug discovery using ab initio fragment molecular orbital (FMO) method [1], refinement of thestructure determination is thus needed to evaluate protein-ligand interaction and reaction
mechanism more properly. In this study, we examine an electron density analysis for therefinement by comparing between X-ray and FMO-based electron densities using Crambin protein.
The FMO-based electron densities are prepared as below: First, stating form the high -resolution(0.48Å) X-ray crystal structure of Crambin including hydrogen atoms (PDB -ID: 3NIR), multiple
conformations are obtained by MM and QM optimizations: Next, FMO calculations of these
conformations are carried out to generate FMO-based electron densities at MP2/6-31G* level byusing a development version of BioStation [2] on K-computer. We here reveal position of hydrogenatoms, protonation, and conformation of amino acid by assessing the original X-ray electron
density and the FMO-based ones each other. In addition, the same analysis is performed using the
low-resolution (2.0Å) structure. Finally, we investigate how the optimized conformations based on
low-resolution X-ray electron density, which are used in generally drug design, reproduce theoriginal high-resolution X-ray electron density.
[1] “The Fragment Molecular Orbital Method: Practical Applications to Large Molecular Systems” ed D. G.
Fedorov and K. Kitaura (Taylor & Francis/ CRC Press, Boca Raton, FL, 200 9).
[2] The calculations and visualization of electron density are performed by a development version of
BioStation (download site; http://www.ciss.iis.u-tokyo.ac.jp/dl/index.php.).
Acknowledgment
This research was supported by the grant for “Strategic Programs for Innovative Research” Field No. 4:
Industrial Innovations from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT)’s“Development and Use of Advanced, High-Performance, General-Purpose Supercomputers Project.”C-1-20Development of molecular modeling software
for protein complex animations

1
2

Shuntaro Itou 1

Yutaka Ueno 1,2

shuntaro-i@is.naist.jp

uenoyt@ni.aist.go.jp

Graduate School of Information Science, Nara Institute of Scienc e and Technology,
8916-5 Takayama-chou, Ikoma, Nara 630-0192, Japan
Health Research Institute, AIST Kansai
1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan

Keywords: Protein modeling, Protein Complex animation
For understanding functions of protein molecules the atomic coordinate data allows us to model
the interacting protein complex. In addition to the experimental evidence for the molecularinteractions, there are molecular dynamics studies for analyzing works of a protein complex. Since
these techniques are the analyses by theoretical calculation based on a hypothesis of working
mechanism, it is difficult to discover how the complex is ac tually working. Thus, the demand of
making animated models of the protein complex has increased. Such tasks involving two or moreexisting software tools alternatively are usually not trivial. For these computational problems, westarted a software development of the script editor to facilitate making animations of molecularmodels visualizing molecular simulations and the protein interactions. The implementation of thissoftware was greatly supported by a graphical software development tool, Luxinia. It is also known
as a game engine incorporating Open Dynamics Engine (ODE) for the physical simulation and thegraphics library OpenGL by means of Lua, a quick script ing language. Our software develop ment
is ongoing for the molecular model arrangement functions required for creating animations, atime-line editor, and a simulation facility, while the function for the pho torealistic rendering isomitted at first. Our development aims to clarify necessary functions required for making moleculeanimations for the researchers in molecular biology. The efficiency in improving and extending thesoftware is reinforced by the scripting language that allows adding custom functions for individual
researchers to animate protein molecules to fit their experimental results easily.

C-1-21

Prediction of Protein-ligand Binding Affinities Using
Molecular Simulations
Noriaki Okimoto 1
okimoto@gsc.riken.jp

Yoshinori Hirano 1
hirano@riken.jp

Atsushi Suenaga 2
atsushi.suenaga@aist.go.jp

Makoto Taiji 1

taiji@riken.jp

1
2

QBiC, RIKEN, 1-6-5 Minatojima-minami, Chuo-ku, Kobe, Hyogo 650-0047, Japan
AIST, molprof, 2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan

Keywords: molecular dynamics simulation, protein-ligand binding affinity
Accurately predicting the binding affinities of small-molecule ligands to target proteins is one of
the ultimate goals of structure-based drug design. A variety of computational methods have beendeveloped and tested in reproducing experimental binding data for different target systems. The
computational methods range from molecular docking to free energy calculations using molecular
dynamics (MD) simulations.
In this study, we have studied the calculation of protein–ligand binding affinities by using MD
simulations with external perturbations. Especially we focused on the steered MD simulations 1-3 .
Steered MD simulation induces unbinding of ligand and conformational changes in protein on timescales accessible to the simulations. Time-depepndent external forces are applied to a system, andthe responses of the system are analyzed. We have validated the conditions of steered MD
simulations toward the practical use of the drug discovery.

[ 1] Nicolini, P.; Frezzato, D.; Gellini, C.; Bizzarri, M.; Chelli, R., Toward quantitative estimatesof binding affinities for protein-ligand systems involving large inhibitor compounds: a steered
molecular dynamics simulation route. J Comput Chem 2013, 34, (18), 1561-76.
[2] Park, S.; Schulten, K., Calculating potentials of mean force from steered molecular
dynamics simulations. J Chem Phys 2004, 120, (13), 5946-61.
[3] Wriggers, W.; Schulten, K., Investigating a back door mechanism of actin phosphate releaseby steered molecular dynamics. Proteins 1999, 35, (2), 262-73.

C-1-22
A proposal for peptide inhibitors to block the ligand-binding
pocket of urokinase receptor: ab initio molecular simulations
T. Mizushima 1, T. Kasumi 1, K. Araki 1, H. Kobayashi 2 and N. Kurita 1,**e-mail: kurita@cs.tut.ac.jp
1

Toyohashi University of Technology, 2 Nara Medical University

Keywords: peptide inhibitor, cancer invasion, urokinase-type plasminogen activator, urokinase receptor,
fragments molecular orbital calculation, ligand docking, binding energy, specific interaction
1. Introduction

A variety of proteases were found to play important roles in cancer invasion and metastasis. In
particular, binding of urokinase-type plasminogen activator (uPA) to uPA receptor (uPAR) existingon the surface of a cancer cell is considered as a trigger for cancer invasions. It is thus expected thatthe blocking of the binding can inhibit cancer invasion efficiently. Based on the X-ray crystal
structure of uPA-uPAR complex, it was elucidated which amino acid residues of uPA are important
for the binding, and several peptides having these residues were synthesized as potent inhibitors [1].
In our previous molecular simulations [2,3], the specific interactions between uPA and uPAR
were investigated at an electronic level by ab initio fragment molecular orbital (FMO) calculations.
The results are comparable to the experiment [1] and highlight some important residues of uPA,which have strong attractive interactions with uPAR. In the present study, we constructed novel
peptides composed of the important residues and investigated their specific interactions to uPAR by
the ab initio FMO calculations, with solvating water molecules considered explicitly.
2. Results and discussion

We first constructed 6 types of peptides composed of 5~10 residues and docked each of them tothe uPA-binding domain of uPAR by a protein-ligand docking program. These docked structures
were fully optimized in water by classical molecular mechanics method, and binding energy
between the peptide and uPAR was investigated by the ab initio MP2/6-31G method in FMO.
Figure 1 shows the optimized structure of the solvated complex of uPAR with our proposed peptide.
This peptide has the largest binding energy to uPAR among
our proposed peptides. In addition, it was elucidated that
Glu36, Glu134 and Glu135 residues of uPAR have large
contribution to the peptide binding. Based on the results
simulated, we furthermore mutated some residues of thepeptide in silico and searched for the novel peptide inhibitors
having larger binding affinity to uPAR. Such peptides are
expected to be more potent inhibitors for cancer invasions.
The details of the results will be shown at the conference.
[1] C. Barinka, et al., J. Mol. Biol., 363(2006) 482.
Fig.1 Our proposed peptide
blocking the uPAR pocket[2] S. Tsuji, et al., J. Mol. Graph. Model., 29(2011) 975.
[3] T. Kasumi, et al., Molecular Simulations, 2013, in press.

C-2-01
Protein-Ligand Docking Using Artificial Bee Colony
Algorithm
Shota Uehara 1

Kazuhiro Fujimoto 1

uehara@eniac.scitec.kobe-u.ac.jp

fujimoto@ruby.kobe-u.ac.jp

Shigenori Tanaka 1
Tanaka2@kobe-u.ac.jp

1

Department of Computational Science, Graduate School of System Informatics, Kobe
University, 1-1, Rokkodai, Nada-ku, Kobe, Hyogo 657-8501, Japan

Keywords: Protein-ligand docking, Structure-based drug design, Artificial bee colony algorithm
Protein-ligand docking is one of the most significant issue in structure-based drug design (SBDD).
Generally, this docking is considered as an optimization problem which specifies the energeticallystable conformation of the ligand at the binding site. However, it is very difficult to identify thecorrect pose because of many optimization parameters with high correlation. In previous studies, it
has been reported that popular docking programs (Glide [1] , GOLD [2] , FlexX [3] , AutoDock [4] , etc.)
can identify the correct docking pose with an accuracy of only abo ut 60% [5] . In this work, we
attempted to apply Artificial Bee Colony algorithm [6] (ABC) to docking. ABC is an optimization
algorithm based on the intelligent behavior of honey bee swarm, which has a higher global ability
than other algorithms such as Simulated Annealing (SA), Genetic Algorithm (GA), Particle Swarm
Optimization (PSO). The performance of the ABC for docking is evaluated for several
protein-ligand complexes including some highly flexible ligands in comparison with otheralgorithms. The results reveal that the ABC might be more suitable for docking than others inparticular for dealing with highly flexible ligands.

[1] Friesner, R. A., Banks, J. L., Murphy, R. B., et al., Glide: A new approach for rapid, accurate
docking and scoring. 1. Method and assessment of docking accuracy, Journal of Computational
Chemistry, 47:1739-1749, 2004.
[2] Jones, G., Willett, P., Glen, R. C., et al., Development and validation of a genetic algorithm forflexible docking, Journal of Molecular Biology, 267:727-748, 1997.
[3] Rarey, M., Kramer, B., Lengauer, T., et al., A fast flexible docking method using an incremental
construction algorithm, Journal of Molecular Biology, 261:470-489, 1996.
[4] Morris, G. M., Goodsell, D. S., Halliday, R. S., et al., Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energy function, Journal of Computational
Chemistry, 19:1639-1662, 1998.
[5] Plewczynski, Dariusz., Lazniewski, Michal., Augustyniak, Rafal., et al., Can We Trust Docking
Results? Evaluation of Seven Commonly Used Programs on PDBbind Database, Journal ofComputational Chemistry, 32:742-755, 2011.
[6] Karaboga, Dervis and Basturk, Bahriye, A powerful and efficient algorithm for numericalfunction optimization: artificial bee colony (ABC) algorithm, Journal of Global Optimization,
39:459-471, 2007.

C-2-02
A new approach for constructing CYP2C9 pKi estimation
scheme with ligand-receptor interaction effects
Sumie Tajima 1
tajima@hulinks.co.jp
1

Yasuo Inoue 1
inoue@hulinks.co.jp

Hulinks Inc., 5-14 Nihonbashi Hakozaki-cho, Chuo-ku, Tokyo 103-0015, Japan

Keywords: CYP2C9 pKi, property estimation scheme, Field Point pharmacophores, QSARHigh quality Quantitative Structure Activity Relationship (QSAR) models are important in the
drug discovery process. In general, it depends on the researcher's target whether a model is the bestfor them. Therefore, drug researchers sometimes need to construct their original estimation scheme.
StarDrop [1] can provide the powerful tool for producing original high quality QSAR model [2] . We
attempted to get a novel high quality CYP2C9 pKi model by using StarDrop. StarDrop can produce
330 descriptors which consist of a total of 321 SMARTS based descriptors and 9 whole molecule
properties but also add user's original descriptors. It is our purpose to find a new approach forconstructing CYP2C9 pKi estimation scheme with ligand-receptor interaction effects . For getting
the descriptors including these effects, we used the Field Point technology [3] of Cresset [4] . Field
points are the local extrema of the electrostatic, van der Waals and hydrophobic potentials of themolecule. They can be thought of as extended pharmacophores, with the advantages that theirposition is directly calculated from the molecule’s physical properties . We will present how to get
the descriptors of Field Point pharmacophores and the details of our original models.
[1] http://www.optibrium.com/[2] J. Comp.-Aided Mol. Desi., 2008, 22, 431-440[3] J. Chem. Inf. Model., 2006, 46, 665-676[4] http://www.cresset-group.com/

C-2-03
Electronic Similarity of Molecules
for Evaluation, Classification, and Discovery
Manabu Sugimoto* 1

Ryo Iwane 1

sugimoto@kumamoto-u.ac.jp	
 1

135d8311@st.kumamoto-u.ac.jp

Graduate School of Science and Technology, Kumamoto University. 2-39-1, Kurokami,
Chuo-ku, Kumamoto 860-8555, Japan

Keywords: Electronic structure calculation, Molecular Similarity, Electronic Similarity
Numerical expression of molecules is important for their quantitative description. By
quantification, we can apply a variety of statistical methods to observe quantitative characteristics
of molecules, which are expected to facilitate evaluating their values, classifying them, anddiscovering their derivatives. Intrinsic structures and properties of molecules are due to theirelectronic structures, it is natural to quantify molecules on the basis of electronic-structure
calculations.
In this presentation, we suggest a method of defining molecular similarity by applying ab initio
electronic-structure calculations. It will be shown that reasonable classification of molecules ispossible for the trial set shown in Scheme 1. When a reference molecule is taken as lutein A, thecarotenoid molecules are top-ranked as shown in Table 1. The present computational method is
also applied to investigate bioisosterism of functional groups.
Scheme 1.
(a)$lutein$A$(b)$lutein$B$(c)$myxol

(d)$alloxanthin(e)$astaxanthin

(f)$ethylene

(g)$ethane(h)$cyclohexane

(i)$benzene(j)$phenol (k)$p9xylene(l)$stylene(m)$naphthalene(n)$hexane (o)$hexanol (p)$hexanone

Table 1. Ranking of electronic similarity of molecules in Fig. 1.
Rank

Compd.

Similarity (%)

Rank

Compod.

1

lutein A

100.0

92-hexanone

Similarity (%)
31.5

2

lutein B

98.0

10

phenol

30.5

3

myxol

91.9

11

1-hexanol

29.7

4

alloxanthin

91.8

12

benzene25.5

5astaxanthin

77.9

13

hexane24.9

6p-xylene

37.7

14

ethane20.4

7naphthalene

37.3

15

ethylene

20.1

8stylene37.2

16

cyclohexane

19.2

C-2-04
Understanding polypharmacology and promiscuous
chemotypes on LSKB
Atsushi Midorikawa
midorikawa@w-fusion.co.jp,

Tetsuya Takasaki

Toshiro Kimura

takasaki@w-fusion.co.jp,

kimura@w-fusion.co.jp

Kozo Kawahara
kkawahara@w-fusion.co.jp

World Fusion Co., Ltd., 2-15-15 Nihonbashi Ningyocho, Chuo-ku, Tokyo
103-0013, Japan

Keywords: knowledge, Open Data Source, Polypharmacology, Molecular Framework, Workflow
Toward disease related target, it is important to classify the drug target(genes/proteins) based on
its function, and the subtype has often different ligand recognition.
Knowledge of SAR and related studies is used for recognition of ligand structures ; In many cases,
we try to find common core and/or scaffold from the analysis and use the infor mation.
Bajorath et al., reported promiscuous chemotypes by analysis of polypharmacology between drugs
and targets, using the chemical structures, scaffolds and molecular frameworks[3]. And also aseries of compounds, which have common scaffold, is specific toward a target class in same caseand they describe the benefit of activity cliffs for the understanding SAR[1,2].
In the poster, Life Science Knowledge Bank(LSKB)[4] is well-organized database system storing
the binary relationship each combination of the terms (Gene/Protein/Disease/Tissue/chemical)
mined from Medline and extracted information from various Open Data Sources. And thewell-organized compound structure database is also available in LSKB; the chemical contents isbuilt as non-redundant (NR)chemical structures from PubChem[5], ChEMBL[6], ZINC[7] andligand structures collection taken from PDB protein-ligand complexes (where the information
extracted from PDB[8]). And it associates LSKB knowledge dataset. In LSKB version 4.2,
compound databases stores 70M NR compounds and, molecular frameworks associated with eachcompounds.
The latest release provides protein classification i.e., EC number, GPCR, Kinase. and registration
function of interest gene set in private experimental data. We show you the benefit of combination,
LSKB and workflow tool Pipeline Pilot[9] by several instances for understanding
polypharmacology and SAR Matrix generation.
[1] Hu, Y. and Bajorath,J., Polypharmacology Directed Compound Data Mining: Identification ofPromiscuous Chemotypes with Different Activity Profiles and Comparison to Approved Drugs, J.
Chem. Inf. Model. 2010, 50, 2112?2118[2] Hu, Y. and Bajorath,J., Molecular Scaffolds with High Propensity to Form Multi-Target Activity
Cliffs, J. Chem. Inf. Model. 2010, 50, 500-510[3] Bemis, G.W. and Murcko,M. A., The Properties of Known Drugs. 1. Molecular Frameworks, J.
Med. Chem. 1996, 39, 2887-2893[4] World Fusion Co., Ltd., 2-15-15 Nihonbashi Ningyocho, Chuo-ku, Tokyo 103-0013, Japan[5] http://pubchem.ncbi.nlm.nih.gov/[6] https://www.ebi.ac.uk/chembldb/[7] http://zinc.docking.org/[8] http://www.rcsb.org/pdb/home/home.do[9] Accelrys, 10188 Telesis Court, San Diego CA 92121, USA

C-2-05
Ensemble Docking Simulation for β 2 Adrenergic Receptor
Using Elastic Network Model
Tomoyuki Iwamoto 1
tomoyukii@protein.osaka-u.ac.jp

Shigeru Endo 3
endo@sci.kitasato-u.ac.jp

Hiroshi Wako 2
wako@waseda.jp

Yu Takano 1
ytakano@protein.osaka-u.ac.jp

Haruki Nakamura 1
harukin@protein.osaka-u.ac.jp
123Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
School of Social Sciences, Waseda University, 1-6-1 Nishiwaseda, Shinjyuku-ku, Tokyo
169-8050, Japan
Department of Physics, Faculty of Science, Kitasato University, 1-15-1 Kitasato, Minami-ku,
Sagamihara-shi, Kanagawa 252-0373, Japan

Keywords: Elastic Network Model, Normal Mode Analysis, Ligand Docking, In Silico Screening
G protein-coupled receptors (GPCRs) are membrane proteins involved in signal transduction
pathways, and so many of them are target molecules for drug discovery of various human diseases.
Although more and more crystal structures of GPCRs have been determined with particular agonists
and antagonists, it is now widely known that drug docking simulations based on such rigid crystal
structures do not always succeed because of their very flexible structural features. Therefore, we
adopted an ensemble docking method to take into account the protein flexibility in drug docking andscreening.
In an ensemble docking method, each compound in the library including drug candidates is docked at
the pockets of the individual receptor conformations, which were produced by molecular simulations.
Molecular Dynamics (MD) simulation now becomes standard for producing putative different
conformations of GPCRs, but the dynamic aspects of GPCRs can be captured by a much simpler modelsuch as an elastic network model (ENM).
In this study, we generated the conformation polymorphism of β 2 adrenergic receptor by ENM withthe successive Normal Mode Analysies (NMA), based on the X-ray crystal structure (PDBID: 2RH1).
Various structual models were built by moving the atom positions following several representativemodes, which were calculated by ENM-NMA. Then, docking simulations were made for those virtual
models. The models based on the selected particular five modes showed the best scores for agonist andantagonist screenings. Compared to the initial crystal structure, virtual model provided 20% better
screening efficiency.

C-2-06
Investigation of protein-ligand interactions
Yoshinori Hirano 1

Noriaki Okimoto 1

hirano@riken.jp

okimoto@riken.jp

Makoto Taiji 1
taiji@riken.jp

1

Laboratory for Computational Molecular Design, Computational Biology Research Core
Quantitative Biology Center (QBiC), RIKEN, Hyogo, 651-0047, Japan

Keywords: Molecular docking, Protein-ligand interaction
Finding therapeutic drugs is challenging matter. Many methodologies have been applied to thismatter (i.e. drug design). In addition to the experimental techniques, various computational
approaches are utilized at the different stages of drug design. In the early stage, compound
libraries are screened in order to increase potential compounds. Due to select the compound fromthe huge number of compounds in relatively short time, molecular docking methods are routinely
used. The molecular docking has already proven to be a successful virtual screening tool forseveral target proteins, but it is less reliable because the calcu lation of binding free energy between
target protein and ligand is not so accurate. The problems with molecular docking have been
widely mooted: protein dynamics and solvation effects are ignored, the energy functions areinaccurate, and so on. Therefore more effective computational method is necessary for rational
drug design.
Molecular dynamics (MD) simulations can treat both proteins and ligands in a flexible manner,
directly estimate the effect of explicit water molecules, and provide more accurate binding affinity,
although their computational costs and times are significantly greater than those of moleculardocking. We report an effective method for computational screening; this method is a combinationof molecular docking and molecular dynamics simulations. The proposed method showed a higher
and more stable enrichment performance than the molecular docking method used alone. We willuse quantum chemical (QC) calculations as final filter of drug screening.
[1] Okimoto N. et al., PLoS Comput Biol 5(10): e1000528.

C-2-07
Predicting bioactivity of compound-drug target protein
pairs using support vector regression models reflecting
ligand efficiency
Nobuyoshi Sugaya 1
sugaya@pharmadesign.co.jp

1

Drug Discovery Department, Research & Development Division, PharmaDesign, In c.,
2-19-8, Hatchobori, Chuo-ku, Tokyo 104-0032, Japan

Keywords: Support vector regression, G-protein coupled receptor, Protein kinase, Ion channel,
ligand efficiency
Predicting bioactivity of compounds to drug target proteins using machine learning met hods is
one of the most intensively studied area in drug discovery and development. Although many
previous machine learning studies have succeeded in predicting novel ligand -protein interactions
with high performance, all of the previous studies to date ha ve been heavily dependent on thesimple use of raw bioactivity data of ligand potencies measured by IC50, EC50, Ki, and Kd
deposited in databases. In our previous study [1], we have showed that, using support vector
machines, binary classification models based on training data reflecting one of the representativesof ligand efficiency, Binding Efficiency Index (BEI) [2] can offer better performance in classifying
active and inactive compound-protein pairs than models based on training data reflecting IC50 or
Ki. In this study, we report that this result holds also when support vector regression (SVR) models
are applied to bioactivity data.
Utilizing bioactivity data measured by IC50 in GPCRSARfari ver. 2, KinaseSARfari ver. 4, and
ChEMBL 14 databases [3], we retrieved bioactivity data associated with G protein -coupled
receptors, protein kinases, and ion channels and created four types of training data; IC50 -based,
pIC50-based, BEI-based, and Surface Efficiency Index (SEI) -based. Values of pIC50, BEI, and SEI
were transferred from observed values of IC50 in the databases. The number of instances in the
training data is shown in Table 1. To represent compound -protein pairs in the training data, threekinds of compound descriptors (MACCS, 2D descriptors in MOE, a nd OpenBabel FP2) and single
protein descriptor (frequencies of dimmers of amino acid in protein sequence) were used. FromGPCRSARfari ver. 3, KinaseSARfari ver. 5.01, and ChEMBL 15 databases, we collected newly
added bioactivity data and used the data as validation data for evaluating the performance of theconstructed SVR models. Objective comparisons of the performance of the SVR models showedthat their prediction capabilities follow an order of SEI > BEI > pIC50 > IC50 as a whole. This
result is independent of compound descriptors used and drug target protein families. The
superiority of ligand efficiency-based SVR models may be partially attributed to distinct
distribution patterns of pIC50s, BEIs, and SEIs, showing narrower range of BEIs than pIC50 s and
SEIs than BEIs.
[1] Sugaya, N., Training based on ligand efficiency improves prediction of bioactivities of ligands
and drug target proteins in a machine learning approach , Journal of Chemical Information andModeling, in press.
[2] Abad-Zapatero, C. and Metz, J. T., Ligand efficiency indices as guideposts for drug discovery,
Drug Discovery Today, 10:464-469, 2005.
[3] Gaulton, A., et al., ChEMBL: a large-scale bioactivity database for drug discovery, Nucleic
Acids Research, 40:D1100-D1107, 2012.

C-2-08
CzeekD: Fragment-based de novo Drug Design System
for Drug Discovery

12

Tatsuya Yoshikawa 1

Chisato Kanai 1

Yoshihiro Yamamoto 1

yoshikawa@k-ct.jp

kanai@k-ct.jp

yamamoto@k-ct.jp

Ryuta Murakami 1

Yasushi Okuno 2

murakami@k-ct.jp

okuno@pharm.kyoto-u.ac.jp

Kyoto Constella Technologies Co., Ltd., 558 Yamabushiyama-cho, Takoyakushi-sagaru,
Muromachi-dori, Nakagyo-ku, Kyoto 604-8156, Japan
Department of Systems Biosciences for Drug Discovery, Graduate School of Pharmaceutical
Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501,
Japan

Keywords: FBDD, de novo drug design, PSO, CGBVS, RECAP
For early stages in drug development, it is a critical issue to find lead compounds with novel
scaffolds. However, the set of all possible small organic molecules has been estimated to consist of
more than 10 60 compounds, it is difficult to figure out the best scaffolds from among the vastchemical space through experimental synthetic approaches.
We have developed a computational approach to efficiently generate lead compounds with novel
scaffolds using fragment chemical library. The fragment-based drug de novo design system with afast stochastic optimization algorithm, called “CzeekD”, has following functions:– Generation of novel scaffolds in vast chemical space is achieved by combination of building
blocks in fragment library.
– It is easy to synthesize the designed chemical structures because of using basic chemical
reactions, i.e. RECAP rule.
– Chemical Genomics-Based Virtual Screening (CGBVS) is implemented as a scoring function,
which can efficiently estimate the possibility of molecular interactions between the generated
compounds and the target proteins.
– Particle Swarm Optimization (PSO) algorithm was newly developed to swiftly explore
druggable molecular seeds and to efficiently find out diverse structures among the vastchemical space.
As a result of bioactivity evaluation of the designed compounds through organic synthesis and invitro assay, we successfully identified novel active compounds with highly hit rate (20－40%) for
GPCR proteins. This result suggested that CzeekD provides a powerful solution to guide medicinal
chemists into the discovery of novel bioactive molecules.
[1] Hartenfeller M, Proschak E, Schüller A, and Schneider G., Concept of combinatorial de novo
design of drug-like molecules by particle swarm optimization, Chem Biol Drug Des, 72:16-26.
2008.
[2] Lewell XQ, Judd DB, Watson SP, and Hann MM., RECAP–retrosynthetic combinatorial
analysis procedure: a powerful new technique for identifying privileged molecular fragmentswith useful applications in combinatorial chemistry, J Chem Inf Comput Sci, 38:511-22, 1998.
[3] Yabuuchi H, Niijima S, Takematsu H, Ida T, Hirokawa T, Hara T, Ogawa T, Minowa Y,
Tsujimoto G, and Okuno Y., Analysis of multiple compound-protein interactions reveals novel
bioactive molecules, Mol Syst Biol, 7:1-12, 2011.
[4] J. Kennedy and R. C. Eberhart, Particle swarm optimization, Proc. IEEE International
Conference on Neural Networks, 1942-1948, 1995.

C-2-09
Mixed Fragment-based screening of WNK1 inhibitor
Nae Saito

Yukio Tada

nae-saito@mol.f.u-tokyo.ac.jp

tada-yukio@mol.f.u-tokyo.ac.jp

Takayoshi Okabe

Hirotatsu Kojima

Tetsuo Nagano

tokabe@mol.f.u-tokyo.ac.jp

kojima@mol.f.u-tokyo.ac.jp

tlong@mol.f.u-tokyo.ac.jp

Open Innovation Center for Drag Discovery, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan

Keywords: Fragment screening, WNK1, SPR
Pseudohypoaldosteronism type II, known as a rare autosomal dominant disorder, is considered tobe caused by overexpression of WNK [with no lysine(K)]1 or mutations in WNK4. Thus far, there
have been only a few reports regarding the specific inhibitor for WNK1 or WNK4. We therefore
explored lead compounds for WNK1 by mixed fragment -based drug discovery wherebycompetition experiment using surface pla smon resonance (SPR) enables us to select better leadcompound in the early stages.
Mobility shift assay, by which inhibitory activity for phosphorylations of WNK1 was estimated,
was examined in order to select hit compounds out of 9,000 fragments. Then we selectedcompounds having structural similarity to the hit compounds, and their binding abilities, including
those of hit compounds, to WNK1 were tested using SPR. The competition assay by SPR was
carried out using a single or a mixture of the candidate c ompounds.
Figure 1 shows competition assay about combination of candidate compounds 1(1,3-isoquinolinediol) and 2 [N-(4-methyl-5-oxo-4,5-dihydro[1,2]dithiolo[4,3-b]pyrrol-6-yl)], and
3 (1,3-isoquinolinedione) and 4 (indirubin-3’-oxime). Compounds 1 and 2 were found to bind to
the same site in WNK1 because the response of their mixture was almost the same as that of 2. In
contrast, as shown in the right side of Figure 1, the response of the mixture of compounds 3 and 4
were the sum of those of the two. This clearly indicates compound 3 and 4 bind to the different
sites in WNK1. The binding site of these compounds will be discussed using a docking study.
H
N
HO

O

N
N
OH
1

S

O
S
HN

2

O
HN
O

N
HO

3

4

O

Fig. 1 Competition experiments using candidate compounds 1-4.

NH

C-2-10
Identification of novel chemical agents with antibacterial
activity against Mycobacterium by in silico structure-based
drug screening
Yuji Koseki 1 , Tomohiro Kinjo 1 Maiko Kobayashi 1 , Atsumi Yamada 1 , Koji Morita 1 , Kento Yamaguchi 1
Mitsuru Kitamura 2 and Shunsuke Aoki 1, 3, **Corresponding author aokis@bio.kyutech.ac.jp
12

3

Department of Bioscience and Bioinformatics, Graduate school of Computer Scienceand Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuk-shi,
Fukuoka 820-8502, Japan
Department of Applied Chemistry, Kyushu Institute of Technology, 1-1 Sensui-cho,Tobata-ku, Kitakyushi-shi, Fukuoka 804-8550, Japan
Biomedical Informatics R&D Center, Kyushu Institute of Technology, 680-4 Kawazu,
Iizuka-shi, Fukuoka 820-8502, Japan

Keywords: Antibiotics, Enoyl-acyl carrier protein reductase (InhA), in silico structure-base drug
screening (SBDS), Mycobacterium, Protein-ligand interaction fingerprint analysis (PLIF)
The increasing prevalence of drug-resistant tuberculosis (TB) (i.e. multidrug-resistant TB:
MDR-TB, extensively drug-resistant TB: XDR-TB), which is resistant to several effective
antibiotics, presents a major global health threat [1]. The enoyl-acyl carrier protein reductase of
Mycobacterium tuberculosis (InhA) is a key enzyme of the mycobacterial type Ⅱ fatty acidbiosynthesis pathway (FAS-Ⅱ). In this pathway, the InhA plays major role in the production of
mycolic acid and is known as a target protein for first-line drug isoniazid (INH), an effective
antibiotics for TB chemotherapy [2].
In this study, we attempted to identify novel chemical compounds specifically targeting the InhA.
We performed a hierarchical in silico SBDS [3,4] using InhA crystal structure data (PDB ID:
2H7I) and the virtual chemical library (ChemBridge, CA) including 154,118 chemicals. We thenevaluated the antibiotic effects of the candidate chemicals, and we found two hits (KE3 and KE4),
which were able to inhibit the growth of model mycobacteria strains (M. vanbaalenii and M.
smegmatis). We also performed similarity analysis to identify five additional chemicals(KES1-KES5) with similar structures to the active chemicals from ChemBridge compound library,containing 461,383 compounds. The most potent inhibitors (KE4 and KES4) do not have any toxic
effects in model intestinal bacteria (E. coli BL21 and JM109 strains) and mammalian cells (MDCKand SH-SY5Y cells). Moreover, we also confirmed that these chemicals directly inhibit the
enzymatic activity of InhA.
In conclusion, the structural and experimental information regarding these novel chemicalcompounds is likely to be useful for the hit-to-lead optimization of new antibiotics for thetreatment of TB. Furthermore, our screening methodology presented in this study could contributeto the further identification of novel hit compounds for other candidate medicinal drugs.
References[1] Global Tuberculosis Control: WHO Report 2011, ISBN 978 92 4 156438 0[2] H. Lu, et al., Acc. Chem. Res. 2008 (41), 11-20.
[3] Y. Koseki, et al., Eur. J. Med. Chem. 2013 (60), 333-339.
[4] T. Kinjo, et al., J. Chem. Inf. Model. 2013 (53), 1200-1212.

C-2-11
Identification of novel potential antibiotics for Staphylococcus
by Structure-Based Drug Screening
Maiko Kbayashi 1 , Tomohiro Kinjo 1 , Yuji Koseki 1 and Shunsuke Aoki 1, 2 , **Corresponding author aokis@bio.kyutech.ac.jp
12

Department of Bioscience and Bio informatics, Graduate school of Computer Scienceand Systems Engineering, Kyushu Institute of Technology, 680 -4 Kawazu, Iizuk-shi,
Fukuoka 820-8502, Japan
Biomedical Informatics R&D Center, Kyushu Institute of Technology, 680 -4 Kawazu,
Iizuka-shi, Fukuoka 820-8502, Japan

Keywords: in silico Structure-Based Drug Screening (SBDS), Staphylococcus, Dihydrofolate
reductase (DHFR), Protein Ligand Interaction Fingerprint (PLIF)
The emergence and spread of multidrug-resistant Staphylococcus aureus (S. aureus) increased
the morbidity and mortality of the infected patients worldwide [1, 2]. Therefore, it is importanceto develop new antibacterial drugs against the multidrug-resistance S. aureus. In this study, we
attempted to identify novel potential antibiotics targeting S. aureus dihydrofolate reductase(saDHFR), which is an essential coenzyme for DNA replication.
We performed a hierarchical in silico Structure-Based Drug Screening (SBDS) [3] with crystal
structure of saDHFR (PDB ID: 2W9G [4]) and virtual chemical compound library (154,118
chemical compounds, ChemBridge, SanDiego, CA). We identified five candidate chemicalcompounds predicted to have high binding affinity with active site of saDHFR. We thenexperimentally tested whether these candidate chemical compounds inhibit the growth ofStaphylococcus epidermidis (S. epidermidis), a model bacteria strain. A chemical compound(KB1) exhibited growth inhibitory effect on S. epidermidis. Moreover, we found five KB1
analogs from a library including 461,383 chemical compounds (CemBridge, SanDiego, CA). FourKB1 analogs (KBS1-KBS4) inhibited the growth of S. epidermidis. The hit compounds (KB1 andKBS2-KBS4) in this study do not have toxic effects on model enterobacteria (Escherichia coli
BL21 and JM109 strains) and mammalian cell (Madine-Darby Canine Kidney cell). Furthermore,
we evaluated binding modes of hit chemical compounds using Protein Ligand Interaction
Fingerprint (PLIF) analysis and Ligand Interaction (LI) analysis. Results of PLIF and LI
analyses predicted that these hit chemical compounds are able to have inhibitory effects on growthof the resistance strains with mutated saDHFRs, similar to wild-type saDHFR.
In conclusion, structural information of four candidate chemical compounds identified in thisstudy (KB1, KBS2-KBS4) will likely contribute to development of new drugs for S. aureus.

References[1] Bai H., et al., PLoS One., 2012 (7), e29886.
[2] Iwase T., et al., Nature, 2010 (465), 346-349.
[3] T. Kinjo, et al., J. Chem. Inf. Model. 2013 (53), 1200-1212.
[4] Holly H., et al., PROTEINS, 2009 (76), 706-717.

C-2-12

Design of transfection reagents in RNAi therapeutics bychemoinformatics approach
Yu Sakai 1

Hiromasa Kaneko 2

y_sakai@chemsys.t.u -tokyo.ac.jp

hkaneko@chemsys.t.u -tokyo.ac.jp

Seiichi Ohta 3

Taichi Ito4s-ohta@chemsys.t.u-tokyo.ac.jp

taichi@m.u-tokyo.ac.jp

Kimito Funatsu 5
funatsu@chemsys.t.u-tokyo.ac.jp
125

Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan
34
Center for Disease Biology and Integrative Medicine, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan

Keywords: Chemoinformatics, RNAi, Transfection reagent
RNAi is a natural biological process involving gene silencing or regulation with siRNA and
expected to be applied in the therapeutics of gene disorders. The delivery of siRNA into thecell is demonstrated using cationic lipid. The lipid is called ‘Transfection reagents’ and thedevelopment of new reagents has recently advanced. Some relation exists between thechemical structures of the reagents and their properties, but it is not clear yet
quantitatively and the development of new reagents depends on the experim ental
perception.
In this study we collected the data of the transfection reagents from the literature 123 andconstructed statistical models between the structures of the reagents and their suppression
efficiency of gene expression in order to predict the chemical structures of high efficient
reagents in future.

[1] K. T. Love, et al., PNAS, 107, 1864-1869, 2010
[2] K. P. Mahon, et al., Bioconjugate Chem ., 21, 1448-1454, 2010
[3] A. Akinc, et al., nature biotechnology , 26, 561-569, 2008

C-2-13

Toward Crowdsourcing Evaluation of
Synthetic Accessibility
1

Yukino Baba 	  	 yukino_baba@mist.i.u-tokyo.ac.jp

12
3

Tetsu Isomura 2
isomura.tetsu@mx.kaiteki-institute.com

Hisashi Kashima 1

Hirofumi Nakano 3kashima@mist.i.u-tokyo.ac.jp

h-nakano@mol.f.u-tokyo.ac.jp

Department of Mathematical Informatics, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8656, JapanThe KAITEKI Institute, Inc., 1-1-1 Marunouchi, Chiyoda-ku, Tokyo 100-8251, Japan
Open Innovation Center for Drug Discovery, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan

Keywords: Synthetic accessibility, Crowdsourcing, Human computation
A rapid method for the assessment of synthetic accessibility for a vast number of chemicalcompounds is expected to bring about a breakthrough in the drug discovery. Although several
computational methods have been proposed for estimating the synthetic accessibility [1-3], they do
not meet the requirements of both quick computation time and high estimation accuracy. In thefield of computer science, crowdsourcing has received considerable attention for being an efficientand scalable approach for solving complicated tasks that are difficult for computers but relatively
easier for humans. Using crowdsourcing platforms such as Amazon Mechanical Turk [5], one caneasily outsource various professional and non-professional tasks to a large group of people online.
Jobs on crowdsourcing are typically decomposed into small and simple tasks that can be solved inparallel by different workers.
As the first step toward creating a crowdsourcing platform where the synthetic accessibility of anumber of compounds is assessed by people with different levels of expertise and backgrounds, weinvestigate the consistency among experienced medicinal chemists in assessing synthetic
accessibility. Unlike previous studies [2, 4], we prepared the following three compound groups:
Already existing compounds, ones having substructures that are hard to be synthesized, and ones
having novel substructures. We picked ten compounds from each group and asked the experienced
medicinal chemists to rate the synthetic accessibility of each compound on a five-point scale
within several minutes. Results show that there is a high correlation between the assessments given
by a subset of the chemists.
[1] Baber, J. C., Feher, M.: Predicting Synthetic Accessibility: Application in Drug Discovery andDevelopment. Mini Reviews in Medicinal Chemistry, 4:681-692, 2004.
[2] Boda, K., Seidel, T., Gasteiger, J.: Structure and Reaction based Evaluation of Synthetic
Accessibility. Journal of Computer-Aided Molecular Design, 21:311-325, 2007.
[3] Ertl, P., Schuffenhauer, A.: Estimation of synthetic accessibility score of drug-like molecules
based on molecular complexity and fragment contributions, Journal of Cheminformatics, 1:8,
2009.
[4] Takaoka, Y., Endo, Y., Yamanobe, S., Kakinuma, H., Okubo, T., Shimazaki, Y., Ota, T.,
Sumiya, S., Yoshikawa, K.: Development of a Method for Evaluating Drug-Likeness and Ease of
Synthesis Using a Data Set in Which Compounds Are Assigned Scores Based on Chemists'
Intuition. Journal of chemical information and computer sciences, 43:1269-1275, 2003.
[5] http://mturk.com

C-3-01
Identification of genes and pathways involved in DNA
methylation in hepatocellular carcinoma
Sufeiya Subati 10376com@tmd.ac.jp

Takashi Kohda 3

tkohda.epgn@tmd.ac.jp

Kaoru Mogushi 1
mogushi@bioinfo.tmd.ac.jp

Shinji Tanaka 4shinji.msrg@tmd.ac.jp

Mahmut Yasen 2
mahmut@bioinfo.tmd.ac.jp

Hiroshi Tanaka 1
tanaka@bioinfo.tmd.ac.jp

1

Department of System Biology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo,
Tokyo 113-8510, Japan2
Department of Pathology, Japanese Foundation for Cancer Research, 3-8-31 Ariake, koto ward,
Tokyo 135-8550, Japan3
Department of Epigenetics, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo,
Tokyo 113-8510, Japan
4
Department of Hepato-Bukuary-Pancreatic Surgery, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo, Tokyo 113-0034, Japan

Keywords: DNA methylation, Hepatocellular carcinoma, DNA microarray
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide.
Despite major efforts to improve diagnosis and treatment of HCC, therapeutic options remain
limited [1] . A role for DNA methylation in the regulation of gene expression was hypothesized many
years ago. Recent genetic studies suggest that tumor suppressor genes (TSGs) are often silenced
during carcinogenesis through epigenetic modification caused by metylation of promoter CpG
island [2] . Therefore, detailed investigation of genes inactivated by DNA methylation in HCC isnecessary for the development of novel treatment options for HCC.
In this study, we aimed to identify candidate genes commonly methylated in HCC. We treatedeight HCC cell lines by the DNA demethylating agent 5 -aza-2'-deoxycytidine (DAC).
Comprehensive gene expression analysis revealed that 489 probe sets had more than two-foldup-regulated in at least six DAC-treated cell lines compared to the control cell lines. Moreover,Gene set enrichment analysis (GSEA) of DAC-treated cells suggested that down-regulation of
immune response pathways were possibly induced by DNA methylation. Interestingly, chemokine(C-X-C motif) ligand 2 (CXCL2) was included in the 44 probe sets and is associated with immune
response. Because immune surveillance is an important host system to inhibit carcinogenesis andthereby down-regulation of CXCL2 might cause immune escape of cancer cells, CXCL2 can beone of the key molecules for cancer progression by DNA methylation.
Our results suggest that these genes and pathways may play an important role incarcinogenesis and disease progress of HCC through inactivation by DNA methylation. Notably,
the candidate gene CXCL2 might be an important molecule for further investigation o f immune
escape in HCC. In our validation studies, we showed that the expression of CXCL2 was
down-regulated in HCC tissue as compared to non-tumor tissues. In addition, CXCL2 expression
was significantly up-regulated by DAC treatment in HCC cell lines. This suggests that CXCL2
might be an important molecule for further investigation of aberrant methylation in HCC.
References[1] Tischoff I, Tannapfe A. DNA methylation in hepatocellular carcinoma. World J Gastroenterol.
2008;14:1741-8.
[2] Partha M Das and Rakesh Singal. DNA methylation and cancer. Journal of Clinical oncology.
2004;22:4632-42.

C-3-02
1

H NMR-based metabolomics of plasma and dialysate
from hemodialysis patients
Masako Fujiwara1, Itiro Ando1, Hiroshi Sato1,
fmasako@m.tohoku.ac.jp, sprite@mail.pharm.tohoku.ac.jp, hsymhs2i@m.tohoku.ac.jp,

Yutaka Imai1, Kazuhisa Takeuchi1,2
rinsyo@mail.pharm.tohoku.ac.jp, kazut2i@koujinkai-med.or.jp
1

Graduate School of Pharmaceutical Sciences, Tohoku University,
2

Koujinkai Hemodialysis Clinic

Keywords: Alanine, Creatinine, dialysate, Lactate, Pyruvate, TCA cycle,
1

H NMR observation of plasma and dialysate from hemodialysis (HD) patients was subjected
for profiling of metabolites. Our previous work on 1H NMR metabolomics of plasma from HD
patients has revealed lactate increment after treatment.1) Lactate increment suggested impairedenergy metabolisms in HD patients. Blood sample collection is limited in HD patients becauseof anemia. Then we used dialysate, which is non-invasive for patients and without limitations ofcollection. In analytical aspect, plasma samples were less reproducible because plasma isheterogeneous mixture of proteins, lipids, organic acids and other small metabolites. On theother hand, dialysate has good reproducibility for its composition of only small metabolites, andalso ensures adequate quantification of metabolites by 1H NMR. We have also verified dialysate
as a surrogate for blood in measuring small metabolites during HD, by quantitative 1H NMR. 2)
In this study, 600 dialysate samples from 20 patients were collected in time course during HD
sessions, and measured 1D single-pulse spectra by 600 MHz NMR (ECA, JEOL Ltd.)
spectroscopy. The main metabolites were quantified by their peak integrations on the spectra.
These concentrations in time course revealed to have unique pattern to patient in every HD
session. The finding has potential information for future personalized therapy.
In all of patients, creatinine exhibited monotonous decay and valine showed plateau toward
the end of the session. While patients derived from chronic-glomerular-nephrites exhibited
significant increment of lactate together with alanine and pyruvate at the middle during HD
sessions, which indicated rather amount of productions from the body.. HD treatments rapidly
remove electrolytes, water, and small molecules including bioactive necessities and nutrients aswell as uremic toxins from the blood. As the response to the HD stress, compensative reaction
to maintain homeostasis may occur followed by their productions into blood. The increment oflactate, pyruvate and alanine suggested accelerated glycolysis and/or disturbances of metabolic
pathway at the entrance to TCA cycle.
We will discuss the relation between metabolic profile in each patient and one’s etiology, andthe possible revisions of HD therapies.
1) Fujiwara M. et al Pattern recognition analysis for 1H NMR spectra of plasma fromhemodialysis patients. Anal. Bioanal. Chem. 2009, 394: 1655-1660
2) Ando I, Fujiwara M. et al, 1H NMR Spectroscopic Quantification of Plasma Metabolites in
Dialysate during Hemodialysis Magn.Res. Med.Sci. 2013, 12, No.2, 129-135

C-3-03
Epigenome-wide discovery of ovarian and breast cancer
specific DNA methylation markers
Hitomi Hasegawa 1
hitomi.hasegawa@genedata.com

Dominik Mertens 1
dominik.mertens@genedata.com

Andreas Biegertirst 1
andreas.biegert@genedata.com

Jane Hayward 2
jane.hayward.09@ucl.ac.uk

Shahzia Anjum 2s.anjum@ucl.ac.uk

Marc Flesch 1
marc.flesch@genedata.com

Tamas Rujan 1
tamas.rujan@genedata.com

1
2

Harri Lempiäinen 1
harri.lempiainen@genedata.com

Arnd Brandenburg 1
arnd.brandenburg@genedata.com

Michael Remmerte 1
michael.remmert@genedata.com

Allison Jones 2
allison.jones@ucl.ac.uk

Martin Widschwendter 2
m.widschwendter@ucl.ac.uk

Jens Hoefkens 1
jens.hoefkens@genedata.com

Timo Wittenberger 1
timo.wittenberger@genedata.com

Genedata AG Margarethenstrasse 38, 4053 Basel, Switzerland
Department of Women’s Cancer, University College London, Elizabeth Garrett Anderson
Institute for Women’s Health, London, UK

Keywords: Next Gene Sequencing, Omics data, DNA Methylation, Epigenomics, Clinical
Bioinformatics
Breast and ovarian cancers pose huge and unsolved challenges to the medical profession. The EU
FP7 consortium EpiFemCare develops blood tests based upon DNA methylation technology tofacilitate early detection and prediction of therapeutic outcome of these cancers. In phase 1 of the
project Infinium® HumanMethylation450 BeadChip technology is used to assess the methylation
status of ~485’000 sites in cancer and control tissues. In parallel, Reduced Representation Bisulfite
Sequencing (RRBS) is used to identify and confirm cancer specific methylated circulating DNA inmatching serum samples. Using Genedata Expressionist® for Genomic Profiling, we haveestablished an automated bioinformatics pipeline for the detection of cancer specific Differentially
Methylated Regions (DMRs) that most likely fulfill the strict specificity criteria of a serum based
test. The most promising DMRs are taken forward to clinical assay development (phase 2) andvalidation in thousands of serial samples from prospective clinical trials (phase 3).

C-3-04
Analysis of Human Immunodeficiency Virus Type 1(HIV-1) CRF07_BC in China Using BEAST
Asiya Hapaer 1,3

Fengrong Ren 1

0430bio@tmd.ac.jp

ren@bioinfo.tmd.ac.jp

Yoshihito Niimura 2aniimura@mail.ecc.u-tokyo.ac.jp

1

2

3

Hiroshi Tanaka 1
tanaka@bioinfo.tmd.ac.jp

Department of Bioinformatics, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-8510, Japan
Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan
Center for Disease Control and Prevention, Xinjiang Uyghur Autonomous Region, China

Keywords: HIV-1, Recombinant virus, Phylogeographic analysis
It has been reported that HIV-1 subtypes B' and C from Thailand and India, respectively, wereintroduced into Yunnan province of China in the late 1980s, via drug-trafficking routes andresulted in the initial outbreak of CRF07_BC virus among injection drug users in this region. Since
then, this recombinant virus spread to many other regions and became one of main HIV-1 subtypes
in mainland China nowadays. However, the contradictory results have been reported on the origin
and transmission route of this virus. We conducted a comprehensive analysis to clarify this issue.
We retrieved the viral samples from more than one public databases and created five datasets:
1) env gene, 2) gag gene of subtype C origin, 3) gag gene of subtype B origin, 4) pol gene ofsubtype C origin and 5) pol gene of subtype B origin. These datasets included more locations (15provinces), more sampling time points (1996-2011) and the longer genome regions of viral genes.
Moreover, the most recent version of a Bayesian inference method for phylogeographic analysis
was employed in the study.
The results showed that the origin of the CRF07_BC virus was Yunnan, but the transmission
routes in China are quite complicated. The virus spread to other regions not only from Xinjiang but
also from Yunnan, Sichuan etc. Our results suggest that it is not proper to simply conclude that thevirus spread only from Xinjiang or Yunnan.
[1] Liu, J. and C. Zhang, Phylogeographic analyses reveal a crucial role of Xinjiang in HIV-1
CRF07_BC and HCV 3a transmissions in Asia. PLoS One, 6(8): p. e23347, 2011.
[2] Meng, Z., et al., A new migration map of HIV-1 CRF07_BC in China: analysis of sequencesfrom 12 provinces over a decade. PLoS One, 7(12): p. e52373, 2012.

C-3-05
Computational Approach to Validate Long-range Chromatin
Association between Estrogen Receptor Alpha Proteins andCandidate Genes of Breast Cancer MCF-7 within Different
Human Reference Genomes
Vutha Phav 1chitha.civil@gmail.com
1

Akihiko Konagaya 1
kona@dis.titech.ac.jp

Department of Computational Intelligence and Systems Science, Tokyo Institute ofTechnology, 4259-J3-25 Nagatsuta, Midori, Yokohama, 226-8502, Japan.

Keywords: epigenetics, estrogen receptor alpha binding site, human reference genome sequence
To investigate mechanism of estrogen receptor alpha (ER-α) associated with gene expression, In
particular, we studied computational long-range effects by mapping estrogen receptor alphabinding sites (ER-αBSs) on 37 breast cancer candidate genes with the aim to reveal the most
associated genes along the transcription start sites. Then, we compared result of the computational
long-range chromatin interaction analysis with pair-end tag (ChIA-PET) on different humanreference genome sequences 17 (hg17), 18 (hg18) and 19 (hg19) [1, 2].
By obtaining numerical coordinates of ER-αBSs, we could see a more completely computational
result with ChIA-PET workflow on hg19 by detecting more number of ER-αBSs associated withbreast cancer candidate genes within the same range of the transcription start sites. By
investigating ER-α on different human reference genome sequences, we could determine
coordinates of ER-αBSs can be altered to detect different positions of ER-αBSs based on
association with breast cancer candidate genes such GREB1 and SGK3. In this study, we obtained
3500, 3488 and 3501 ER-αBSs respectively from hg17, hg18 and hg19. We found common 3487
ER-αBSs between hg17 and hg18, common 3425 ER-αBSs between hg17 and hg19, and common
3426 ER-αBSs between hg18 and hg19. However, we only found 3412 common ER-αBSs amonghg17, hg18 and hg19. Therefore, within our comparative study, we could detect different positions
and number of ER-αBSs associated with candidate genes on a single map, as we had not expected
so far. To specifically differentiate the coordinates of ER-αBSs generated by computational
ChIA-PET workflows on hg17, hg18, and hg19, we also reported all different coordinates of
ER-αBSs associated with detectable breast cancer candidate genes.
Within our comparative study, we found that ChIA-PET workflow really depends on humanreference genomes and this would lead to detect different numbers of ER-αBSs; moreover,different positions of ER-αBSs from breast cancer candidate genes can also be confirmed within
our research study.
[1] GuoLiang L, Han Y, Fabianus Hendiryan M, Stoyan V, Vinsensius V, Pramila Nuwantha A,
Yusoff Bin M, Hong-Sain O, Wing-Kin S, et al: ChIA-PET tool for comprehensive chromatininteraction analysis with paired-end tag sequencing. Genome biology 2010, 11:R22.
[2] Vutha PHAV, Akihiko Konagaya. Comparison of computational approaches to estimate
long-range chromatin interaction between human breast cancer candidate genes and estrogen
receptor alpha proteins. Proceeding of InCob 2013 international conference; 2013, 20-22;
Taicang, China.

C-3-06

Association of CDKN2A/B, ADTRP and PDGFD
polymorphisms with coronary atherosclerosis in Japan

1
2
3

Sariya Dechamethakun 1

Shinobu Ikeda 1

Tomio Arai 2

dsariya.epi@mri.tmd.ac.jp

sikeda.epi@mri.tmd.ac.jp

arai@tmig.or.jp

Noriko Sato 1

Masaaki Muramatsu 1

Motoji Sawabe 3

nsato.epi@tmd.ac.jp

muramatsu.epi@mri.tmd.ac.jp

m.sawabe.mptmd.ac.jp

Department of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental
University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology,35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan
Section of Molecular Pathology, Graduate School of Health Care Sciences, Tokyo Medical and
Dental University, Yushima, Bunkyo-ku, Tokyo 113-0034, Japan

Keywords: coronary artery disease (CAD), myocardial infraction (MI), pathology
Background: Genome-wide association studies have identified a series of susceptibility loci forcoronary artery disease (CAD). Our study attempted to replicate the results for eight of these loci,
CDKN2A/B (rs1333049), ADTRP (rs6903956), PDGFD (rs974819), TCF21 (rs12190287),
COL4A1-A2 (rs4773144), HHIPL1 (rs2895811), ADAMTS7 (rs4380028), and UBE2Z (rs46522), in
pathologically defined atherosclerosis of the coronary artery. Methods: Autopsy cases of elderly
Japanese subjects were enrolled in the JG-SNP study (n=1536). Polymorphisms were genotyped,
and their association with coronary stenosis index (CSI) and pathologi cal myocardial infraction(MI) was investigated. Potential combinatorial effects of susceptibility loci were also investigated.
Results: Among the eight loci tested, three gave a sign of positive association. CDKN2A/B showed
the most robust association with CSI and MI (p=0.007 and OR=1.843, 95% CI 1.293-2.629,
p=0.001, for CC+CG vs. GG). ADTRP showed association with CSI and MI, but the risk allele was
opposite from the original report (p =0.008 and OR=1.652, 95% CI 1.027-2.656, p =0.038 for GG
vs. AA+AG). PDGFD showed a suggestive association with CSI in females , but not in males (p=0.023). CDKN2A/B, and ADTRP were significantly associated with severity of CSI, in acase-control setting (top 75% vs. the rest: OR=1.683, 95% CI 1.219-2.323, p =0.002 for CC+CG vs.
GG, OR=1.839, 95% CI 1.172-2.886, p =0.008 for GG vs. AA+AG, respectively). The cumulativerisk allele counting of CDKN2A/B, ADTRP, and PDGFD indicated that increasing number of risk
alleles associated with higher CSI (p <0.001). Conclusions: Our data confirms the association of
CDKN2A/B with CAD, and suggests a different associated risk allele of ADTRP. PDGFD shows agender specific association to CAD. The combination of multiple risk alleles may associate withhigher risk of CAD.
[1] Schunkert, H., et al., Large-scale association analysis identifies 13 new susceptibility loci forcoronary artery disease, Nature Genetics, 6;43(4):333-8, 2011.
[2] Peden, J.F., et al., A genome-wide association study in Europeans and South Asians identifies
five new loci for coronary artery disease, Nature Genetics, 6;43(4):339-44, 2011.
[3] Wang, F., et al., Genome-wide association identifies a susceptibility locus for coronary arterydisease in the Chinese Han population, Nature Genetics, 6;43(4):345-9, 2011.

C-3-07
PoSSuM Updates and Integration With ChEMBL
For Application of Drug Reuse
Kazuyoshi Ikeda 1,2,3
ikeda@level-five.jp

	
  	
 

Kenji Mizuguchi 2
kenji@nibio.go.jp
1

2
3

	
  	
 

Jun-Ichi Ito 2
junichi@nibio.go.jp

Kentaro Tomii 3
k-tomii@aist.go.jp

Drug Discovery Informatics Group, System Solution Division, LEVEL FIVE Co.,Ltd.,
Shiodome Shibarikyu Bldg., 1-2-3 Kaigan, Minato-ku, Tokyo 105-0022, Japan.
National Institute of Biomedical Innovation, 7-6-8 Saito Asagi, Ibaraki, Osaka, Japan.
Computational Biology Research Center (CBRC), National Institute of Advanced Industrial
Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.

Keywords: Ligand binding site, Pocket similarity, Open drug discovery data, Drug reuse, Database
PoSSuM is a large-scale database for finding similar ligand-binding sites in proteins [1], which isfreely available via our web-interface (http://possum.cbrc.jp/PoSSuM). As of Aug. 2013, PoSSuM
stores over four millions of data points including both known small molecule-binding pockets andputative ones. Detection of structurally similar sites of known small-molecule drugs gives ususeful information for rational drug design such as drug-reuse and off-target prediction. For the
purpose, we have integrated PoSSuM pocket similarity data with ChEMBL open drug discovery
data [2], and have developed a new function for supporting the user to retrieve potential binding
pockets for a given small-molecule drug, in addition to a number of improved web interfaces.
First, we selected orally bioavailable small-molecule drugs from the release of ChEMBL16, andmatched the compound structures with the latest PDB ligand structures using IUPAC standard
InChIKey. In PoSSuM, 4,282 sites where 193 unique small-molecule drugs bound were found.
Second, we annotated 3D protein-structures in PDB using the ChEMBL target information for
drugs and drug candidates, and classified the protein structures based on its drug development
stage. For this purpose, we extracted 7,547 protein-targets from ChEMBL16 and joined those to
the FDA-approved drug targets from DrugBank [3]. The protein-targets were classified into thefollowing classes: A) Approved drug targets, B) Hit-to-lead stage, C) Bioactive targets and D)
Unknown. Then, we combined the drug target classification with PoSSuM pocket similarity data,
and built a pipeline for automatically identifying potential new drug targets having a pocket
similar to known small-molecule drug binding sites.
We identified in total 154,191 binding sites, from 10,255 protein structures, which are
structurally similar to the focused 4,282 binding sites, covering 182 unique small-molecule drugs
deposited in ChEMBL16. Users can retrieve all of the identified similar sites and can visualize 3D
superpositoin between them on our new web-interface which will be available soon. We also
conducted a preliminary study to compare between PDB-scale pocket similarity and genomic-scalebioactive data for surveying relations between the similarity score and binding affinities.
In this presentation, we will show new functions of the PoSSuM database, utility forstructure-based drug design, and discuss the possibility and limitation for application of drug
discovery purpose.
[1] Ito, J., et al., PoSSuM: a database of similar protein-ligand binding and putative pockets,
Nucleic Acids Res, 40(Database issue):D541-D548. 2012.
[2] Gaulton, A., et al., ChEMBL: a large-scale bioactivity database for drug discovery, Nucleic
Acids Res, 40(Database issue):D1100–D1107. 2012.
[3] Knox C., et al., DrugBank 3.0: a comprehensive resource for 'omics' research on drugs,
Nucleic Acids Res, 39(Database issue):D1035-D1041. 2011.

C-3-08
Phasing haplotypes of HLA genes from Next Generation
Sequencing data at individual level
Chenxi Zhao1,
zhao.bio@tmd.ac.jp

Nay Chi Htun1,

Noriko Sato1,

nhtun.epi@mri.tmd.ac.jp

Masaaki Muramatsu

nsato.epi@mri.tmd.ac.jp

1muramatsu.epi@mri.tmd.ac.jp

1

Department of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Keywords: Next Generation Sequencing, Human leukocyte antigen, Haplotype
Objective: Haplotype information of an individual is valuable for practical applications of
personalized medicine, such as pharmacogenomics. For instance, haplotypes of HLA (humanleukocyte antigen) genes, which are essential for immune functions, are reported to be
associated with Stevens–Johnson syndrome (SJS), a drug induced severe adverse reaction of
the skin. In order to facilitate pharmacogenomics application of NGS (Next Generation
Sequencing) data, we employed these data to define haplotypes of HLA genes.
Method: Whole-Genome Sequencing (WGS) and Whole-Exome Sequencing (WES) data
was collected from public database. Initially, we focused on HLA-B haplotypes, which are
involved in SJS induced by Carbamazepine and Allopurinol. Fastq data was mapped against
hg19 reference genome and HLA-B gene data was extracted. Genotypes and haplotypes of
HLA-B were detected by applying the linkSNPs program [1]. Integrative Genomics Viewer(IGV) was used for visualizing short reads.
Results: Haplotype phasing results showed that, 22 haplotype clusters were found. Combined
information from the short reads, all 7 exons of HLA-B gene can be phased into 4 major parts,
with 16 possible haplotype combinations. Protein blast result of the 16 combinations above
showed that 10 candidate haplotypes showed the highest scores. We further used haplotype
frequency table, to rank the probability of haplotype combinations in the individual [2]. As aresult, we inferred the most likely HLA-B gene haplotypes.
Conclusion: Haplotype information of HLA genes from an individual NGS data is an
important issue in personalized pharmacogenomics. As a practical example, the method
introduced in this study may be useful in estimation of adverse reaction risks of SJS.
[1] Sasaki E, Sugino RP, Innan H, The linkage method: a novel approach for SNP detection and haplotype reconstruction from a single diploid
individual using next-generation sequence data, Mol Biol Evol, 30(9):2187-2196, 2013.
[2] Maiers M, Gragert L, Klitz W, High-resolution HLA alleles and haplotypes in the United States population, Hum Immunol, 68(9):779-788,
2007.

C-3-09
Dynamic evolution of forkhead transcription factors andchanges in their DNA-binding sites
So Nakagawa 1,2

1

2

3

Stephen S. Gisselbrecht 3

so.nakagawa@tokai-u.jp

sgisselb@genetics.med.harvard.edu

Julia M. Rogers 3

Tadashi Imanishi 1

juliarogers@fas.harvard.edu

imanishi@tokai-u.jp

Daniel L. Hartl 2

Martha L. Bulyk 3

dhartl@oeb.harvard.edu

mlbulyk@receptor.med.harvard.edu

Department of Molecular Life Science, Tokai University School of Medicine, 143
Shimokasuya, Isehara, Kanagawa 259-1193 Japan
Department of Organismic and Evolutionary Biology, Harvard University, 16
Divinity Avenue, Cambridge, MA 02138, USA
Division of Genetics, Department of Medicine, Brigham and Women’s Hospitaland Harvard Medical School, Boston, MA 02115, USA

Keywords: transcription factor binding site motif, protein–DNA interactions, multigene family
Forkhead box (Fox) proteins are transcription factors (TFs) related with various diseases. The
Fox family, defined by a highly conserved winged helix DNA-binding domain (DBD), hasdiverged into dozens of subfamilies in animals, fungi, and related protists. We have used a
combination of maximum likelihood phylogenetic inference and unbiased functional assays of
DNA binding capacity to explore the evolution of DNA binding specificity within the Fox family.
We present converging evidence that similar alternative sequence preferences have arisen
repeatedly and independently in the course of Fox evolution. The vast majority of DNA binding
specificity changes we observed are not explained by alterations in the known DNA-contacting
amino acid residues conferring specificity for canonical Fox binding sites. Intriguingly, we havefound Fox DBDs that retain the ability to bind very specifically to two completely distinct DNA
sequence motifs. We propose an alternate specificity-determining mechanism whereby
conformational rearrangements of the DBD broaden the spectrum of sequence motifs that a TF canrecognize. DNA binding bispecificity suggests a new source of modularity and flexibility in gene
regulation and may play an important role in the evolution of transcriptional regulatory networks[1]. In this conference, we will also discuss changes in Fox domain sequences and their bindingsites by estimating ancestral sequences of several Fox subfamilies.

[1] Nakagawa S, Gisselbrecht SS, Rogers JM, Hartl DL, Bulyk ML., DNA-binding specificity
changes in the evolution of forkhead transcription factors, Proceedings of the National Academyof Sciences of the United States of America,110(30):12349-12354, 2013.

C-3-10
Applications of an integrated data warehouse system toinvestigate complex biological systems
Yi-An Chen 1

Lokesh P. Tripathi 1chenyian@nibio.go.jp

lokesh@nibio.go.jp

Kenji Mizuguchi 1
kenji@nibio.go.jp

1

National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi,
Ibaraki-City, Osaka, 567-0085, Japan.

Keywords: Data integration, Target prioritization, Data warehouse, Drug discovery
We have developed TargetMine, an integrated data warehouse for the purpose of early-stage drug
discovery [1]. TargetMine is based on InterMine, a flexible and effective framework for combining
biological data from various repositories, which enables navigating through diverse biological data
within a single user-friendly interface. TargetMine incorporates a wide range of biologicalassociations including pathways, Gene Ontology annotations, protein structures, chemicalcompounds and different types of biomolecular interactions. Furthermore, TargetMine includes
multiple application programming interfaces, thereby permitting complex pipeline analysis. We
have proposed several protocols and also implemented accessory tools to enable streamlined data
analysis using TargetMine. These approaches have been effectively employed for target selectionin protein-protein interaction (PPI) network-based analyses of Hepatitis C virus (HCV)
pathogenesis [2,3,5] and lung tumorigenesis [4]. The academic version of TargetMine is freely
available at http://targetmine.nibio.go.jp .
[1] Chen YA, Tripathi LP, Mizuguchi K (2011) TargetMine, an Integrated Data Warehouse for
Candidate Gene Prioritisation and Target Discovery. PLoS ONE 6(3): e17844.
[2] Tripathi LP, et al. (2010) Network based analysis of hepatitis C virus Core and NS4B protein
interactions. MolBiosyst 6: 2539-2553.
[3] Tripathi LP, et al. (2012) Proteomic analysis of hepatitis C virus (HCV) core proteintransfection and host regulator PA28γ knockout in HCV pathogenesis: A network-nased study. J
Proteome Res 11: 3664-3679.
[4] Ihara S, et al. (2012) Inhibitory roles of signal transducer and activator of transcription 3 inanti-tumor immunity during carcinogen induced lung tumorigenesis. Cancer Res.72, 2990-2999.
[5] Tripathi LP, et al. (2013) Understanding the Biological Context of NS5A-Host Interactions in
HCV Infection: A Network-Based Approach. J Proteome Res.

C-3-11

Exploring Development Origin of Health and Disease(DOHaD) hypothesis employing publicly available data
Tay Zar Kyaw 1

Noriko Sato 1

tayzaraction@gmail.com

nsato.epi@mri.tmd.ac.jp

Masaaki

Muramatsu 1

muramatsu.epi@mri.tmd.ac.jp

1

Department of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental
University, MRI Bldg. F-2, 3-10 Kanda-Surugadai 2, Chiyoda-ku, Tokyo 101-0062, Japan

Keywords: Low birth-weight, Obesity
Background: DOHaD is a concept initially proposed by David Barker that environmental
influences during fetal development may predispose the individual towards specific phenotype or
diseases. Many epidemiological and animal studies have been undertaken to prove this hypothesis
and study its molecular mechanism. However, the impact of this concept to public health is not
fully described in Japan and other Asian countries.
Objective: As for a preliminary survey, we searched for clues that sustain DOHaD hypothesis, andfind if there are correlation between low birth-weight and increasing trend of obesity in children
and/or adult of Japan and other Asian countries.
Materials and Method: Publicly available data and literature search were employed. These include
Japan`s school health data, Japan`s National Health and Nutrition Survey, WHO World Health
Statistics data and other related databases. The correlation of the rate of low birth-weight during
1970-2000 with 5 years interval and the rate of obesity at their age of 10 years was analyzed byplotting the prevalence of obesity against prevalence of low birth-weight. Simple linear regression
and Pearson`s correlation coefficient was used to determine the strength of correlation in variables.
Results: Among eight Asian countries surveyed, Japan and Korea were encountering a significant
increasing trend of low birth-weight. We observed a tendency of positive correlation between rateof low birth-weight and rate of obesity at 10-year-old, but it was not statistically significant.
(p=0.15)
Conclusion: The cause and outcomes of increasing rate of low birth-weight in Japan and otherdeveloped countries warrant further research studies. We are currently investigating birth-weight
and other intrauterine factors that associate with DOHaD hypothetical outcomes among Japanese
population.
[1] Barker, D.J.P., Developmental origins of adult health and disease, Journal of Epidemiology andCommunity Health, 58:114-115, 2004.
[2] Whitaker, R.C. and Dietz, W.H., Role of the prenatal environment in the development ofobesity, J Pediatr, 132:768-76, 1998.
[3] Takimoto, H., Tetsuji, Y., Nobuo, Y. and Fukuoka, H., Increase in low-birth-weight infants inJapan and associated risk factors, 1980-2000, J. Obstet. Gynaecol. Res., 31:314-322, 2005.
[4] http://www.mhlw.go.jp/bunya/kenkou/kenkou_eiyou_chousa.html[5] http://www.e-stat.go.jp/SG1/estat/List.doC-3-12
Correlation of annotation terms in heterogeneous databases
and its application to Gene Set Enrichment Analysis(GSEA) and ontology construction
Katsuhiko Murakami 1

Tadashi Imanishi 1, 2

k-murakami@aist.go.jp

t.imanishi@aist.go.jp

1

Molecular Profiling Research Center for Drug Discovery,
National Institute of Advanced Industrial Science and Technology,
2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan
2
Tokai University School of Medicine, Tokai University,
143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan

Keywords: Gene Set Enrichment Analysis, Database, Annotation, Correlation, Ontology
Characterization of a given gene set, such as "gene set enrichment analysis (GSEA)" [1], has
become an important task in omics era. In such analyses, ann otations are used as if they wereindependent, despite that some annotations are correlated each other. To interpret complex
multiple annotations, we comprehensively examined correlation among each annotation for humangenes. We selected ten gene annotation (gene family, Gene Ontology, InterPro, KEGG pathway,
protein-protein interaction, SCOP, SOSUI membrane protein prediction, OMIM, tissue specificity
of gene expression, and subcellular localization) from the integrated human gene database,
H-InvDB [2,3]. For all pairs of the terms, the correlations were evaluated using Fisher's exact(two-side) test with Bonferroni correction. As a result, we found 99,813 and 653 pairs withpositive and negative correlation respectively. Many of the positive relationships were synonyms,
such as "SCOP g.44.1.1 (RING finger)" and "IPR001841 (Zinc finger, RING -type)". We foundother pairs with relevant but not apparent relationships, such as "GO:0006470, protein
dephosphroylation" and "hsa04940: Type I diabetes mellitus". We a lso obtained negative
relationships. Many of them seemed unlikely to co-occur in a gene and related to subcellular
localization, such as extracellular and nuclear. Those information will help to refine predictive
annotation, or perhaps include multifunctions of the genes. By integrating these annotation
relationships together with other integrated databases, such as UniProt, we can re -evaluate
complex annotations of GSEA results and produce a new summary report of the gene set, as well asa construction of an integrated ontology..
[1] Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A,
Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: aknowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad
Sci 102(43):15545-50, 2005
[2] Takeda J, Yamasaki C, Murakami K, Nagai Y, Sera M, Hara Y, Obi N, Habara T, Gojobori T,
Imanishi T., H-InvDB in 2013: an omics study platform for human functional gene and transcript
discovery. Nucleic Acids Research 41(Database issue): D915-9, 2013[3] http://www.h-invitational.jp/

C-3-13
Analysis of abnormalities in gene expression and splicing
patterns of spinocerebellar ataxia type 6 knockin miceusing RNA-seq

1

Junya Hagiwara 1

Masaki Morioka 2

Kaoru Mogushi 2

ma120065@tmd.ac.jp

morioka-tky@umin.ac.jp

mogushi@bioinfo.tmd.ac.jp

Kei Watase 3

Hidehiro Mizusawa 4

Hiroshi Tanaka 2

keinuro@tmd.ac.jp

h-mizusawa.nuro@tmd.ac.jp

tanaka@bioinfo.tmd.ac.jp

Department of Computational Biology, 2 Department of Bioinformatics, 3 Center for Brain
Integration Research, and 4 Department of Neurology and Neurological Science, Tokyo Medical
and Dental University, Bunkyo -ku, Tokyo 113-8510, Japan

Keywords: RNA-seq, spinocerebellar ataxia type 6, alternative splicing
Spinocerebellar ataxia (SCA) is a neurodegenerative disease characterized by a loss of motor
coordination and balance capability. SCA is one of the polyglutamine (poly -Q) diseases including
subtypes of SCAs and Huntington disease. Among them, SCA6 is late -onset autosomal dominantneurodegenerative disease caused by a poly-Q expansion in CACNA1A gene that encodes Cav2.1
voltage-gated calcium channel subunit. 1 Since there is no treatment for this congenital disease, thedevelopment of novel therapies is strongly needed. It has been reported that the poly -Q expansion
causes Purkinje cell degeneration, but the detailed pathogenesis pathway is still unclear. Therefore,
we developed a knockin mouse model that had a 118 CAG repeat tract (118Q) in CACNA1A andinvestigated abnormalities in gene expression and splicing patterns in this model mice. 2
In this research, we aimed to analyze genes differentially expressed and spliced between
cerebellar tissues of a wild-type mouse and an SCA6 model mouse. We obtein 1.6 hundred million
reads (16 billion bases) from the wild-type and 1.5 hundred million reads(15.3 billion b ases) fromthe SCA6 model mouse using HiSeq 2000 (Illumina).The TopHat package 3 was used for thealignment of the short reads to the mm10 reference genome, followed by Cufflinks 4 for transcript
assembly RNA expression and alternative splicing analysis.We investigated several genes andpathways that might be associated with pathogene sis of SCA6 such as degeneration of Purkinjecells.
[1] Schöls L, Bauer P, Schmidt T, Schulte T, Riess O. Autosomal dominant cerebellar ataxias:
clinical features, genetics, and pathogenesis. Lancet Neurol. 2004;3(5):291-304.
[2] Unno T., Wakamori M, Koike M, Uchiyama Y, Ishikawa K, Kubota H, Yoshida T, Sasakawa H,
Peters C, Mizusawa H, Watase K. Development of Purkinje cell degeneration in a knockinmouse model reveals lysosomal involvement in the pathogenesis of SCA6. Proc Natl Acad Sci U
S A. 2012;109(43):17693-8.
[3] Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq.
Bioinformatics 2009;25(9):1105-11[4] Trapnell C, Williams BA, Pertea G, Mortazavi AM, Kwan G, van Baren MJ, Salzberg SL, Wold
B, Pachter L. Transcript assembly and quantification by RNA -Seq reveals unannotatedtranscripts and isoform switching during cell differentiation Nature Biotechnol 2010;28(5):511-5

C-3-14C-3-15
PathAct: a novel method for pathway analysis
using gene expression profiles

1

Kaoru Mogushi 1

Hiroshi Tanaka 1

mogushi@bioinfo.tmd.ac.jp

tanaka@bioinfo.tmd.ac.jp

Department of Bioinformatics, Medical Research Institute, Tokyo Medical and Dental
University, 24F M&D Tower Bldg., 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan

Keywords: DNA microarray, Gene expression profile, Pathway analysis
Gene expression profiling by microarray analysis provides a huge amount of biologicalinformation and has been widely used in biological and clinical research. We developed PathAct 1 ,a novel method for pathway analysis to investigate the biological and clinical implications of thegene expression profiles. The advantage of PathAct in comparison with the conventional pathway
analysis methods is that it can estimate pathway activity levels for individual patient quantitatively
in the form of a pathway-by-sample matrix using the median polish algorithm 2 . This matrix can beused for further analysis such as hierarchical clustering and other analysis methods.
To evaluate the feasibility of PathAct, comparison with frequently used gene -enrichment analysis
methods was conducted using two public microarray datasets. The dataset #1 was that of breast
cancer patients, and we investigated pathways associated with triple -negative breast cancer by
PathAct, compared with those obtained by gene set enrichment analysis (GSEA) 3 . The dataset #2
was another breast cancer dataset with disease-free survival (DFS) of each patient. Contribution byeach pathway to prognosis was investigated by our method as well as the Database for Annotation,
Visualization and Integrated Discovery (DAVID) 4 analysis. In the dataset #1, four out of the six
pathways that satisfied p < 0.05 and FDR < 0.30 by GSEA were also included in those obtained by
the PathAct method. For the dataset #2, two pathways ("Cell Cycle" and "DNA replication") out offour pathways by PathAct were commonly identified by DAVID analysis.
Thus, we confirmed a good degree of agreement among PathAct and conventional methods.
Moreover, several applications of further statistical analyses such as hierarchical cluster analysis
by pathway activity, correlation analysis and survival analysis between pathways were conducted.
Therefore, PathAct is a promising tool for pathway -level investigation and interpretation of thecomprehensive gene expression data.
[1] Mogushi K, Tanaka H. PathAct: a novel method for pathway analysis using gene expression
profiles. Bioinformation. 2013;9(8):394-400.
[2] Hoaglin DC, Mosteller F, Tukey JW, eds. Understanding robust and exploratory data analysis.
Vol. 3. New York: Wiley, 1983.
[3] Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A,
Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: aknowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad
Sci U S A. 2005;102(43):15545-50.
[4] Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID:
Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003;4(5):P3.

C-3-16
RNA-Sequencing-Based Screening of Long Non-Coding
RNAs Targeted by Steroid and Xenobiotic Receptor
Kuniko Horie-Inoue 1

Kotaro Azuma 2khorie-tky@umin.ac.jp

kazum-tky@ umin.ac.jp

Kazuhiro Ikeda 1
ikeda@saitama-med.ac.jp

Satoshi Inoue 1.2
INOUE-GER@h.u-tokyo.ac.jp
1

Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka,
Saitama 350-1241, Japan
Departments of 2 Geriatric Medicine and 3 Anti-Aging Medicine, Graduate School of
Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Keywords: long non-coding RNA, steroid and xenobiotic receptor, osteoblast, RNA -sequencing
Steroid and xenobiotic receptor, SXR, is a nuclear receptor that plays an impor tant role in thedetoxification of bile acids and chemical compounds [1]. We have previously showed that vitamin
K 2 -liganded SXR is a transcriptional regulator of bone marker genes [2,3]. Recent advance ingenomic technology enables to identify numerous long non-coding RNAs (lncRNAs), which haveno obvious coding capacities with their length >200 bp and widely transcribed in spati otemporal
specific manners [4]. While biological functions of some lncRNAs have been characterized, few
lncRNAs have been identified in the context of nuclear receptor-regulated gene network.
Here we screened functional lncRNAs upregulated by SXR agonists in human osteoblastic
MG63 cells overexpressing SXR, based on RNA-sequencing analysis. Among RefSeq transcripts
with >1.5-fold up-regulation or < 0.67-fold down-regulation by either rifampicin or vitamin K 2
versus vehicle, ~10% were identified as NR non-coding transcripts and half of the NR transcripts
were up-regulated by either SXR ligand (except transcripts down-regulated by the other ligand).
Based on the recent genomic data, one third of the up-regulated NR transcripts were lncRNAs
except non-coding variants of coding-genes. Interestingly, known lncRNA HOTAIR [5] wasinvolved in the group of putative SXR target lncRNAs. We cons ider that this integrative screen of
lncRNAs will reveal the RNA-mediated transcriptional regulation of SXR, and will provide useful
information for the potential use of lncRNAs as new osteoblastic biomarkers and therapeutictargets for bone diseases such as osteoporosis.
[1] Sonoda, J., Rosenfeld, J.M., Xu, L., Evans, R.M., and Xie, W., A nuclear receptor-mediatedxenobiotic response and its implication in drug metabolism and host protection , Current Drug
Metabolism, 4:59-72, 2003.
[2] Tabb, M.M., et al., Vitamin K2 regulation of bone homeostasis is mediated by the steroid andxenobiotic receptor SXR. Journal of Biological Chemistry, 278:43919-43927, 2003.
[3] Ichikawa, T., Horie-Inoue, K., Ikeda, K., Blumberg, B., and Inoue, S., Steroid and xenobiotic
receptor SXR mediates vitamin K2-activated transcription of extracellular matrix-related genes
and collagen accumulation in osteoblastic cells. Journal of Biological Chemistry,
281(25):16927-34, 2006.
[4] FANTOM consortium, The transcriptional landscape of t he mammalian genome, Science,
309:1559-1563, 2005.
[5] Rinn, J. L., et al., Functional demarcation of active and silent chromatin domains in human
HOX loci by noncoding RNAs, Cell, 129:1311-1323, 2007.

C-3-17The Inferring Method of the large scale regulatory networkfor omics studies
Asako Komori 1

1
2
3

Yukihiro Maki 2

a-komori@brs.kyushu-u.ac.jp

ykhrmaki@tos.bbiq.jp

Isao Ono 3

Masahiro Okamoto 1, 2

isao@dis.titech.ac.jp

okahon@brs.kyushu-u.ac.jp

Department of Bioinformatics, Graduate School of Systems Life Sciences , Kyushu University
West-wing 804, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Synthetic Systems Biology Research Center, Kyushu University, Westwing 812, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Graduate School of Computational Intelligence and Systems Science, Tokyo Institute ofTechnology, G5-8, 4259, Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8503, Japan

Keywords: Inverse problem, S-system, System identification, Real-coded genetic algorithms
Recent advances in measurement techniques have provided a large number of time series
data simultaneously. These data are important for understanding complex systems such as
gene regulatory networks and metabolic pathways required to infer networks. We haveproposed an efficient inferred method, the hybrid method, for such problems using
S-system, the real-coded genetic algorism (RCGA) along with adaptive real-coded ensemble
crossover (AREX) combined with the just generation gap (JGG) and the modified Powellmethod. At this present, we have succeeded in optimizing more than 270 real -valued
parameters using this method. Although the hybrid method is powerful compared withconventional numerical optimization methods, it is still insufficient for inferring large
networks with more than 10 components. In this study, we attempted to infer the 30
components network, which requires optimizing more t han 900 parameters, by applying
our improved inferring method.

[1] Savageau, M. A., Biochemical Systems Analysis: A Study of Function and Design in Molecular
Biology, Addison-Wesley, 1976.
[2] Akimoto, Y., Nagata, Y., Sakuma, J., Ono, I., Kobayashi, S., Proposal and Evaluation of
Adaptive Real-coded Crossover AREX. Journal of Japanese Society for Arti-ficial Intelligence,24(6): 446-458, 2009.
[3] Powell, M.J.D., An Iterative Method for Finding Stationary Values of a Function of Several
Variables. The Computer Journal, 5: 147-151, 1962.
[4] Komori, A., Maki, Y., Nakatsui, M., Ono, I., and Okamoto, M., Efficient Numerical
Optimization Algorithm Based on New Real-Coded Genetic Algorithm, AREX + JGG, andApplication to the Inverse Problem in Systems Biology. Applied Mathematics, 3(10A):
1463-1470, 2012.

C-3-18
Development of an Ontology for Periodontitis
Asami Suzuki 1

Takako Takai-Igarashi 2

nduh-a-suzuki@tky.ndu.ac.jp

Hiroshi Tanaka 2

1
2

General Dentistry, The Nippon Dental University Hospital at Tokyo, 2-3-16
Fujimi, Chiyoda-ku, Tokyo 102-8158, Japan
Department of Computational Biology, Graduate School of Biomedical Science, Tokyo Medical
and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan

Keywords: Periodontitis, Ontology, Multifactor disease, Systemic diseases, Biomedical process
Periodontitis is a multifactor inflammation disease which increases risks for many systemic
diseases such as diabetes and cardiovascular disorders. Despite accumulation of biomedical
literatures on periodontitis pathology, there has been no ontology for the biomedical concepts on
the process of pathological progression and clinical treatments of periodontitis
We build an ontology for periodontitis by extracting conceptual terms from the articles retrieved
from PubMed database with the keywords specific to molecular pathology of periodontitis. The
ontology includes pathological processes in periodontitis specified by the ir activators, suppressors,
locations and participants. In particular osteo-immune processes were specified in terms of
relationships to systemic diseases.
We believe this ontology promotes bioinformatics analyses of data obta ined from periodontal
lesions[1] Suzuki, A., Takai-Igarashi, T., Numabe, Y. and Tanaka, H., Development of a database andontology for pathogenic pathways in periodontitis, In Silico Biology, 9:233-243, 2009.
[2] Grant, M.M., What do ’omic technologies have to offer periodontal clinical practice in the
future?, Journal of Periodontal Research, 47:2-14, 2012.
[3] Harvey, A. and Bruno, G.L., Inflammatory mechanisms linking peri odontal diseases tocardiovascular disease, Journal of Periodontol, 84:S51-S69, 2013.
[4] Gerard, J.L., Amy, L. and Frank A.S., Periodontal systemic associations: review of theevidence, Journal of periodontal, 84:S8-S19, 2013.
[5] Day-Richter, J., Harris, A.M., Haendel, M., The Gene Ontology OBO-Edit Working Group andLewis, S., OBO-Edit-an ontology editor for biologists, Bioinformatics, 23:2198-2200, 2007

C-3-19
Improving the visual interpretation of experimental databy a more informative display of confidence intervals
Daniel Berrar ,berrar.d.aa@m.titech.ac.jp

Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan

Keywords: Standard error, confidence interval, visual interpretation
The clear and effective communication of information is of paramount importance in science.
Commonly, experimental results are depicted in figures, and the observed values (such as means,for example) are frequently accompanied by bars. These bars sometimes reflect standard error of
the mean (SEM), standard deviation (SD), or a confidence interval (CI). Their visual interpretation,
however, is far from being trivial, particularly in more complex study designs [ 1]. A clear andeffective visual representation is therefore challenging [ 2, 3 ]. Here, we discuss the involved
problems and argue against displaying SEM bars because they easily invite misinterpretations. We
recommend displaying adjusted confidence intervals in figures.

[1] Cumming G. and Finch S. (2005) Inference by eye: confidence intervals and how to readpictures of data. American Psychologist 60(2):170−180.
[2] Belia S., Fidler F., Williams J., Cumming G. (2005) Researcher misunderstand confidence
intervals and standard error bars. Psychological Methods 10(4):389−396.
[3] Estes W.K. (1997) On the communication of information by displays of standard errors andconfidence intervals. Psychological Bulletin & Review 4(3):330−341.

C-3-20
A comprehensive selection of single nucleotide
polymorphisms to predict genetic risks of developing
common chronic diseases in Japanese
Akio Ametani 1

Yuji Hamamoto 1

a.ametani@infobio.co.jp

Shungo Deshimaru 1
s.deshimaru@infobio.co.jp

y.hamamoto@infobio.co.jp

Naoko Matsumoto 1
n.matsumoto@infobio.co.jp

Naoki Kojima 2

Shunsuke Kanada 2

naoki.kojima@xicafe.jp

shunsuke.kanada@xicafe.jp

Takuya Ohsugi 2
takuya.ohsugi@xicafe.jp

Masaaki Muramatsu 3
muramatsu.epi@mri.tmd.ac.jp

1
2

3

InfoBio Co.,Ltd., 2-2-1 Koraku, Shigaku-House Bldg, 5F, Bunkyo-ku, Tokyo 112-0004, JapanNPO Xicafe (For the Children of the Children of the Children), 6-8-22 Okusawa, Setagaya-ku,
Tokyo158-0083, Japan
Laboratory of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental
University 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Keywords: SNPs, Database, Relative risk, Genetic susceptibility to disorders
Recently a number of susceptible loci for common chronic diseases and related disorders havebeen identified by genome-wide association studies (GWAS). Although the effect size of each SNP
differs among different ethnics, abundant literatures allow us to assess each risk in a certain ethnic
group. We here report a system to determine genetic risks of developing various disorders among
Japanese people, using relevant allele frequencies and odds ratios of selected SNPs.
First we surveyed published papers for GWAS and smaller-scale studies conducted
predominantly in Japan, and built a database of disease-susceptible SNPs, curating positions on thehuman genome, gene names, allele frequencies, odds ratios, p-values, and calculated relative risk
to the general population. SNPs verified by replication studies, meta-analysis or multiple studies
were preferentially chosen and used for calculating genetic risks. Linkage disequilibrium wasconsidered to remove duplicate SNPs in a susceptible locus. As a result, 128 SNPs were selected
for a total of 28 common diseases and related disorders. Among them 93 SNPs were from theresults of GWAS with replication studies. Other 26 SNPs were from studies for more than 300
cases followed by replication or another studies, where statistical significance was found andp-values were less than 0.05. Remaining 9 SNPs were from smaller-scale studies with somereliability. Finally 1 to 14 SNPs per disorder were employed for risk calculation of these 28
disorders. Next, segregation ratios of each genotype and relative risks to the general population
were calculated and integration of relative risks for each genotype was plotted.
The database with selected SNPs should be useful for predicting risks of developing disorders forJapanese individuals and are ready for the validation of the system.

C-4-01
Precise estimation of the contribution of CYP enzymes tooverall metabolism from in vivo information: applicationof Cluster Newton Method
Kenta Yoshida 1,2

6.kurabu.pasu@gmail.com

1
2
3

Kazuya Maeda 1

kmaeda@mol.f.u-tokyo.ac.jp

Hiroyuki Kusuhara 1

kusuhara@mol.f.u-tokyo.ac.jp

Yuichi Sugiyama 3

Akihiko Konagaya 2

ychi.sugiyama@riken.jp

kona@dis.titech.ac.jp

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7 -3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology,
4256 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, JapanRIKEN Research Cluster for Innovation, 1-6, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045,
Japan

Keywords: drug–drug interactions, pharmacokinetics, PBPK model, membrane transporters
Drug–drug interactions (DDIs) are a major cause of adverse drug reactions and subsequent
withdrawal of new drugs from the market. For DDIs mediated by the inhibition of drug
metabolisms, there are two key determinants of the degree of these interactions: inhibition
constants (K i ) of coadministered drugs and fraction metabolized (f m) by individual metabolic
enzymes in the overall metabolism of substrate drugs.
In this study, we performed physiologically-based pharmacokinetic (PBPK) analyses of DDIs to
determine “in vivo” f m values. For the precise determination of f m values, analyses ofpharmacokinetic alterations in both parent drug and metabolites are essential. Since thepharmacokinetic analyses of metabolites are difficult with conventional parameter optimization
methods owing to the lack of information on feasible initial parameters, we utilized Cluster
Newton Method [1-2] to estimate solution spaces of pharmacokinetic paramete rs.
PBPK analyses were performed for the DDIs involving lansoprazole, lidocaine, or oxycodone,
whose major clearance pathways are CYP2C19 and CYP3A4, CYP1A2 and CYP3A4, or CYP2D6
and CYP3A4, respectively. DDI data were retrieved from the University of Washington
Metabolism and Transport drug interaction database (http://www.druginteractioninfo.org). As aresult, f m of each CYP enzyme was determined with small variations, only when we included
metabolites’ pharmacokinetic alterations in the analyses . Application of the proposed method may
improve in vitro-in vivo extrapolations of f m values, which can lead to the accurate preclinicalprediction of DDIs involving the inhibition of metabolisms.
[1] Aoki, Y., et al., Cluster Newton Method for Sampling Multiple Solutions of an
Underdetermined Inverse Problem: Parameter Identification for Pharmacokinetics , NII
Technical Report, NII, NII-2011-002E, 2011.
[2] Yoshida, K., et al., Estimation of feasible solution space using Cluster Newton Method:
application to pharmacokinetic analysis of irinotecan with physiologically -based
pharmacokinetic models , BMC Systems Biology, 7(Suppl 2):S3, 2013.

C-4-02
Development of the Discriminant KY-methods for
Toxicity Screening
Kohtaro Yuta
k-yuta@insilicodata.com

In Silico Data, Ltd. 5-19-5 Akitu, Sin-narasino, chiba 275-0025, Japan

Keywords: KY-methods, Discriminant KY-methods, Toxicity prediction, In silico screening
The KY-methods are powerful data analysis methods which enables a perfect classificationeven if overlapping between classes of samples is very high and number of samples is quite
large.
KY-methods have been developed on the premise that applied on toxicity prediction research
field. The special features of the KY-methods are as follows. This approach can overcome
various difficult data analysis conditions.
These are conditions which make extremely difficult to do a data analysis. For example,
number of sample is very large, the spread of the sample space is large because structure
diversity of compounds is quite large, and more overlapping between classes in the sample
space is large.
On the toxicity prediction research fields, it have been almost impossible to achieve highclassification value by simple application of multivariate analysis / pattern recognition methods
by conventional approaches.
Basics of the KY-methods are the multi-step operations that repeat classification and sample
re-construction.
Currently, three different discriminant analysis methods have been developed in accordancewith the basics of the KY-methods. In this report, briefly discuss the current development statusof the KY-methods.

[1] K-step Yard sampling (KY-methods); Kohtarou Yuta, US Patent NO.:US7725413 B2 May 25,
2010.
[2] http://insilicodata.com/pdf%20lists/2%20Model%20KY.pdf[3] Kohtaro Yuta, Development of the “K-step Yard sampling method” and Apply to the ADME -T
In Silico Screening, The 34-th SAR symposium , K06, Niigata, Japan, 2005.
[4] Kazuhiro Sato, etc., Skin sensitization study by a new qualitative structure -toxicity
relationships (QSTR) approach: K-step Yard sampling (KY) methods, Journal of Oral Tissue
Engin, 9(3):167-173, 2012.

C-4-03
Quantitative Structure-Pharmacokinetic Relationship(QSPkR) Analyses of Opioids
Yoshihiro Uesawa

uesawa@my-pharm.ac.jp

Miyuki Ishii

Hajime Kagaya
kagayah@my-pharm.ac.jp

Department of Clinical Pharmaceutics, Meiji Pharmaceutical University, 2-522-1
Noshio, Kiyose, Tokyo 204-8588, Japan

Keywords: Opioid, QSPkR, QSAR, half-life, mean residence time, elimination rate constant[Introduction] The prediction of the pharmacokinetic properties of opioids for which it isdifficult to gain pharmacokinetic parameters, is useful in a variety of situations. In clinical practice,evaluating the mean residence time (MRT) and terminal half -lives (t 1/2 ) of active metabolites from
opioids is necessary to understand the exact duration of activity. Pharma cokinetic knowledge isalso important in the care of poisoned patients with compounds that are modified opioids
structures such as designer drugs. Therefore, the construction of prediction models for MRT as
well as t 1/2 was attempted with QSPkR analyses.
[Methods] After the construction of a dataset, consisting of opioids with pharmacokinetic
parameters, a number of descriptors including physico-chemical, quantum-chemical and structuralparameters were calculated from each optimized structure. A small number of parame ters with
importance for the prediction of MRT were selected and used for the construction of the
MRT-prediction model by artificial neural network analyses .
[Results and Discussion] A pharmacokinetic dataset with 26 kinds of opioids such as morphine,
phentanyl, and methadone was constructed. Remifentanyl was eliminated because of becoming an
obvious outlier in the construction process of the MRT prediction model. LogP and Bond
Information Content index (BIC) were the important parameters related to MRT. As a result of thefeature selection, artificial neural network model for MRT prediction was constructed with two
structural descriptors with high generalization capability verified by cross -validation and external
validation. This model is applicable for estimating terminal t 1/ 2 because it was able to predict from
MRT with a high accuracy in opioids.

C-4-04

Proposal of Toxicity Risk Index (TRI)
for Warning Idiosyncratic Drug Toxicity
Takashi Mizuma 1, 2

	
  	
  	
  	
  	
 

	
  	
  	
  	
  	
  	
  	
  	
  	
  tmizuma@cc.matsuyama-u.ac.jp
	
  	
  	
  	
  	
  	
  	
  	
 

Mitsuru Hashimoto2

hashimot@cc.matsuyama-u.ac.jp

1

2

	
 

Miyoshi Morimoto 1
	
 

	
 

Kazuo Samizo 1 	
 

	
  	
 

sammy@toyaku.ac.jp	
  	
 Shin Ohta 1

ohtas@toyaku.ac.jp

School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi,
Hachioji, Tokyo 192-0392, Japan
College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama,
Ehime 790-8578, Japan

Keywords: Pharmacokinetics, Toxicokinetics, Black box warning, Drug toxicity, Covalent binding,Idiosyncratic drug toxicity
Drug toxicity impedes not only its clinical use but also drug development. In the present study, a
toxicity risk index (TRI), which is an index for warning idiosyncratic drug toxicity (IDT), wasdevised. The TRI of drugs was defined as a function of dose, pharmacokinetic parameters, andtoxicokinetic data from covalent binding experiment. Twenty drugs classified into three categories
by a report [1], were studied with TRI. The three categories were BBW (drugs with a block box
warning for IDT), WNG (drugs without a black box warning but with a warning for IDT) and
SAFE (drugs without any warning). The TRIs of drugs classified as SAFE were distinctly different
from those classified as BBW. The TRI of SAFE drugs were lower than 0.456 (nmol/mg protein).
In contrast, the TRI of the BBW drugs were higher than 1.10 (nmol/mg protein). These results
warned us that a drug candidate, where the TRI is higher than 1.0 nmol/mg protein, should be
categorized as a BBW drug. Thus, TRI may be useful for decision-making in drug development
and its clinical use, and is proposed for warning IDT. Further study is now going on for obtaining a
cut-off value with a statistical meaning.
[1] Nakayama, S., Atsumi, R., Takakusa, H., Kobayashi, Y., Kurihara, A., Nagai, Y., Nakai, D.,
Okazaki, O., A zone classification system for risk assessment of idiosyncratic drug toxicity
using daily dose and covalent binding, Drug Metabolism and Disposition, 37: 1970-1977, 2009.

C-4-05
Improvements to the Cluster Newton Method
for Underdetermined Inverse Problems- Parameter Identification for PharmacokineticsPhilippe Joseph Gaudreau 2 ,Khin Moh Moh Latt 1 ,khinmohmohlatt@gmail.com

Yasunori Aoki 4 ,yasunori.aoki@farmbio.uu.se

1
2
3
4
5
6

pjgaudre@ualberta.ca

Hassan Safoui 5 ,andhsafouhi@ualberta.ca

Ken Hayami 3 ,hayami@nii.ac.jp

Akihiko Konagaya 6
kona@dis.titech.ac.jp

Asian Institute of Technology, Pathumthani, Thailand
University of Alberta, Alberta, Canada
National Institute of Informatics, Tokyo, Japan
Uppsala University, Uppsala, Sweden
University of Alberta, Alberta, Canada
Tokyo Institute of Technology, Yokohama, Japan

Keywords: Cluster Newton method; Underdetermined inverse problem; Beta distribution;
Pharmacokinetics Electronic structure calculation, Ligand docking, Protein modeling
The Cluster Newton method (CN method) has proved to be very efficient at finding multiple
solutions to underdetermined inverse problems. In the case of pharmacokinetics, underdetermined
inverse problems are often given extra constraints to restrain the variety of solutions. In this paper,
we propose an algorithm based on the two parameters of the Beta distribution to find families ofsolution near a solution of interest. This allows for a much greater control of the variety ofsolutions that can be obtained with the CN method. In addition, this algorithm facilitates the taskof obtaining pharmacologically feasible parameters. Moreover, we also make some improvements
to the original CN method including an adaptive margin of error for the perturbation of the target
values and the use of an analytical Jacobian in the resolution of the forward problem.
[1] Aoki, Y., Hayami, K., De Sterck, H., and Konagaya, A., Cluster Newton method for sampling
multiple solutions of an underdetermined inverse problem: Parameter identification forpharmacokinetics, SIAM Journal on Scientific Computing (accepted for publication) (Preliminary
version available as NII Technical Report, NII-2011-002E, National Institute of Informatics, Tokyo,
2011, at http://www.nii.ac.jp/TechReports//11-002E.html.)[2] Gaudreau, P., Hayami, K., Aoki, Y., Safoui, H., and Konagaya, A., Improvements to thecluster Newton method for an underdetermined inverse problem,(submitted, also to appear as a
NII Technical Report ).

C-5-01
New Tools for HT Engineering, Cloning & Expression ofMultispecific Next-generation Antibodies
Futoshi Hashino 1

Maria Wendt 2

futoshi.hashino@genedata.com

Maria.Wendt@genedata.com

Peter Ernst 2
peter.ernst@genedata.com

Christopher Smith 3
christopher.smith@genedata.com

Sebastian Schlicker 3

Hans Peter Fischer 2

Sebastian.Schlicker@genedata.com

Hans-Peter.Fischer@genedata.com

1
2
3

Genedata K.K., Higashi-Gotanda 1-7-11-404, Shinagawa-ku, Tokyo 141-0022, Japan
Genedata AG, Margarethenstrasse 38, 4053 Basel, Switzerland
Genedata Inc., 750 Marrett Road, One Cranberry Hill, Suite 403, Lexington, MA 02421

Keywords: antibody screening, research workflow optimization, data management, automa tion,
hit-selection, high-throughput, in-silico engineering/reformatting, humanization, database
Next-generation antibodies promise greater clinical efficacy and safety for a variety of severe humandiseases. As a consequence, today’s biotechs are faced with generating and testing vastly increased
numbers of diverse alternative antibody molecule formats. A significant bottleneck is the establishmentof high-throughput molecular biology and cloning processes required for systematically generating andvalidating DNA constructs to express the desired molecules. Here, we present a new workflow system
for addressing the specific challenges for these antibody molecules produced in large -scale engineering
campaigns. The diversity of new molecule formats (e.g. bi - and n-specifics: KinH BsIgG, Fab-scFv,
IgG-scFv, Dual V domain IgG, etc.) and relevant cloning strategies pose workflow challenges that
require tailored in silico cloning and automation tools for molecule design, DNA synthesis andverification; supporting expression and purification sample management; as well as an integrated
processing of assays and analytics testing results. We present concrete examples of next -generation
antibody workflows using the Genedata Biologics™ platform, including the high -throughput
engineering of bispecifics.
Genedata Biologics™ is the first fully integrated workflow platform for comprehensively supporting
the biologics R&D process, including that of next -generation antibodies. The focus of the platform lies
on supporting complex laboratory workflows around mol ecule generation, with its built-in
biologics-specific business logic, integrated data management and automation.
[END]

C-5-02

Installation of orthogonality to the interface that assembles
two modular domains in the Tetrahymena group I ribozyme
Yoshiya Ikawa 1,2,3
yikawa@cstf.kyushu-u.ac.jp

	
  	
 

Hiroyuki Furuta 1,2
hfuruta@cstf.kyushu-u.ac.jp

Takahiro Tanaka 1t.tanaka.075@s.kyushu-u.ac.jp

1

2

3

Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu
University, Moto-oka 744, Nishi-ku, Fukuoka 819-0395, Japan
International Research Center for Molecular Systems, Kyushu University, Moto-oka 744,
Nishi-ku, Fukuoka 819-0395, Japan
Synthetic Systems Biology Research Center, Kyushu University, Maidashi 3-1-1,
Higashi-ku, Fukuoka 812-8582, Japan

Keywords: Ribozyme, GNRA Tetraloop, Tetraloop Receptor, Kissing Loop
Two modular elements (P5abc and ΔP5) in the Tetrahymena group I ribozyme can be separated
physically to generate a two-piece ribozyme derivative consisting of a separately prepared P5abc(P5 RNA) and the rest of the intron (ΔP5 RNA) [1]. Molecular recognition in the interface
assembling P5 RNA and ΔP5 RNA is strong and specific, and the catalytic ability of the two-pieceribozyme is comparable to that of the parent unimolecular ribozyme [2].
In this study, we designed alternative tertiary interactions participating in the assembly of P5 andΔP5. Using alternative interactions, orthogonality was successfully introduced into the P5/ΔP5assembly interface. The artificial P5/ΔP5 assembly interface would expand the utility of thebimolecular complex as a structural platform for RNA nanotechnology and RNA synthetic biology[3].
[1] van der Horst, G., Christian, A., and Inoue, T., Reconstitution of a group I intron self-splicing
reaction with an activator RNA, Proc. Natl. Acad. Sci. USA., 88:184-188, 1991.
[2] Doherty, E.A., Herschlag, D., and Doudna, J.A., Assembly of an exceptionally stable RNA
tertiary interface in a group I ribozyme, Biochemistry, 38:2982-2990, 1999.
[3] Tanaka, T., Furuta, H., and Ikawa, Y., A two-piece derivative of a group I intron RNA as aplatform for designing self-assembling RNA templates to promote peptide ligation, J. Nucleic
Acids, 2012, art. ID 305867, 2012.

C-5-03
Robust in vitro affinity maturation strategy based oninterface-focused high-throughput mutational scanning
Yasuhiro Fujino 1
Fujino.Yasuhiro@mb.mt-pharma.co.jp
Risako Fujita 2
Fujita.Risako@mv.mt-pharma.co.jp
Kouichi Wada 3
Wada.Kouichi@me.mt-pharma.co.jp
Kotomi Fujishige 4
Fujishige.Kotomi@mb.mt-pharma.co.jp
Takashi Kanamori 5
kanamori@k.u-tokyo.ac.jp
Lindsey Hunt 6
lhunt@sapidyne.com
Yoshihiro Shimizu 7
yshimizu@riken.jp
Takuya Ueda 8
ueda@k.u-tokyo.ac.jp
Advanced Medical Research Department, Mitsubishi Tanabe Pharma Corporation,
1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033 Japan

1-4

6 Sapidyne Instruments, 700 W. Diamond St. Boise, ID 83705 USA
7 Laboratory for Cell-Free Protein Synthesis, Quantitative Biology Center, RIKEN,

2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan5, 8

The Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The
University of Tokyo, FSB-401, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan

Keywords: Protein engineering; Affinity maturation; Mutation scanning; High-throughput
sequencing; Ribosome display
Abstract
Development of protein therapeutics or biosensors often requires in vitro affinity maturation.
Here we report a robust affinity engineering strategy using a custom designed library. The
strategy consists of two steps beginning with identification of beneficial single amino acidsubstitutions then combination. A high quality combinatorial library specifically customized to agiven binding-interface can be rapidly designed by high-throughput mutational scanning of
single substitution scanning libraries. When applied to the optimization of a model antibody Fabfragment, the strategy created a diverse panel of high affinity variants. The most potent variant
achieved a 2110-fold affinity improvement to an equilibrium dissociation constant (Kd) of 3.45 pM
with only 7 amino acid substitutions. The method should facilitate affinity engineering of a wide
variety of protein-protein interactions due to its context-dependent library design strategy.
[1] Fujino,Y., Fujita, R., Wada, K., Fujishige, K., Kanamori, T., Hunt, L., Yoshihiro, S., Takuya, U.,
Robust in vitro affinity maturation strategy based on interface-focused high-throughput
mutational scanning. Biochem. Biophys. Res. Commun. 428:395-400, 2012

C-5-04
In silico analysis of interactions between the ingredient molecules
contained in cold medicines and HLA-A*02:06
Hideto Isogai 1
hi_r_2007@yahoo.co.jp

Chie Sotozono 3
sotozon@koto.kpu-m.ac.jp

Hiroko Miyadera 2

Mayumi Ueta 3

miyadera@m.u-tokyo.ac.jp

mueta@koto.kpu-m.ac.jp

Shigeru Kinoshita 3

Katsushi Tokunaga 2

shigeruk@koto.kpu-m.ac.jp

tokunaga@m.u-tokyo.ac.jp

Noriaki Hirayama 1
hirayama@is.icc.u-tokai.ac.jp
12

3

Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 147
Shimokasuya, Isehara, Kanagawa 259-1143, Japan
Department of Human Genetics, School of International Health, Graduate School of Medicine,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Department of Ophthalmology, Kyoto Prefectural University of Medicine,Hirokoji
Kawaramachi, Kmigyo-ku, Kyoto 602-0841, Japan

Keywords: adverse drug reactions, Stevens-Johnson syndrome, cold medicine, docking
simulations, HLA-A*02:06
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe drug
hypersensitivities with high mortality. Typical over-the-counter drugs of cold medicines aresuggested to be causative. As multiple ingredients are generally contained in cold medicines, it isof particular interest to investigate which ingredients are responsible for SJS/TEN. However,experimental examination of causal relationships between SJS/TEN and a particular drug molecule
is not straightforward. Significant association between HLA-A*02:06 and SJS/TEN with severe
ocular surface complications has been observed in Japanese. We have undertaken in silico docking
simulations between the HLA-A*02:06 molecule and various ingredients contained in cold
medicines available in Japan. We use the composite risk index (CRI) that is the absolute value ofthe binding affinity multiplied by the daily dose to assess the potential risk of the adverse reactions.
The drugs which have been recognized as causative drugs of SJS/TEN in Japan have revealed
relatively high CRI and the association between SJS/TEN and HLA-A*02:06 has been
qualitatively verified. The results have also shown that some drugs whose links to SJS/TEN havenot been clinically recognized in Japan show the high CRI and suggested that attention should be
paid to their adverse drug reactions.

C-5-05

Analysis of the Drug Prescribing Patterns of Physiciansin Patients with both Cardiac and Renal Failure
Masanori Shiro 1

Shotaro Akaho 1

morita.mizuki@aist.go.jp

Mizuki Morita 1

shiro@ni.aist.go.jp

s.akaho@aist.go.jp

Hideki Asoh 1

Toshihiro Kamishima 1

Eiji Aramaki 2

h.asoh@aist.go.jp

mail@kamishima.net

eiji.aramaki@gmail.com

Koiti Hasida 3
hasida.koiti@i.u-tokyo.ac.jp

1

2
3
4
5

Takahide Kohro 4,5kohro-tk@rc5.so-net.ne.jp

National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono,
Tsukuba, Ibaraki 305-8568, Japan
Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, JapanThe University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, JapanThe University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Keywords: Clinical data analysis, Physician prescribing patterns, Markov decision process
Cardiac and renal functions are physiologically related through interactions between systemic
circulation regulation by the heart and extracellular fluid volume control by the kidney. Due to theincrease in the number of patients with hypertension and/or diabetes, the incidence of coexistentcardiac failure and renal failure is increasing. However, evidence for the establishment ofguidelines for treating patients with coexistent cardiac and renal failure is currently insufficientbecause patients with such complications are generally excluded from clinical trials. As a result,
such patients are generally treated on an empirical basis.
In the present study, we analyzed clinical data to clarify the current medical treatment process in
hospital. We evaluated the medical records of patients with cardiac failure from the database of
The University of Tokyo Hospital. We modeled a series of treatments by physicians using theMarkov decision process (MDP), wherein the laboratory data of the patient were employed as thestate and the following drug prescription by the physician was employed as the action.
We identified the prescribing patterns of physicians according to the cardiac and renal state of thepatients. Moreover, we also determined the overall trend of the subsequent state as a result of eachprescription. Although most drugs were used under the recommended conditions according to the
package inserts, these guidelines were not followed for certain other drugs. Only the present andsubsequent states were considered in this study, but we are planning to perform further studies to
identify more specific prescribing patterns according to the series of past states and changes in thetrend of states.
[1] Bongartz, L.G., Cramer, M. J., Doevendans, P. A., Joles, J. A. and Braam, B., The severe
cardiorenal syndrome: ‘Guyton revisited’, Eur Heart J, 26:11-17, 2005.
[2] Shlipak, M. G., Pharmacotherapy for heart failure in patients with renal insufficiency, Ann
Intern Med, 138:917-924, 2003.
[3] Asoh, H., et al., Modeling medical records of diabetes using Markov decision processes,
Proceedings of the 30th International Conference on Machine Learning, ICML, 28, 2013.
[4] Bellman, R., A markovian decision process, Journal of Mathematics and Mechanics, 6:679-684,
1957.

C-5-06
Biophysical Approach of Fragment Screening to inhibit theheme transfer system of Pathogenic bacteria
Satoru Nagatoishi 1

Takuya Shimomura 2

nagatoishi@bioeng.t.u-tokyo.ac.jp

Ryota Abe 2

Jose Manuel Martinez Caaveiro 1
jose@bioeng.t.u-tokyo.ac.jp

Kouhei Tsumoto 1,2,3
tsumoto@bioeng.t.u-tokyo.ac.jp
12

3

Department of Bioengineering, Graduate School of Engineering, The University of Tokyo,
4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The
University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Keywords: Pathogenic bacteria, Heme transfer system, PPI, FBDD
Acquisition of iron from host sources is of vital importance to many pathogenic bacteria during
the course of infection. The process of obtaining heme from the host by Gram-positive pathogens
often involves binding of heme or hemoproteins by bacterial receptor proteins which then deliver
the heme to a membrane transporter for translocation to the cytoplasm [1]. Interestingly, several
pathogenic species of Gram-positive bacteria display surface proteins implicated in heme capturethat contain more than one NEAT domain [2]. The heme capture protien has NTD, NEAT1, andNEAT2 domains. NTD has a hemoglobin-binding ability and both NEAT1 and NEAT2 has aheme-binding ability. However, their detail analysis and interaction among them is not done. We
have studied the interaction with hemoglobin and its heme transfer system of Shr, and revealed that
NTD can be a candidate of the target protein to inhibit the heme transfer.
Here we carried out the fragment-based drug screening to inhibit the interaction between
hemoglobin and NTD. First hit screening of about 2000 compounds fragment library wereconducted in surface plasmone resonance (SPR). After the 124 hit candidates were obtained in SPR
screening, the competition assay of the candidates were carried out in SPR and we obtained 21
compounds that inhibit the interaction between hemoglobin and NTD. To validate the quality ofthe selected compounds in SPR, dose-dependent binding assay to NTD in SPR and thermal
inhibition assay in isothermal titration calorimetry (ITC) were conducted [3]. In these hit
validation, we obtained consequently some compounds that can inhibit specifically the PPI bybinding to NTD.
[1] Nobles, C.L. and Maresso, A.W., The theft of host heme by Gram-positive pathogenic bacteria,
Metallomics, 3:788-796, 2011.
[2] Andrade, M.A., Ciccarelli, F.D., Perez-Iratxeta, C. and Bork, P., NEAT: a domain duplicated ingenes near the components of a putative Fe3+ siderophore transporter from Gram-positive
pathogenic bacteria, Genome Biology, 3:research0047, 2002.
[3] Kobe, A., Caaveiro, J.M., Tashiro, S., Kajihara, D., Kikkawa, M., Mitani, T. and Tsumoto, K.,
Incorporation of rapid thermodynamic data in fragment-based drug discovery, Journal of
Medicinal Chemistry, 56:2155-2159, 2013.

C-5-07
Inhibitor Screening of Capsular Polysaccharide
Synthesizing Enzyme CapF from Staphylococcus aureus
Koichiro Nakano 1
kk126434@mgs.k.u-tokyo.ac.jp

Takamitsu Miyafusa 1
t-miyafusa@aist.go.jp

Satoru Nagatoishi 2

Jose Manuel Martinez Caaveiro 2

nagatoishi@bioeng.t.u-tokyo.ac.jp

jose@bioeng.t.u-tokyo.ac.jp

Kouhei Tsumoto 1,2,3

tsumoto@bioeng.t.u-tokyo.ac.jp

12
3

Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The
University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Department of Bioengineering, Graduate School of Engineering , The University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo ,4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Keywords: FBDD, Enzyme inhibition, Staphylococcus aureus, SPR, ITC
The enzyme CapF is essential for synthesizing capsular polysaccharide of Staphylococcus aureus,
and reduces its substrate by oxidizing NADPH [1,2]. Interestingly, the affinity of CapF for
NADPH is approximately 100-hold higher than that for NADP + . In addition, it has been reported
that NAPDH binding to Ps3αHSD [3] or SDRvv [4] form helical structures, that have a similarfunction of the enzyme activity in CapF. Therfore, we had a hypothesis that the recognition abilityof CapF is caused by its loop structure of CapF near the NADPH-binding site of one. Here, we
performed a small-compoud screening to search a inhibitor for synthesizing capsularpolysaccharide by regulating the significant recognition ability of CapF based on the hypothesis.
To find the specific binder to CapF, a hit screening based on a Fragment library (FBDD) is carriedout using surface plasmon resonance and enzyme activity assay. We obtained some compounds ineach screening assay. These compounds were characterized by the some assays such as
does-dependent binding assays and calorimetric analyses.
[1] Miyafusa, T., Caaveiro, J.M., Tanaka, Y. and Tsumoto, K., Crystal structure of the enzyme
CapF of Staphylococcus aureus reveals a unique architecture composed of two functional domains,
Biochemical Journal, 443:671-680, 2012.
[2] Miyafusa, T., Caaveiro, J.M., Tanaka, Y., Tanner, M.E. and Tsumoto, K., Crystal structure of
the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus, Bioscience
Reports, 33:463-474, 2013.
[3] Buysschaert, G., Verstraete, K., Savvides, S.N. and Vergauwen, B., Structural and biochemical
characterization of an atypical short-chain dehydrogenase/reductase reveals an unusual cofactor
preference, The FEBS Journal, 280:1358-1370, 2013.
[4] Nakamura, S., Oda, M., Kataoka, S., Ueda, S., Uchiyama, S., Yoshida, T., Kobayashi, Y. andOhkubo, T., Apo- and holo-structures of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas
sp. B-0831. Loop-helix transition induced by coenzyme binding. The Journal of Medicinal
Chemistry, 281:31876-31884, 2006.

C-5-08
Functional analysis of Liposome binding peptide selectedby cDNA display
Yuki Yoshikawa

Shota Kobayashi Miho Suzuki

Koichi Nishigaki

Naoto Nemoto
nemoto@fms.saitama-u.ac.jp

Graduate School of Science and Engineering, Saitama University
255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan

Keywords: mRNA/cDNA display, Liposome membrane-binding peptide, In vitro selection
cDNA display is a genotype-phenotype linking method that a peptide is conjugated with its
encoded cDNA via a puromycin-linker using a cell-free translation system [1]. cDNA display
enables to perform in vitro selection from a library of 10 12 peptide molecules. We previously
obtained some kinds of high-affinity peptide aptamers against interleukin-6 receptor by thismethod [2]. In this study, we tried in vitro selection to obtain peptides that bind with liposome
which is artificial lipid bilayer from random peptide (30 residues in length) library. After six
rounds of selection, the random sequences have been converged a unique consensus amino acids
sequence. Then, we designed and synthesized a peptide modified a fluorescein at its N-terminus bychemical-synthesis and examined whether the peptide can interact with liposome membrane with aconfocal laser scanning microscopy. Interestingly, the interaction between the peptide andliposome might depend on the method of preparing liposome. In this presentation, we will show
the results and discuss about the interaction between the peptide and liposome in detail.
Fig.1 Interaction between peptides and liposome.
(a)Giant unilamellar liposomes were preparedwith dioleoylphosphatidylcholine (DOPC)
and incubated peptides (6µM) with for 2h
at 25℃.
(a)Differential interference contrast (DIC)
image of liposomes.
(b) Fluorescence microscopy image ofliposomes (with fluorescent dye labeled
peptides). The image was taken from a
confocal laser scanning microscopy.

(b)[1] Mochizuki, Y., et al., ACS Comb. Sci., 13, 478-485 (2011)[2] Nemoto, N., et al., Biochem Biophys Res Commun., 421, 129-133 (2012)

C-5-09

The CADU Alliance N3 Project1

2

Tsuguchika Kaminuma 1

Masaki Morioka 2kaminuma@join-ica.org

msmorioka-tky@umin.ac.jp

Kaoru Mogushi 2
mogushi@bioinfo.tmd.ac.jp

Masumi Yukawa 1

Naomi Komiyama 1

Hiroshi Tanaka 2yukawa@join-ica.org

komiyama@join-ica.org

tanaka@bioinfo.tmd.ac.jp

Institute for Cyber Associates, 1-11-29 Kugenuma-Fujigaya, Fujisawa-city, Kanagawa
251-0031, Japan
Department of Bioinformatics, Division of Medical Genomics, Medical Research Institute,Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan

Keywords: CBI, Quantum Computing, Open Courseware, Education of Genomics
The history of the Chem-Bio Informatics Society goes back to 1981. The term Chem-Bio
Informatics (CBI) had been coined to cover various interdisciplinary areas for studying the effects
of chemicals on biosystems at the molecular level. Since then, drug discovery has been the most
attractive research area of the CBI, and over the years, it has been a force for the advancement andsophistication of computational methods, programming systems, databases, knowledge-bases, andarts of applying these methodologies and computational resources. Yet now it seems that thisdiscipline has lost its vital energy to offer good new jobs, to attract investors, and to draw theattention of the general public.
We propose new frontiers, which should need new professionals as a workforce, and new
partnerships for cultivating these frontiers in order to revitalize this discipline. First, we proposethat since CBI consists of such interdisciplinary areas as natural sciences, informatics, andcomputing, it is natural to include physics here. Although information and computing had not been
subjects of physics for a long time, the emerging new fields of quantum information and quantum
computing have drastically changed this situation. It is time to consider, for example,
computational chemistry simulation by quantum computers. Second, we propose to include
applications of smart phones or tablets, and cloud services to CBI. Some examples were welldiscussed in several papers. Third, we propose to pay special attention to educating many
professionals working in health and medical fields.
The last proposal must be considered most seriously, since such educational needs are urgent andtremendously important. It is urgent because very rapid advances of sequencing technology are
drastically reducing the costs and time required for genetic and genomic testing so that such tests,
which require some genomic competency, may be accepted in any service sectors in health andmedical cares. This means that, not only basic researchers in clinical medicine, especially those
who are dealing with cancer, but also many clinical professionals such as primary care physicians,
nurses, pharmacists, nutritionists, and others must improve their competencies and literacy ofgenomics. There will be a need to recruit many bioinformaticians to take part in this task.
Moreover such a career extension might open new chances for them. Open courseware based on
the Internet would be a very strong tool for this purpose.
Right now we are proposing to implement some of these proposals in the context of activities of
the CADU Alliance. We hope such efforts might contribute to revitalize this discipline.

C-5-10Light-induced membrane potential regulates bone cellfunction; development of the light-responsive bone cells
Takuya Notomi 1
notomph@tmd.ac.jp

1

Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo, 113-8510, Tokyo, Japan

Keywords: ion channel, osteoblast, calcium
The PTH and VD 3 are not only osteogenic reagents but also major regulator for
osteoclastogenesis. These molecules transiently increase bone resorption by increment of theexpression level of RANKL, whereas their mechanism of RANKL -intracellular transportation(RANKL-iTP) remains unclear. The RANKL-iTP depends on lysosomal vesicles whose fusion isrelated to increment of intracellular Ca 2+ concentration ([Ca 2+ ] i ) following depolarizing membranepotential (MP). Thus, MP could regulate osteoblastic function a nd RANKL-iTP. So far little is
known about the role of MP on bone cells, because it is hard to control temporal patterns of MP.
Here, by novel system of light-responsive osteoblastic MP, we show that the transiently increasing[Ca 2+ ] i following depolarization regulates PTH- and VD 3 - induced RANKL-iTP.
In membrane fraction of RANKL-GFP expressed MC3T3-E1 cell, PTH and VD 3 increase
membrane-bound RANKL (mbRANKL) at 10 min (more than 300% increment compared tocontrol), however those mRNA expression level were unchanged at same time. Confirming bytime-lapse imaging, the addition of PTH and VD 3 translocated RANKL-GFP to cell membranewithin 10 min. In previous reports, PTH and VD 3 transiently increase [Ca 2+ ] i following
depolarization. The [Ca 2+ ] i spike was diminished by depolarization-activated calcium channel
blocker (Diltiazem: Dil) or ER-dependent [Ca 2+ ] i blocker (Thapsigargin: Tg). The increment of
mbRANKL by PTH and VD 3 was also decreased by Dil and Tg. These results showed that
depolarization-dependent change of [Ca 2+ ] i affected PTH- and VD 3 -induced RANKL-TPi.
To address whether depolarization controls RANKL -TPi, we developed a novel system oflight-responsive osteoblastic MP by channel-rhodopsin wide receiver (ChWR). MPs in ChWR
stably expressed cell were depolarized immediately (<20 msec) after light -stimulus (ΔMP: 16.3 ±1.1 mV) and were returned to the pre-stimulus potential after the off-stimulus. Light induced
depolarization increase [Ca 2+ ] i and mbRANKL at 10 min in similar to addition of PTH and VD 3 . In
co-culture of RAW267 and RANKL-GFP expressed MC3T3-E1 cell, TRAP activities wereincreased 4- fold by light stimulus and the development of mature osteoclasts was observed.
We showed that depolarizing MP is related to osteoblastic function. Our results indicates thatRANKL-iTP is regulated by [Ca 2+ ] i following depolarization of MP. The mechanism of PTH - andVD 3 -induced RANKL-iTP would have benefits for developing PTH- and VD 3 -like medicine.

C-5-11E. coli Host Engineering for Efficient Enzyme Discovery
Miyuki Tsukuda 1
miyuki-tsukuda@aist.go.jp

1

2

	
  	
 

Kentaro Miyazaki 2
miyazaki-kentaro@aist.go.jp

Bioproduction Research Institute, National Institute of Advanced Industrial Science andTechnology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517,
Japan
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The
University of Tokyo, 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517,
Japan

Keywords: E. coli, Ribosome, Functional Metagenomics
The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial rolein translating mRNA-encoded information into proteins. Because of the ribosome’s structural andmechanistic complexity, it is believed that each ribosomal component coevolves to maintain its
function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they
form the structural core of the ribosome. However, using E. coli Δ7 (null mutant of operons) as ahost, we have recently shown that an active hybrid ribosome whose 16S rRNA has been
specifically substituted with that from non–E. coli bacteria can be reconstituted in vivo.
To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality,
we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E.
coli Δ7. Various functional 16S rRNA genes were obtained from the Gammaproteobacteria andBetaproteobacteria lineages (with minimal identity of ~80%) [1].
Based on this discovery, we investigated how ribosomal mutation affects gene expression. To thisend, we used a series of reporter genes that are poorly expressed in native E. coli host. They werethen introduced into mutant E. coli library that carry metagenomically retrieved 16S rRNA genes,
and the expression levels were evaluated. As a result, some mutants expressed gene more
efficiently than the native E. coli host. Thus ribosome engineering may be a feasible approach tosolving the expression problem in metagenomic library screening, and should accelerate the
enzyme discovery process [2,3].
[1] Kitahara, K., Yasutake, Y., and Miyazaki, K. Mutational robustness of 16S ribosomal RNA,
shown by experimental horizontal gene transfer in Escherichia coli. Proceedings of the National
Academy of Sciences USA 109:19220-19225, 2012[2] Hosokawa-Okamoto, R., and Miyazaki, K. Escherichia coli host engineering for efficient
metagenomic enzyme discovery. in "Metagenomics: Current Innovations and Future Trends" (ed.
Diana Marco, pp. 241-252, Horizon Scientific Press), 2011
[3] Uchiyama, T., and Miyazaki, K. Functional metagenomics for enzyme discovery: challenges toefficient screening. Current Opinion in Biotechnology 20:616-622, 2009

C-5-12

Methylome diversification through changes in the sequencespecificity of DNA methyltransferases
Yoshikazu Furuta 1,2 , Hiroe Namba 1 , Tomoko F. Shibata 3 , Tomoaki Nishiyama 4 , Shuji Shigenobu 3,5 ,Yutaka Suzuki 1 , Sumio Sugano 1 , Mitsuyasu Hasebe 3,5 , Ichizo Kobayashi 1,2

1

Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo,

Minato-ku, Tokyo, Japan
2

Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan

3

National Institute for Basic Biology, Okazaki, Japan

4

Advanced Science Research Center, Kanazawa University, Kanazawa, Japan

5

Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies,

Okazaki, Japan

Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes
with each strain carrying a unique repertoire. Previous genome comparison works suggested that thesemethyltransferases often change DNA sequence specificity through movement of amino-acid sequencesin the target recognition domains between genes and within a gene (Domain Movement). By
Single-Molecule Real-Time sequencing technology, we detected methylated DNA sites throughout
several closely related genomes. Number of methylated sites varied between strains and had correlation
with number of methyltransferase genes in each strain. We successfully deduced DNA sequence motifs
for methylation and assigned each of them to a specific amino-acid sequence group of target
recognition domains in the specificity determinant genes. Overall, the methylome turned out to be quite
variable among the closely-related strains, although there are hypermethylated loci conserved in all thestrains. Hypomethylated sites were also found on genomic island regions. As expected from theireffects on gene expression, knockout of a specificity gene led to changes in the transcriptome. These
results provide evidence for proposed mechanisms of sequence-specificity change in the DNA
methyltransferases and lend support to the concept of epigenetics-driven adaptive evolution.

C-5-13Site-specific Cellular Modification by Cre-incorporating
Integrase-defective Retroviral Vectors
Takuya Shimomura 1

Shuohao Huang 2

Yoshinori Kawabe 1

2TE12131R@s.kyushu-u.ac.jp

shhuang001@hotmail.com

kawabe@chem-eng.kyushu-u.ac.jp

1

2

Akira Ito 1

Masamichi Kamihira 1,2

akira@chem-eng.kyushu-u.ac.jp

kamihira@chem-eng.kyushu-u.ac.jp

Department of Chemical Engineering, Faculty of Engineering, Kyushu University,
744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku,
Fukuoka 819-0395, Japan

Keywords: Integrase-defective retroviral vectors, Cre/loxP, Site-specific cellular modification
Retroviral vectors have been employed in various biotechnology fields such as gene therapy [1],generation of transgenic animals [2] and induced pluripotent stem cells (iPSCs) [3]. However, thenearly random integration property may cause problems such as insertional mutagenesis and genesilencing. Therefore, the development of targeted transgene integration system is desired formodification of retroviral vectors. We have achieved retroviral insertion of transgene into a
pre-determined site of chromosome in CHO cells using integrase-defective retroviral vectors(IDRVs) and Cre-recombinase mediated cassette exchange (RMCE) [4]. In this case, founder cells
were transfected with a Cre expression plasmid prior to retroviral transduction. To apply
site-specific gene modification for hard-to-transfected cells, it is desired to encapsulate both geneof interest and Cre protein into the retroviral virion. In this study, we attempted to generate novel
hybrid IDRVs, which can deliver both viral genome and enzymatically active Cre.
First, we designed a fusion protein-expression vector (pGPM-Cre), in which a Cre gene wasinserted into the gag-pol precursor genes (pGPM). Cre-incorporating IDRV particles (Cre-IDRV)
were produced by 293FT cells co-transfected using IDRV producing plasmids (pQMSCV/NE,
pGPM and pVSV-G) and a pGPM-Cre plasmid. Western blot analysis revealed that Cre wasincorporated into retroviral virions. A Cre-IDRV encoding marker genes (ATG-deleted-Neo r/IRES/EGFP) flanked by wild-type and mutant loxP sites was infected into founder CHO cells(CHO/F17), in which a single copy of an expression unit containing a CMV promoter, an ATG
codon and marker genes (ATG-deleted-Hyg r /IRES/DsRed) flanked by corresponding loxP sites wasintroduced into the genome. We observed appearance of G418-resistant colonies expressing GFP.
Moreover, site-specific integration of transgene was confirmed by PCR and sequence analyses.
These results indicate that bioactive Cre protein was incorporated into virions and site-specificrecombination between retroviral DNA derived from IDRV and cellular genome was mediated by
Cre-RMCE. The Cre-IDRV system may expand the applicability of retroviral vectors.
[1] Qaism et al., Progress and prospects: Gene therapy for inherited immunodeficiencies. Gene
Therapy, 16:1285-1291, 2009.
[2] Kamihira M et al., Production of chimeric monoclonal antibodies by genetically manipulated
chickens. Journal of Biotechnology, 141:18-25, 2009.
[3] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors. Cell, 126:663-676, 2006.
[4] Huang S et al., Cre recombinase-mediated site-specific modification of a cellular genome using
an integrase-defective retroviral vector. Biotechnology and Bioengineering,107:717-729, 2010.

C-5-14
Construction of artificial sensor to regulate gene
expression in the cyanobacterium Synechocystis sp. PCC
6803
Misaki Kawaguchi 1
s1330329@u.tsukuba.ac.jp

Yoshihiro Shiraiwa 2shiraiwa.yoshihir.ft@u.tsukuba.ac.jp

Iwane Suzuki 2
iwanes6803@biol.tsukuba.ac.jp

12

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8572, Japan
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305 -8572,
Japan

Keywords: Chimeric sensor; Environmental response; Ethylene; Histidine kinase
The cyanobacterium Synechocystis sp. PCC6803 is a model photosynthetic prokaryote, and isincreasingly being recognized as a promising resource in the production of useful substances suchas bioactive compounds [1] and biofuels [2], by genetic manipulations. Thus, it is inevitable that
researchers will develop artificial systems to regulate gene expression.
Two-component signal transduction system is a major gene expression system in Synechocystis.
One of the components of this system, a histidine kinase (Hik), includes two portions, asignal-input domain and a kinase domain, and linker domain between them. Substitution of thesignal-input domain of a Hik with that from another Hik may alter the stimulus perceived by theformer Hik to the later Hik [3].
In this study, we substituted the signal-input domain and the linker domain of SphS, aphosphate-deficient sensor, with those from ethylene sensors from Arabidopsis thaliana, i.e. ETR1,
ETR2, ERS1, ERS2, and EIN4, to construct ethylene-responsible sensor. The activity of resulting
five chimeric sensors was evaluated by the activity of alkaline phosphatase (AP), originallyregulated by SphS.
Results showed that the cells carrying ERS1-SphS or ERS2-SphS did not expressed AP activity,
whilst cells carrying ETR1-SphS, ETR2-SphS, or EIN4-SphS expressed AP activity under standard
conditions. However, none of the chimeric constructs r esponded to ethylene treatment.
Then, we deleted 1 to 7 amino acid residues in the linker domain of ETR1-SphS to construct
functional sensor. These linker-deleted mutants showed no responses to ethylene, but periodic
changes in AP activity were shown corresponded to the number of deleted amino acids.
Currently, our experiments are focused on determining the suitable conditions under which thechimeric sensors will respond to ethylene in the cyanobacterial cells.
[1] Guerrero, F., Carbonell, V., Cossu, M., Correddu, D., Jones, PR., Ethylene Synthesis andRegulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803, Plos ONE , 2012[2] Schirmer, A., Rude, MA., Li, X., Popova, E., del Cardayre, SB., Microbial Biosynthesis of
Alkanes, Science, 2010[3] Shimura, Y., Shiraiwa, Y., Suzuki, I., Characterization of the Subdomains in the N-Terminal
Region of Histidine Kinase Hik33 in the Cyanobacterium Synechocystis sp. PCC 6803, Plant
Cell Physiol., 2012

C-5-15
Development of an ON/OFF-transcriptional control systemfor plural genes to express sequentially
Yuri Samata
s13mb107@mail.saitama-u.ac.jp

Kei Asai
asai@mail.saitama-u.ac.jp

ADivision of Life Science, Graduate School of Science and Engineering, Saitama University,
255 Shimo-Ohkubo, Sakura-ku, Saitama, Saitama 338-8570, Japan.

Keywords: Sigma factor, transcription, stress response
The gene expression in either a bacterial cell or a eucaryotic cell is important bio-technology
performed frequently. Generally expression is induced by adding an inducer, which is usually hard
to be inactivated or removed, and depression after induction is difficult.
Sigma factors rule the DNA-binding specificity of RNA polymerase to the promoter, and are aconserved protein family among bacteria. The protein family of sigma factors is further divided
into a subfamily, Extra-Cytoplasmic Function (ECF) sigma factors. The activity of ECF sigma
factors is regulated by binding specifically to cognate anti-sigma factor proteins on a simple
principle by protein-protein interaction. Anti-sigma factor proteins localize to the cytoplasmic
membrane and response to various external and environmental stress.
ON/OFF control of gene expression is thought to be performed reversibly by some environmental
stress (temperature, pH, the oxidation state, etc.). We have tried to use these plural
sigma-anti-sigma control systems in order to control a lot of gene clusters consecutively.
In Bacillus subtilis, a Gram-positive sporulating soil bacterium, seven ECF sigma factors havebeen identified. The SigW protein is a well-studied ECF sigma factor in Bacillus subtilis. It isregulated by anti-sigma factor, RsiW. Their coding genes constitute an operon, whose transcription
is induced in response to various cell-wall antibiotics, alkaline shock, and other stresses affecting
cell-envelope homeostasis. SigM, is strongly involved in cell envelope integrity and regulated with
YhdL and YhdK anti-sigma proteins. Expression and activity of SigM are elevated under acid, heat,salt, superoxide and cell envelope stresses.
We investigated whether the SigW and SigM could be used for ON/OFF control system by using
pH exchange of medium used for B. subtilis cell culturing.

C-5-16
Which has a better chemotaxis controller,
E. coli or Paramecium?
Shun-ichi Azuma 1
sazuma@i.kyoto-u.ac.jp

1

Graduate School of Informatics, Kyoto University
Gokasho, Uji 611-0011, Japan

Keywords: chemotaxis, E. coli, Paramecium, control performance
In this presentation, we briefly review our recent result on the performance of the internal
controllers generating the chemotaxis [1]. We show two performance indeces to capture thechemotaxis. Based on them, the performance is evaluated for two controller models, which are ofmodel organisms for the chemotaxis, Escherichia coli and Paramecia. It is disclosed that thecoli-type controller achieves the chemotaxis fast but roughly, while the paramecium-type
controller achieves it slow but precisely.
[1] S. Azuma, et al., Performance Analysis of Chemotaxis Controllers, 52nd IEEE Conference on
Decision and Control, to appear (2013)

C-5-17t-Riboregulator: Regulation of Nonsense Suppression
by Modulating 3’ Processing of Suppressor tRNA

1

Yasunori Doi 1

Atsushi Ogawa 1

z844022m@mails.cc.ehime-u.ac.jp

a-ogawa@ccr.ehime-u.ac.jp

Pro teo -Scie nce Ce nter, Ehi me U ni ver si t y, 3 Bu n k yo -c ho , Mat su ya ma, E hi me 790-8577, J ap an

Keywords: Riboregulator, Suppressor tRNA, Nonsense suppression, tRNA processing
Recently, we rationally evolved in-vitro-transcribed, unmodified, and non -aminoacylated
amber suppressor tRNAs toward high er suppression efficiency in wheat germ extract(WGE). [1] The artificial evolution was conducted in the following four steps : [1st] anticodon
alteration of natural tRNAs; [2nd] chimerization of efficient suppressors in the first
generation; [3rd] investigation and optimization of the effective parts in the second
generation; [4th] combination of the optimize d parts in the third generation . The suppression
efficiency of the suppressor obtained in the last generation was approximately 60% (85% in thepresence of an eRF1 aptamer), which was 2.4-fold higher than that of the best suppressor in
the first generation.
In this study, we employed this highly evolved suppressor and an amber-mutated gene to evaluatetRNA processing, especially terminal (5’ and 3’) processing, in WGE. Specifically, we preparedvarious kinds of terminal-premature tRNAs based on the evolved suppressor and compared theirsuppression efficiencies to that of the mother tRNA. As a result, 3 ’ processing including CCA
addition was found to rapidly proceed in WGE in contrast to extremely slow 5 ’ processing.
However, 3’ processing was effectively inhibited by distorting the tRNA structure with adistortion-inducing sequence in the 3’ end. We therefore used the structure-distorted suppressor todevelop a novel gene regulation system, named “t-riboregulator“, wherein nonsense suppression(i.e., expression of full-length protein) is controlled by a modulator (nucleic acids) that restores thetRNA structure and the 3’ processing activity of the structure-distorted suppressor. The
t-riboregulator system is expected to be used as an alternative of or in combination with normal
mRNA-based riboregulators or other gene regulators such as riboswitches to construct artificial
gene circuits in synthetic biology.
[1] Ogawa, A., et al., Improvement of in vitro-transcribed amber suppressor tRNAs toward higher
suppression efficiency in wheat germ extract , Organic & Biomolecular Chemistry, 9 : 8495-8503,
2011.

C-5-18
DNA Modification Enzymes Utilizing
Sequence-Specificity of Zinc Finger Domains
Wataru Nomura

Hirokazu Tamamura

nomura.mr@tmd.ac.jp

tamamura.mr@tmd.ac.jp

Institute of Biomaterials and Bioengi neering, Tokyo Medical and Dental University, 2-3-10
Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Keywords: DNA binding, DNA methylation, Gene editing
Artificial zinc finger proteins (ZFPs) consist of Cys 2 -His 2 -type modules composed of about 30
amino acids with a  structure that coordinates a zinc ion. ZFPs that recognize specific DNA
target sequences can substitute for the binding domains of enzymes that act on DNA to create
designer enzymes, such as nucleases, recombinases [1], and methylases [2], with programmablesequence specificity. Genome editing and modification by the enzymes could be applied for many
fields of basic research and medicine. Some zinc f inger nucleases (ZFN) are currently in humanclinical trials that aim at therapeutic gene editing. In this presentation, ZFN pairs targetingpromoter region of human telomerase reverse transcriptase (hTERT) were designed andconstructed. Expressed zinc fingers were purified in vitro and the DNA binding properties were
evaluated. Highly active zinc fingers were utilized for ZFN binding domain. The DNA cleavage of
ZFNs was performed in vitro. Furthermore, the digestion of promoter region in the endogenous
sequences was successfully confirmed. The telomerase activity in the cell treated by ZFN was
reduced suggesting the digestion by ZFN affects to the expression of hTERT. There are few reports
about the strategy with ZFN targeting the promoter region of the target gen e. The present results
give a new insight into the gene targeting and editing by sequence -specific nucleases. In addition,
our current accomplishments on zinc finger enzymes , such as the zinc finger recombinase (ZFR)
design and ZFN targeting replication sites of EB virus, will be presented.
[1] Nomura, W., Masuda, A., Ohba, K., Urabe, A., Ito, N., Ryo, A., Yamamoto, N., and Tamamura,
H., Effects of DNA binding of zinc finger and linkers for domain fusion on catalytic activity ofsequence-specific chimeric recombinases determined by a facile fluorescent system,
Biochemistry, 51:1510-1517, 2012.
[2] Nomura, W., and Barbas, C. F., III, In vivo site-specific DNA methylation with a designed
sequence-enabled DNA methylase, J. Am. Chem. Soc., 129:8676-8677, 2007.

C-5-19

Discovery of a Potent and Selective FLT3 Kinase Inhibitor by
Fragment Evolution
Hirofumi Nakano 1

Nae Saito 1

h-nakano@mol.f.u-tokyo.ac.jp

nae-saito@mol.f.u-tokyo.ac.jp

Hirotatsu Kojima 1

Takayoshi Okabe 1

kojima@mol.f.u-tokyo.ac.jp

tokabe@mol.f.u-tokyo.ac.jp

Tetsuo Nagano 1
tlong@mol.f.u-tokyo.ac.jp
1

Open Innovation Center for Drug Discovery, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan.

Keywords: FLT3, acute myeloid leukemia, kinase inhibitor, fragment evolution
FLT3 (FMS-Like Tyrosine kinase 3), a member of class III receptor tyrosine kinase family, is apromising therapeutic target for acute myeloid leukemia (AML). Activating FLT3 mutations are foundin approximately 30% of AML patients and associated to poor prognosis. To discover a novel FLT3
inhibitor, we performed a screening of our in-house fragment library. The screening hit was rapidly
progressed to a potent and selective FLT3 inhibitor.

C-5-20
An approach to reconstruction of cell cycle oscillation of
DnaA activity for replication initiation and transcription
regulation
Masayuki Suetsugu 1,2,3
suetsugu@rikkyo.ac.jp

	
  	
 

Hiroko Kobayashi 1,2

Tsutomu Katayama 2
1

2

3

Department of Life Science, College of Science, Rikkyo University, 3-34-1
Nishi-Ikebukuro, Toshima-ku, Tokyo, 171-8501, Japan
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
JST, PRESTO

Keywords: Chromosome replication, Cell cycle, Escherichia coli
In Escherichia coli, an initiation stage of chromosome replication is a main target to trigger cell
cycle progression. DnaA initiator protein binds to chromosomal origin, oriC, leading to theinitiation reaction. The DnaA activity is regulated by binding of ATP and ADP, and the cellularratio of the ATP-bound form and ADP-form oscillates during cell cycle. The sliding clamp is acomponent of the replication machinery. Following replication initiation, the DNA-loaded sliding
clamps accumulate behind replication forks [1]. The DNA-loaded clamp promotes hydrolysis of
ATP bound on the active DnaA, yielding the inactive ADP-DnaA. Thus, the initiation reaction isregulated in a feedback manner. After completion of replication, the clamps are released from
DNA and the feedback regulation of DnaA is withdrawn. Before the next round of replication
initiation, the ratio of ATP-DnaA is re-accumulated. Chromosomal loci called DARS are involvedin this re-accumulation by promoting a nucleotide exchange reaction from ADP-DnaA to
ATP-DnaA. DnaA also serves as a regulator for transcription of several genes. In some of thesegenes, the transcription regulation activity of DnaA is known to be controlled by its
ATP/ADP-binding. Thus, the oscillation cycle of DnaA activity seems to act globally for cell cycle
progression. We present our approach to reconstruction of the oscillation cycle of DnaA activity
by reproducing the dynamic behavior of the clamp.
[1] Su’etsugu, M. and J. Errington, J., T he R eplicase S liding C lam p D ynam ically A ccum ulates
behind P rogressing R eplication F orks in B acillus subtilis C ells, Molecular Cell, 41,
720-732, 2011

C-5-21
Photodynamic Effect of Single-walled Carbon Nanotubes andits potential for cancer phototherapy
Tatsuya Murakami 1

	
  	
  	
  	
 

Mami Inada 2

murakami@icems.kyoto-u.ac.jp	  	  	
  inada.mami.26u@st.kyoto-u.ac.jp

Hiroshi Imahori 1, 2
imahori@scl.kyoto-u.ac.jp

12Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan
Department of Molecular Engineering, Graduate School of Engineering,
Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan

Keywords: Carbon nanotube, Photodynamic effect, Reactive oxygen species, Cancer cell killing
Single-walled carbon nanotubes (SWNTs) are known to be classified into two types, metallic
and semiconducting ones (m-SWNTs, s-SWNTs), on the basis of their chiralities. SWNTs reveal
photothermal (PTE) and photodynamic effects (PDE), which result in generation of heat andreactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ) and superoxide anion (O 2 •– ),respectively. While PTE of SWNTs has received much attention for cancer therapy, a recent reportsuggests that PDE of carbon nanotubes can be used for protein inactivation throughphotogeneration of superoxide anion 1 . In this study, we enriched m- and s-SWNTs by a gel
chromatography and evaluated their PTE and PDE in detail. Under near-infrared laser irradiation,s-SWNTs generated both ROS much more efficiently than m-SWNTs, showing that s-SWNTs had
higher PDE. For cell studies, s-SWNTs were successfully stabilized with a natural dispersant,high-density lipoprotein (HDL). HDL-stabilized s-SWNT did not show significant cytotoxicity,and caused photo killing of cancer cells through 1 O 2 generation like other photosensitizers undernear-infrared irradiation. This is the first example of observing cancer cell killing by
photodynamic effect of SWNT.

[1] Joshi, A.; Punyani, S.; Bale, S. S.; Yang, H.; Tasciuc, T. B.; Kane, R. S. Nat. Nanotechnol.
2008, 3, 41.
[2] Murakami, T.; Nakatsuji, H.; Inada, M.; Matoba, Y.; Umeyama, T.; Tsujimoto, M.; Isoda, S.;
Hashida, M.; Imahori, H. J. Am. Chem. Soc. 2012, 134, 17862.

C-5-22
Probabilistic Model Based Error Correction of Various
Mutant Sequences Analyzed by the Single-Molecule
Real-Time Sequencing
Takuyo Aita 1

Norikazu Ichihashi1 1,2

aita-takuyo@bio.eng.osaka-u.ac.jp

ichihashi-norikazu@bio.eng.osaka-u.ac.jp

Tetsuya Yomo 1,2,3
yomo@ist.osaka-u.ac.jp
12

3

Exploratory Research for Advanced Technology, Japan Science and Technology Agency,
Yamadaoka 1-5, Suita, Osaka, Japan
Department of Bioinformatic Engineering, Graduate School of Information Science andTechnology, Osaka University, Yamadaoka 1-5, Suita, Osaka, Japan
Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-5, Suita, Osaka,Japan

Keywords: base call error; next generation sequencing; image restoration; quality score;quasispecies; sequence analysis; SMRT
To analyze the evolutionary dynamics of a mutant population in an evolutionary experiment, itis necessary to sequence a vast number of mutants by high-throughput sequencing technologies.
Particularly, we focus on the single-molecule real-time (SMRT) sequencing technology [1],which enables rapid and parallel analysis of multikilobase sequences. However, the observed
sequences as "circular consensus sequences (CCS)" obtained by the SMRT sequencing include
many random errors of base call. Therefore, if the SMRT sequencing is applied to analysis of aheterogeneous population of various mutant sequences, it is necessary to discriminate between
true bases as point mutations and random errors of base call in the observed sequences, and to
subject the sequences to error-correction processes [2]. To address this issue, we have developed
a novel method of error correction based on the Bayesian theory with the Potts model and amaximum a posteriori probability (MAP) estimation. The available information for error
correction is (1) "quality scores" which are assigned to individual bases in the observed
sequences [3] and (2) a spatial distribution of the observed sequences in sequence space [4]. The
computer experiments of error correction of artificially generated sequences supported theeffectiveness of our method, showing that 50-90 % of errors were removed. Interestingly, thismethod is analogous to a probabilistic model based method of image restoration developed in
the field of information engineering [5].
[1] Eid J et al., Real-time DNA sequencing from single polymerase molecules. Science,323,
133-138, 2009.
[2] Zagordi O, Geyrhofer L, Roth V, Beerenwinkel N., Deep sequencing of a geneticallyheterogeneous sample: local haplotype reconstruction and read error correction. J Comput
Biol.,17,417-428, 2010.
[3] Cock P.J., Fields C.J., Goto N, Heuer M.L. and Rice P.M., The Sanger FASTQ file format for
sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Research,
38, 1767–1771, 2010.
[4] Eigen M, Winkler-Oswatitsch R., Statistical geometry on sequence space. Methods Enzymol.,
183, 505-530, 1990.
[5] Tanaka K. and Morita T., Cluster variation method and image restoration problem. Physics
Letters A, 203, 122-128, 1995.

C-5-23
Meta-synthetic metabolism:
Identification of critical intermediates for synthetic aminoacid derivative production
Robert Sidney Cox III 1

Masahiko Nakatsui 1

Hiroki Makiguchi 2

nakatsui@port.kobe-u.ac.jp

makiguchi-hiroki@mki.co.jp

Teppei Ogawa 2

Akihiko Kondo 1

Michihiro Araki 1

ogawa-teppei@mki.co.jp

akondo@kobe-u.ac.jp

araki@port.kobe-u.ac.jp

cox@port.kobe-u.ac.jp

1

2

	
  	
 

Organization of Science and Technology, Kobe University, 1-1 Rokkodai Nada,
Kobe 657-8501, Japan
Mitsui Knowledge Industry Co., Osaka Mitsui-Bussan Bldg. 6F, 2-3-33
Nakanoshima, Kita-ku, Osaka 530-0005, Japan

Keywords: metabolic engineering, synthetic biology, database, chemoinformatics
The growing catalogue of known enzymes gives promise for creating new useful biologicalproducts by combining transgenes in vivo and engineering enzymes with novel catalytic
capabilities [1, 2]. Amino acid production is part of the core metabolism of every free-livingorganism, but derivatives of the standard 20 amino acids are often produced as part of the‘secondary metabolism.’ For example, many neurotransmitters are derivatives of standard aminoacids, and therefore amino acid derivatives are rich targets for drug discovery. Toward engineeringcell factories for the production of useful amino acid derivatives, we have explored the potential
catalytic space of known enzymatic functions.
We began with the rich set of more than 11,000 amino acid-like compounds in the PubChem
database [3], and using these as target molecules, calculated acceptable metabolic pathways fortheir synthesis from glucose. For this we used the annotated enzymes from the KEGG database [4],and identified reaction steps between compounds that are currently present in nature, along withenzymatic steps which might be easily engineered due to high chemical similarity between known
and target compounds. We scored each reaction step by chemical similarity (with 1 being a known
reaction in KEGG), and used these to rank the pathways for producing each of more than 3,000
compounds with identified synthetic pathways.
From the calculated synthetic pathways, we found several compounds which acted as ‘criticalintermediates’ which participated in more than 15 reaction steps. These compounds act asintermediates in the synthesis of multiple identified amino acid derivatives. We also found manyamino acid derivatives which could be synthesized by only a single reaction pathway, relying on aunique enzyme from KEGG for the critical reaction step. Along with these results, we present
some of the interesting amino acids identified from the study, along with possible applications inindustry.
[1] Nielsen, J., and Keasling, J.D., Synergies between synthetic biology and metabolic enginnering,
Nature Biotechnology, Vol. 29:694-695, 2011.
[2] Lee, J.W., Na, D., Park, J.M., Lee, J., Chi, S., and Lee, S.Y., Systems metabolic engineering of
microorganisms for natural and non-natural chemicals, Nature Chemical Biology, Vol. 8:536-546,
2012.
[3] http://pubchem.ncbi.nlm.nih.gov/[4] http://www.genome.jp/kegg/

C-5-24

A Cellular Automaton Approachfor Modeling Chemical Reactions in Protein Synthesis
Daichi Takata 1
er12g030@steng.u-hyogo.ac.jp

Ferdinand Peper 2
peper@nict.go.jp
1

2

Teijiro Isokawa 1
isokawa@eng.u-hyogo.ac.jp

Nobuyuki Matsui 1
matsui@eng.u-hyogo.ac.jp

Graduate School of Engineering, University of Hyogo,
2167 Shosha, Himeji, Hyogo 671-2280, Japan
Brain Networks & Communication Laboratory, National Institute of Information andCommunications Technology, 1-4 Yamadaoka, Suita, Osaka 565-0871 Japan

Keywords: Brownian Cellular Automaton, Protein Synthesis, Self-Reproduction
Self-reproduction is an important topic in the field of cellular automata (CAs), since the original
CA model has been invented with the purpose to formalize biological self-reproduction [1].
Various self-reproducing machines on CAs have been proposed, such as universal constructors [1]and self-reproducing loops [2], resulting in the realization of simpler self-reproduction
mechanisms [3,4]. However, there have hardly been investigations on self-reproduction that
employs biologically realistic self-reproduction, such as chemical reactions in the protein
synthesis.
This paper explores self-reproduction from a more biological point of view by modeling andsimulating the chemical reactions in protein synthesis of biological systems. To this end we adopt a
Brownian cellular automaton (BCA), which is a type of asynchronous CAs in which fluctuations
drive operations. A BCA with an appropriately designed transition function can deal with bothrandom processes, such as proteins flowing over an area, and sequential processes, such assynthesizing messenger RNAs [5]. There are four stages in the process of protein synthesis: thereproduction of DNA strands, the transcription from DNA strands to RNAs, the synthesis ofmessenger RNAs by a splicing process, and the synthesis of proteins. All these processes aremodeled in this paper through the use of BCAs.
[1] Banks, E. R., Universality in cellular automata, IEEE 11th Annual Symposium on Switching andAutomata Theory, 194–215, 1970.
[2] Langton, C. G., Self-reproduction in cellular automata, Physica D, 10:135–144, 1984.
[3] Serizawa, T., 3-state Neumann neighbor cellular automata capable of constructing selfreproducing
machine, IEICE Transaction Japan, J69:653–660, 1986.
[4] Reggia, J. A., Armentrout, S. L., Chou, H.-H., and Peng, Y., Simple systems that exhibit
self-directed replication, Science, 259:1282–1287, 1993.
[5] Peper, F., Lee, J., and Isokawa, T., Brownian Cellular Automata, Journal of Cellular Automata,5:185–206, 2010.

C-5-25
Dynamical control of protein concentration usingsynthetic two-component system
Akifumi Nishida 1

Ryoji Sekine 1

Daisuke Kiga 1

Masayuki Yamamura 1

nisida@es.dis.titech.ac.jp

1

Department of Computational Intelligence and Systems Science , Tokyo Institute of Technology,
J2-51, 4259
Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8503, Japan

Keywords: two-component system, system modeling, dynamical control of protein concentration
To control cells using external input is useful for understanding biological function. For
the analysis of the function, adding a time-invariant perturbation to a component iseffective, but adding a time-variant perturbation is also effective. Dynamical control ofprotein concentration using external input (e.g. inducer or light) have been studied
recently[1][2]. For dynamical control, a control system that converts external input into
output like protein is required, and mathematical model of the system is desirable becauseof time dilation and noise. These studies are recently developed and it is necessary toincrease variety of control systems. By using Cph8/OmpR two -component system that
responds to a red-light stimulus and activate transcription of target gene, we controlled
protein concentration tracking a desired value with mathematical model.
Cph8/OmpR two-component system has Cph8 as sensor protein and OmpR as regulator
protein. Cph8 was synthesized by photoreceptor protein to be able to respond red light inE.coli[3]. In this study, Cph8/OmpR two-component system activates GFP expression underdark condition and can’t activate it under red light condition.
Since the signal transduction mechanism of Cph8 is unknown, we constructed somemathematical models based on a few assumptions. In the process of parameter
identification, we used time evolution of output (protein concentration) in response to
dynamical switching of the input light. Finally, we dynamically controlled GFP
concentration by changing input light pulse cycle. Mathematical model of the sys tem isused to determine the optimum input. We show that Cph8/OmpR two-component system
can be used for dynamical control of protein concentration. These studies would be usefulfor understanding biological function.

[1] Milias-Argeitis A, Summers S, Stewart-Ornstein J, Zuleta I, Pincus D, EI-Samad H, Khammash
M, In silico feedback for in vivo regulation of a gene expression circuit , nature biotechnology,
29:1114-1116, 2011.
[2] Uhlendorf J, Miermont A, Delaveau T, Charvin G, Fages F, Bottani S, Batt G, Hersen P,
Long-term model predictive control of gene expression at the population and single -cell levels,
Proc Natl Acad Sci U S A, 109(35):14271-14276, 2012.
[3] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras
A, Ellington AD, Marcotte EM, Voigt CA, Synthetic biology: engineering Escherichia coli to see
light, Nature, 438(7067):441-442, 2005.

C-5-26
Synthetic cellular membrane systems
Tsutomu Hamadat-hamada@jaist.ac.jp

School of Materials Science, Japan Advanced Institute of Science & Technology
1-1, Asahidai, Nomi, Ishikawa 923-1292, Japan

Keywords: soft interface, cell membrane, liposome, photo-control, nano-particles
In living systems, bio-molecules self-assemble in a predetermined fashion under specificconditions into highly organized supramolecular structures with specific functions. The
constructive and synthetic approach to biological systems is an important and interesting challengethat spans across several disciplines, including physics, chemistry and biology. A closed
two-dimensional surface consisting of a lipid bilayer is an essential compartment of livingorganisms. We have developed a synthetic membrane system that exhibits cellular functions [1].
We utilized a synthetic photosensitive amphiphile to achieve the photo-based manipulation of
the opening and closing of membranes through reversible transitions between sphere and disk
structures. The manipulation is based on the photo-switching of the membrane line tension, as
deduced from the fluctuation of the membrane edge. Furthermore, we demonstrated thecontrollable capture and release of a targeted object into and from a membrane vesicle. This
successful photo-manipulation of mesoscopic membrane structures in a noncontact and reversible
manner may see wider application, such as in micro-reactors and smart drug-delivery systems(SDDS).
We then investigated the association of nano-particles (NPs) on a model membrane surface [3].
We found that lateral heterogeneity in the membrane mediates the partitioning of NPs in asize-dependent manner: small particles with a diameter of ≤200 nm were localized in an orderedphase, whereas large particles preferred a fluidic disordered phase. In terms of the membraneelastic energy, we present a physical model that explains this localization preference of NPs. Thesefindings may lead to a better understanding of the basic mechanisms that underlie the associationof nano-materials within a cell surface. These results help us to design proto-cell systems that takeadvantage of the membrane thermodynamic characteristics to recognize contacting objects.
[1] Hamada, T. & Yoshikawa, K., Cell-sized liposomes and droplets: real-world modeling of living
cells, Materials, 5:2292-2305, 2012.
[2] Hamada, T. et al., Membrane disc and sphere: controllable mesoscopic structures for thecapture and release of a targeted object, J. Am. Chem. Soc., 132:10528-10532, 2010.
[3] Hamada, T. et al., Size-dependent partitioning of nano/micro-particles mediated by membranelateral heterogeneity, J. Am. Chem. Soc., 134:13990−13996, 2012.

C-5-27

Molecular Evolution of a TALE Protein
to Change DNA Binding Manner
Miki Imanishi
imiki@scl.kyoto-u.ac.jp

Shogo Tsuji
tsuji.shougo.27n@st.kyoto-u.ac.jp

Shiroh Futaki
futaki@scl.kyoto-u.ac.jp

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Keywords: Protein-DNA interaction, Molecular evolution, Synthetic biology
Artificial DNA binding proteins binding to desired DNA sequences are useful to control
transcription of various genes and/or gene editing. So, they can be powerful tools for synthetic
biology. Transcription activator-like effectors (TALEs) are sequence-specific DNA binding
proteins secreted by the bacterial pathogen Xanthomonas. Their DNA binding specificity isdetermined by a series of tandem repeats of typically 34 highly conserved amino acids, containing
repeat variable di-residues (RVDs) at positions 12 and 13, that define the base preferences of arepeat. Because of the simple one-to-one base recognition by each repeat, TALEs can be readily
designed to target desired DNA sequences just by replacing the RVDs. Though TALEs have thetarget sequence versatility determined by tandem repeats regions, almost all TALE binding sites
are preceded by a highly conserved 5’ terminal T nucleotide. N-terminal noncanonical repeat (-1 st
repeat) has been thought to interact specifically with the 5’-T. However, the details of the baserecognition mode are still unclear. In this study, we substituted amino acids that correspond to theresidues of the adjoining loops of the -1 st repeat and verified the contributions to the 5’-Trecognition. In addition, we performed directed evolution of the -1 st repeat to bind to non 5’-T
sequences using a bacterial 1-hybrid assay.
We introduced a point mutation in a tryptophan residue at the adjoining loop of the -1 st repeat ofdHax3. The DNA bindings of the mutated dHax3s were examined by luciferase reporter assays in
HeLa cells. Only the substitutant with tyrosine showed a comparable activity to wild type. A
significant decrease in the luciferase activity was observed when the tryptophan residue wassubstituted with each of the other 18 amino acids. In addition, all of the mutants showed a DNA
binding preference to 5’-T. This result suggests that tryptophan and tyrosine play an important rolein the DNA recognition by the -1 st repeat, but the recognition of the 5’-T appears not to be so
simple as the canonical repeat domains. Therefore, we randomized the four amino acid residues in
the loop region and intended to change the recognition pattern of the 5’-terminal nucleotide.
Through a bacterial one-hybrid screening using a reporter vector with a dHax3 binding site startingfrom 5’-C, we obtained highly conserved amino acid sequences of the loop region. The selected
dHax3 mutant showed significantly strong DNA binding activity to the binding sites starting fromnon 5’-T. This result directly indicates the importance of the -1 st repeat on the recognition of the
5’-terminal nucleotide.

C-5-28

A Magnetically Triggered Gene Expression System
Mediated by Heating of Nanoparticles
Masaki Yamaguchi

Akira Ito

3TE12035Y@s.kyushu-u.ac.jp

akira@chem-eng.kyushu-u.ac.jp

Yoshinori Kawabe

Masamichi Kamihira

kawabe@chem-eng.kyushu-u.ac.jp

kamihira@chem-eng.kyushu-u.ac.jp

Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744
Motooka, Nishi-ku, Fukuoka 819-0395, Japan

Keywords: Inducible gene expression, Transcriptional amplification, Magnetite nanoparticles,
Heat shock protein promoter, Tetracycline-responsive transactivator
Gene therapy promises a wide range of applications in medicine, including cancer treatment.
Similar to chemotherapy, the therapeutic efficacy depends on the dosage (gene expression level)
and timing. Thus, controlled gene expression is essential for gene therapy. Synthetic biology
focuses on designing artificial gene expression systems. Numerous gene circuits have beendeveloped to control gene expression. Among them, the tetracycline (Tet)-inducible system [1] andheat shock protein promoter system [2] have been used as effective inducible gene expression
systems. Using a synthetic biological approach, we have previously constructed a heat-inducible
transgene expression system incorporating a transcriptional positive feedback loop mediated by
Tet-inducible system that enhances heat-induced gene expression [3].
Remote activation of target cells to trigger specific gene expression in vivo can provide a useful
research tool and potential means to control gene expression in clinical settings [4]. For thispurpose, nanotechnology is becoming increasingly important in medicine. Magnetite nanoparticles
have been used for drug delivery systems and cancer diagnosis as contrast-enhancement agents inmagnetic resonance imaging. Furthermore, magnetite (Fe 3 O 4 ) particles absorb energy and generateheat in response to an alternating magnetic field (AMF) [5]. Thus, heat generation by AMF
exposure can be a potent tool as a switch to induce target gene expression. Here, we combinedsynthetic biology with nanotechnology to convert a local heating signal using magnetite
nanoparticles and AMF exposure into high-level gene expression at a specific site. We investigated
the in vivo feasibility of this approach for cancer gene therapy using a tumor xenograft model.
[1] Stieger, K., Belbellaa, B., Le, Guiner. C., Moullier, P., and Rolling, F., In vivo gene regulation
using tetracycline-regulatable systems, Advanced Drug Delivery Reviews, 61:527-541, 2009.
[2] Huang, Q., Hu, J. K., Lohr, F., Zhang, L., Braun, R., Lanzen, J., Little, J. B., Dewhirst, M. W.,
and Li, C. Y., Heat-induced gene expression as a novel targeted cancer gene therapy strategy,
Cancer Research, 60:3435-3439, 2000.
[3] Yamaguchi, M., Ito, A., Okamoto, N., Kawabe, Y., and Kamihira, M., Heat-inducible transgene
expression system incorporating a positive feedback loop of transcriptional amplification for
hyperthermia-induced gene therapy, Journal of Bioscience and Bioengineering, 114:460-465,
2012.
[4] Stanley, S. A., Gagner, J. E., Damanpour, S., Yoshida, M., Dordick, J. S., and Friedman, J. M.,
Radio-wave heating of iron oxide nanoparticles can regulate plasma glucose in mice, Science,
336:604-608, 2012.
[5] Gazeau, F., Lévy, M., and Wilhelm, C., Optimizing magnetic nanoparticle design for
nanothermotherapy, Nanomedicine (Lond), 3:831-844, 2008.

C-5-29
A combinatorial library of enzymes for anthocyanin
biosynthesis toward designing of an artificial anthocyanin
operon1
2

Takeshi Yoshizumi 1

Masaru Tomita 1,2

takey@ttck.keio.ac.jp

mt@sfc.keio.ac.jp

Mitsuhiro Itaya 1,2

Kenji Tsuge 1

mita2001@sfc.keio.ac.jp

ktsuge@ttck.keio.ac.jp

Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka,
Yamagata 997-0017, Japan
Faculty of Environment and Information Studies , Keio University, 5322 Endo, Fujisawa,
Kanagawa 252-0882 Japan

Keywords: operon, synthetic biology, anthocyanin, OGAB
Many of biosynthetic pathways for useful chemicals of plant have been tried to transplant inbacterial cell for the purpose of increase production speed and quantity. Construction of aheterologous metabolic pathway in a bacterial cell is, however, still difficult as the number ofrequired enzyme is getting bigger. To investigate a method for construction of metabolic pathway,
we render a biosynthetic pathway of plant specific pigment, anthocyanin as a model. In our former
work, a series of 9 enzymes required for production of peralgonidin, an anthocyanin compounds, inEscherichia coli were assembled in a plasmid as polycistronic operon form. However, any
anthocyanin was detected in an E. coli harboring the operon, although production of naringenin, anintermediate metabolite, was observed. This implies that design for the artificial operon isnecessary to produce peralgonidin. We then focus on relevant four enzymes required for
peralgonidin synthesis from naringenin. Since, in an artificial operon profile of expression level ofthe genes is expected as monotonical decrease from promoter [1, 2], we thus tried in vitro
production of peralgonidin with different amount of the enzymes mimicking possible 24 (=4 !)operons to find out optimal 4-enzyme ratio for production. As a result, specific combinations
showed significant production of peralgonidin. Now we are making several artificial operons
including four enzyme genes with optimal gene orders according to t he enzyme combinations by
Ordered Gene Assembly in Bacillus subtilis (OGAB) method [3].
[1] Nishizaki, T., Tsuge, K., Itaya, M., Doi, N., and Yanagawa, H. , Metabolic engineering of
carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl.
Environ. Microbiol. 73:1355-1361, 2007.
[2] Hiroe, A., Tsuge, K., Nomura, C.T., Itaya, M., and Tsuge, T. , Rearrangement of gene order inthe phaCAB operon leads to effective production of ultrahigh -molecular-weight
poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli, Appl. Environ.
Microbiol. 78:3177-3184, 2012.
[3] Tsuge, K., Matsui, K., and Itaya, M., One step assembly of multiple DNA fragments with adesigned order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res. 31:e133, 2003.

C-5-30

Mathematical modeling and theoretical analysis for thequantitative control of the target gene expression ofsynthetic genetic circuit
Yuki Soma 1souma@brs.kyushu-u.ac.

Yohei Motomura 1

Keigo Tsuruno 1

ktsuruno@brs.kyushu-u.ac.jp
12
3

Mai Murakami 2Masahiro Okamoto 123

okahon@brs.kyushu-u.ac.jp

Shunsuke Yasutake 2
Taizo Hanai 123

taizo@brs.kyushu-u.ac.jp

Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
6-10-1, Hakozaki, Higashi-ku, Fukuoka, Fukuoka, 812-8581, Japan
Graduate School of Systems Life Sciences, Kyushu University,
6-10-1, Hakozaki, Higashi-ku, Fukuoka, Fukuoka, 812-8581, Japan
Synthetic Systems Biology Resrch Center, Kyushu University, 3 -1-1, Maidashi, Higasi-ku,
Fukuoka, Fukuoka, 812-8582, Japan

Keywords: synthetic genetic circuit, synthetic biology,
In recent years, synthetic genetic circuits have been developed as a gene expression
regulatory system in expectation of biotechnological applications . For practical applications ofthem, quantitative control of target genes expression is necessary. For this purpose, various
parameters, such as promoter or RBS strength and copy number of genes, should be adjusted
properly according to characteristics of the circuit. Development of mathematical modeling on thebasis of the experimental results in vivo is important for the efficient and deliberate construction ofdesired circuits. In this study, we designed a simple inducible switch which is composed of twogene expression modules (P LtetO 1 ::gene1 and P L lacO 1 ::tetR (repressor) gene2) and a constitutive
source of LacI repressor (PlacI q ::lacI). The switching of expression from gene1 to gene2 should be
induced by addition of IPTG.
At first, we experimentally evaluated promoter strengt h of the two P L promoters bymonitoring expression level of GFP in Escherichia coli. Next, we built two stochastic models ofeach module and fitted their parameters to the results of the in vivo experiment. The desired
switching behavior was realized by integrating two stochastic models above. Furthermore, theexpression level of gene1 and gene2 showed a positive and a negative correlation with the increasein the copy number of PlacI q ::lacI, respectively. Based on these results, we constructed the genetic
switch and examine its dynamic behavior in vivo. As the results, we obtained the desired switchingand observed the predicted correlation between the copy number of lacI and target genes
expression. For quantitative control of target genes expression, othe r parameters such as promoter
strength or RBS strength can be adjusted in future work.
[1] Callura, J.M., Cantor, C.R., Collins, J.J., Genetic switchboard for synthetic biology
applications, Proc. Natl. Acad. Sci. USA., 10, 5850-5855, 2012.
[2] Zhang, F., Carothers, J.M., Keasling, J.D., Design of a dynamic sensor-regulator system forproduction of chemicals and fuels derived from fatty acid s, Nat. Biotechnol., 25, 354-359, 2012.

C-5-31
Synthetic analysis of the effect of parameter balance on
phenotypic stabilities in a synthetic mutual inhibitory
network
Ryoji Sekine 1

Daisuke Kiga 2

green-dolphin@es.dis.titech.ac.jp

kiga@dis.titech.ac.jp

Masayuki Yamamura 2
my@dis.titech.ac.jp

1

Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology,
Kanagawa 226-8503, Japan

Keywords: mutual inhibition, phenotypic stability, synthetic gene circuit
Mutual inhibitory network, in which two genes inhibit each other, appears in high frequency invarious organisms from bacteria to mammalian: for example, Nanog and Gata6 in embryo [1].
Cells carrying the mutual inhibitory network are known to exhibit either of two distinct phenotypes
where one gene is expressed a lot while the other is slightly when parameters such as theexpression rate constants of the two genes are balanced [2]. However, quantitative knowledge
about the effect of parameter balance on the stability of each phenotype is not experimentallyinvestigated well. In this work, we analyzed the effect of the parameter balance to phenotypicstabilities in a synthetic mutual inhibitory network constituting of CIts and LacI, based on P luxlac
toggle [3]. The net rate constants of the CIts expression in our synthetic mutual inhibitory network
can be tuned by the addition of an inducer N-acyl homoserine lactone (AHL). We measured thephenotypic ratios, determined by the switching rates and growth rates of each phenotype, at
stationary state under various AHL concentrations, and analyzed the relationship between theswitching rate corresponding to the phenotypic stabilities and the balance of the promoter strength.
Our results and approach would be a basis of an experimental system to esti mate and adjust
switching rates and phenotypic ratios in mutual inhibitory networks of interest.
[1] Zhou, J. X., and Huang, S., Understanding gene circuits at cell-fate branch points for rational
cell reprogramming, Trends in Genetics, 27:55-62,2011.
[2] Gardner, T. S., Cantor, C. R., and Collins, J. J., Construction of a genetic toggle switch in
Escherichia coli, Nature, 403:339-342, 2000.
[3] Sekine, R., Yamamura, M., Ayukawa, S., Ishimatsu, K., Akama, S., Takinoue, M., Hagiya, M.,
and Kiga, D., Tunable synthetic phenotypic diversification on Waddington ’s landscape throughautonomous signaling, Proceedings of National Academy of Sciences U. S. A, 108:17969-17973,
2011.

C-5-32

Theophylline-dependent Riboswitch as a Useful Genetic
Tool for Synthetic Biology in Cyanobacteria
Yoichi Nakahira 1
nakahira.yoichi.mu@ehime-u.ac.jp

Atsushi Ogawa 2

Tokitaka Oyama 3oyama@cosmos.bot.kyoto-u.ac.jp

1

2

3

Hiroyuki Asano 3

a-ogawa@ccr.ehime-u.ac.jp

Yuzuru Tozawa 1, 2
tozawa.yuzuru.mx@ehime-u.ac.jp

Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime
790-8577, Japan
Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577,
Japan
Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku,
Kyoto 606-8502, Japan

Keywords: Synthetic biology, Theophylline riboswitch, Translational regulation, Cyanobacteria
Cyanobacteria are favorable cell factories for the production of renewable biofuels and valuable
chemicals because of their ability to capture solar energy. However, biotechnology for cyanobacteria stilllags behind conventional model species such as Escherichia coli or baker’s yeast. In this work, we
employed engineered riboswitches to control translational initiation of target genes in cyanobacterium
Synechococcus elongates PCC 7942. A firefly luciferase reporter assay revealed that three theophylline
riboswitches performed well in Synechococcus. Among the riboswitches, the best one exhibited very low
leaky expression of luciferase and dose-dependent ON/OFF regulation of protein expression by
theophylline. The maximum magnitude of the induction versus basal level was ~190-fold. We also adopted
this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC geneproduct is controlled in a theophylline dose-dependent manner. The result demonstrated that the adequately
adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic
mutant. These suggest that the theophylline-dependent riboswitch system has great promise as a usefulgenetic tool for synthetic biology in cyanobacteria.

[1] Nakahira, Y., Ogawa, A., Asano, H., Oyama, T., and Tozawa, Y., Theophylline-dependentriboswitch as a novel genetic Tool for strict regulation of protein expression in cyanobacterium
Synechococcus elongatus PCC 7942, Plant Cell Physiology, in press, 2013.

C-5-33
Automatic Design with Frequency Characteristics for
Synthetic Gene Oscillators
Takafumi Hayashi 1
t_hayashi@es.dis.titech.ac.jp

1

Masayuki Yamamura 1
my@dis.titech.ac.jp

Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology,
Kanagawa 226-8503, Japan

Keywords: synthetic gene circuit, oscillator, design automation
Oscillation is an essential phenomenon in biological systems. Many activities and life
maintenance of organisms are supported by that phenomenon. For this reason, a deeper
understanding about oscillation in biological system is important in biology. In synthetic biology,
synthetic gene circuit is used as a tool to understand the biological system [1]. In order to get a
deeper understanding of biological systems, the more complex and larger gene circuits are required.
As increasing the number of components, however, difficulty of design becomes higher
exponentially. It is difficult to design in human trial and error or ad-hockery. Therefore, Automatic
design approaches are important [2].
We propose an automatic design method for synthetic gene oscillators. In previous research
related to automation of design, an evaluation is calculated by error of protein concentration in
only time domain. However, that evaluation function might make inadequate landscape to optimize
an oscillate solution because it’ sensitivity for a shift of phase is too strong. The proposed method
is also to use the frequency characteristic in the evaluation function. That function can make more
appropriate landscape to search oscillators. Moreover we propose two-stage design method. In thefirst stage, it optimizes the network structure by frequency characteristics. In the second stage, it
optimizes parameters by error in time domain. As a result of performance examination s, it was
shown that the probability to find an optimal solution by proposed method is higher than previous
methods.
[1] Elowitz, M. Lim, W.A., Build life to understand it, Nature, 2010.
[2] Macdonald J.T., Barnes C., Kitney R.I., Freemont P.S., Stan G.B., Computational design
approaches and tools for synthetic biology, Integrative Biology, 2011.

C-5-34
RNA-protein complexes for designing functional
nanostructured molecules
Yoshihiko Fujita1

fujita.yoshihiko.8c@kyoto-u.ac.jp

Rie Furushima2

12

Hirohisa Ohno1

h-ohno@kuchem.kyoto-u.ac.jp

Tan Inoue1

tan@kuchem.kyoto-u.ac.jp

Graduate School of Biostudies, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto-shi, Kyoto
606-8502, Japan
Dynamical Micro-scale Reaction Environment Project, Exploratory Research for Advanced Technology(ERATO), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, Japan.

Keywords: RNA-protein complex, nanostructure, synthetic biology
Nanostructures consisting of DNA provide promising frameworks for designing nanomachines withdefined shapes and sizes. However, the DNA based structures have problems when applied in vivo. To avoid
such problems including genomic integration or immune responses, several approaches including episomal
technologies have been practiced without sufficient success.
RNA-protein complexes (RNP) can also be employed to design and construct nanostructures. They
have characteristic features as follows. (1) RNA can be designed by using a simple Watson-Crick basepairings like DNA nanostructure and naturally occurring tertiary interactions, (2) proteins can be exploitedfor reinforcement of the RNA structure and to give a function to the RNP, and (3) RNA-protein structures
have potential to be produced and assembled in vivo. We have designed triangular shaped RNP [1].
In this study, we have further exploited the potential use of the RNPs with defined nanostructures.
Various shapes (i. e. square or zigzag) of the RNP have been designed, constructed and visualized by
HS-AFM. Several functional RNA-binding proteins have been successfully attached to the RNAs withdefined structures to improve their physiological functions. Thus, the RNA-protein complexes are potentially
useful for designing novel nanostructures linking the structures to the functions.
[1] Ohno, H.; Kobayashi, T.; Kabata, R.; Endo, K.; Iwasa, T.; Yoshimura, S. H.; Takeyasu, K.; Inoue, T. &Saito, H. (2011), 'Synthetic RNA-protein complex shaped like an equilateral triangle.', Nat Nanotechnol
6(2), 116--120.

C-5-35

Construction of a computational platform formetabolic pathway design
Michihiro Araki 1
araki@port.kobe-u.ac.jp

	
  	
 

1

2

Teppei Ogawa 2

makiguchi-hiroki@mki.co.jp

ogawa-teppei@mki.co.jp

Masahiko Nakatsui 1

Akihiko Kondo 1

nakatsui@port.kobe-u.ac.jp

akondo@kobe-u.ac.jp

Robert Sidney Cox III 1 	
  	
  	
 
cox@port.kobe-u.ac.jp

Hiroki Makiguchi 2

Organization of Science and Technology, Kobe University, 1-1 Rokkodai Nada,
Kobe 657-8501, Japan
Mitsui Knowledge Industry Co., Osaka Mitsui-Bussan Bldg. 6F, 2-3-33
Nakanoshima, Kita-ku, Osaka 530-0005, Japan

Keywords: metabolic engineering, synthetic biology, chemoinformatics
Computational design of metabolic pathways has become an essential and complementary
approach to identify metabolic pathways for the production of chemical products. In order to findthe metabolic pathways of value, it is primarily necessary to utilize comprehensive chemical andenzymatic information to ensure a wide range of the enzymatic reaction, and obtain pathway
candidates including putative compounds and enzymatic reactions. We have developed an efficientalgorithm and a computational tool for the design of extensive metabolic pathways.
The design tool begins the search for metabolic pathways with the initial and last compounds as
queries. In the result viewer, the candidate list of metabolic pathways is presented with ranking
scores, and the changes in the chemical structures in the metabolic pathways can be checked at a
glance. The putative candidates for reactions and compounds are displayed with scores based on
the comparison of structural similarities. The design tool provides a user with various design
options including the number of reactions, reaction steps and so on. Any chemical and reaction
information can be also applied to the design tool, which will be a powerful tool to identify
unknown metabolic pathways of chemical products.

C-5-36

Highly parallel and sensitive method for analyzing
gene expression kinetics
Maasa Yokomori, Osamu Gotoh and Akira Suyama.
yokomori@genta.c.u-tokyo.ac.jp, suyama@dna.c.u-tokyo.ac.jp

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1
Komaba, Meguro-ku, Tokyo 153-8902, Japan

Keywords: synthetic gene circuits, absolute quantification, multiplex measurement
Synthetic biology is a bottom-up approach to a deeper understanding of biological phenomena
by building synthetic gene circuits and studying their kinetics in cells. Analysis of kinetics of
intricate synthetic gene circuits with multiple components plays a crucial role in elucidation of
biomolecular networks and in their bioengineering applications. Since synthetic gene circuits
are integrations of chemical reactions, it is eagerly expected to develop a method for monitoring
temporal changes of gene expression not in terms of relative amounts of transcription products
but in terms of their absolute amounts. Moreover, in the case of analyzing synthetic gene
circuits introduced into living cells, a large number of originally-existing internal genes in thecells may interfere with external genes introduced from the synthetic circuits. Therefore, ahighly multiplex method that can measure both the internal and external genes is required forinvestigating behaviors of such synthetic circuits.
Here, we describe a highly parallel and sensitive method for analyzing gene expression
kinetics by measuring the absolute amounts of their transcripts without reverse transcription
processes[1]. The method determines the absolute mRNA amounts by quantifying
well-designed DNA tag sequences called DNA-Coded Numbers (DCNs) [2] generated byphoto-chemical ligation of pairs of two chemically synthesized DNA oligonucleotides thatspecifically bind to the target mRNA. The converted DCNs are then amplified by PCR andfinally detected on microarrays. The conversion to DCNs also provides advantages such as theuse of target-independent microarrays and no need for sample labeling. The method wasvalidated by using chemically synthesized RNA samples of known quantities and total RNA
samples prepared from mouse liver and retina. The absolute amounts of 43-144 mRNA species
were reproducibly quantified in parallel with high sensitivity (24-60 zmol) from a small amountof total RNA samples (20 ng) in 7 hours.
[1] Yokomori, M., Gotoh, O., Fujimoto, K., Suyama, A. submitted.
[2] Gotoh, O., Murakami, Y., Suyama, A. Multiplex cDNA quantification method thatfacilitates the standardization of gene expression data. Nucleic Acids Res. 39, e70 (2011)

C-5-37
A development of molecular sensor that delivers environmental
information to inside of vesicle-based molecular robots

Koh-ichiroh Shohda 1
shohda@genta.c.u-tokyo.ac.jp

1

Akira Suyama 1
suyama@dna.c.u-tokyo.ac.jp

Graduate School of Arts and Sciences, University of Tokyo, 3-8-1
Komaba, Meguro-ku, Tokyo 153-8902, Japan

Keywords: DNA, lipid, vesicle
In the molecular robotics project, a planned molecular robot that is based on a vesicle or a gel as abody consists of molecular sensors, molecular computers, and molecular actuators. These components
are communicated with single-stranded DNAs each other. We covered a development of molecularsensor that was a conjugated molecule between DNA strands and lipid. [1] This sensor molecule locatedon a surface of vesicle could hybridize with a complementary DNA/RNA strand that represented anenvironmental information. The hybridization caused a shape change of the molecular sensor,
consequently a single-stranded DNA was released into an inner water pool of vesicle. The released
DNA was a trigger for molecular computers encapsulated in the vesicle.

[1] P. Barton, C. A. Hunter, T. J. Potter, S. J. Webb, and N. H. Williams Angew. Chem. Int. Ed. 2002, 41,3878-3881.

C-5-38

DNA computer-based rational construction of artificial genetic circuits
in cell model vesicles containing cell-free protein synthesis system◦

Takamasa Hasegawa1 , Koh-ichiroh Shohda2 , Akira Suyama1,2
1

Department of Physics, 2 Department of Life Sciences, University of Tokyo[hasegawa, shohda]@genta.c.u-tokyo.ac.jp, Suyama@dna.c.u-tokyo.ac.jp

In the field of synthetic biology, the construction of artificial genetic circuits is aimed for understanding natural biological systems and designing new biological parts. However, the rational constructionof artificial genetic circuits large enough to be of practical value is not easy at all. No eﬀective method
for the rational construction has yet been developed. Here, we report a DNA computer-based approachto the construction of artificial genetic circuits, which may have the potential for the rational construction of large artificial genetic circuits of practical value. In this study, a simple artificial genetic circuit was constructed based on a framework of RTRACS, which is a modular DNA computing systemcomprised of modules communicating with each other by using input and output RNA strands (Phys.
Rev. E, 78:041921, 2008). The simple circuit receives input RNA for two inputs and produces output
mRNA coding green fluorescent protein (GFP) as a consequence of the AND operation on the two inputs(Fig. 1). The constructed circuit was encapsulated into a Giant Unilamellar Vesicle (GUV), which is an
artificially-prepared phospholipid-bilayer vesicle and has been recognized as a model to study biologicalcells. Expression of GFP gene controlled by input RNA strands was observed by green fluorescence emitted from GFP translated (synthesized) from the output mRNA using a cell-free protein synthesis system
in a GUV. It was confirmed that the genetic circuit worked successfully in a GUV (Fig. 2). The present
work would be the first small but important step toward the rational construction of more sophisticatedartificial genetic circuits of practical value working in cell model vesicles.

D1

aC1
input RNA A

D7

aC1 aC2 aC3C3 promoter GFP gene

C1 C2

D2

aC1 aC2OFF

Reversetranscription
D8

aC1 aC2 aC3

C1 C2
RNase Hdigestion
D3

C3 promoter GFP geneaC1 aC2ONaC1 aC2 aC3D4

aC1 aC2

C3
input RNA B
mRNA

C2 C3

D5

aC1 aC2 aC3Reversetranscriptionamino acyl-tRNAaC1 aC2 aC3

C3

polypeptide

C2 C3
D6

aC1 aC2 aC3RNase Hdigestion
ribosomeਤ 1 Reaction scheme of constructed module.

ਤ 2 FM observation of GFP-expressed GUVs.

C-5-39
New Bottom-up Construction Strategy of Biomolecular
Circuits Based on Dynamic Robustness Measure
Masaki Inoue 1,2minoue@cyb.mei.titech.ac.jp

Takayuki Arai 2
arai@cyb.mei.titech.ac.jp

Kenji Kashima 3
kashima@sys.es.osaka-u.ac.jp

1
2
3
4

Jun-ichi Imura 2
imura@mei.titech.ac.jp

Kazuyuki Aihara 4
aihara@sat.t.u-tokyo.ac.jp

FIRST, Aihara Innovative Mathematical Modelling Project, JST
Graduate School of Information Science and Engineering , Tokyo Institute of Technology,
2-12-1-W8-1, O-okayama, Meguro-ku, Tokyo 152-8552, Japan
Graduate School of Engineering Science, Osaka University, 1-3, Machikaneyama, Toyonaka,
Osaka 560-8531, Japan
Institute of Industrial Science, the University of Tokyo, 4 -6-1, Komaba, Meguro-ku, Tokyo
153-8505, Japan

Keywords: Synthetic biology, Robustness analysis, Bistability
We analyze the biomolecular switchable memory system created by a bottom -up construction
strategy [1] to reveal the reason to the successful synthesis. The system is represented by a
feedback form. Utilizing the robust bifurcation analysis method for feedback systems [2], which
evaluates the robustness against both of dynamic and parametric perturbations, we can guarantee
the validity of the bottom-up construction. Lastly, we propose a novel construction strategy forsystem-level biomolecular circuits by assembling core modules with high dynamic robustness.
[1] Padirac A., Fujii T., and Rondelez Y., Proceedings of the National Academy of Sciences ,109:E3212-E3220, 2012.
[2] Inoue M., Imura J.-I., Kashima K., Aihara K., International Journal of Bifurcation and Chaos,23, 2013 (in press).

C-5-40
iGEM 2013 Team Tokyo Tech Project: Mutant Ninja Coli
Daisuke Kiga 1

Shotaro Ayukawa 2

kiga@dis.titech.ac.jp

ayukawa@acls.titech.ac.jp

Hiroshi Fujimoto 3

fujimoto.h.ac@m.titech.ac.jp

Ryota Fujiwara 4

fujiwara.r.ac@m.titech.ac.jp

Ema Hatanaka 5hatanaka.e.aa@m.titech.ac.jp

Tatsuhiro Isozaki 6

Tatsuya Kobayashi 3

Masanori Kawabata 7

isozaki.t.ac@m.titech.ac.jp

kobayashi.t.bn@m.titech.ac.jp

mkawabata@suou.waseda.jp

ogino.s.ab@m.titech.ac.jp

saito.k.bb@m.titech.ac.jp

shi.h.ac@m.titech.ac.jp

Yochiku Shi

Shinya Suzuki

Sara Ogino 3

8y_shi@sb.dis.titech.ac.jp

Ken Saito 10

5

suzuki.s.bs@m.titech.ac.jp

Sairi Tomatsu 3

Yuta Wakabayashi 3

tomatsu.s.aa@m.titech.ac.jp

wakabayashi.y.ab@m.titech.ac.jp

Hiroyuki Ohta 9

hohta@bio.titech.ac.jp

Akihiko Konagaya 1

kona@dis.titech.ac.jp

Hengyu Shi 5

Shunta Suzuki 3

suzuki.s.cf@m.titech.ac.jp

Naoki Watarai 5

watarai.n.aa@m.titech.ac.jp

Masayuki Yamamura 1
my@dis.titech.ac.jp

1

Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology,
J2-1707, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8503, Japan
2
Education Academy of Computational Life Sciences, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan3
Department of Bioengineering, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan
4
School of Bioscience, Tokyo Institute of Technology, 4259 Nagatsuta -cho, Midori-ku,
Yokohama-shi, Kanagawa 226-8501, Japan
5
Department of Biological Information, Tokyo Institute of Technology,4259 Nagatsuta-cho,
Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan
6
School of Biotechnology, Tokyo Institute of Technology,4259 Nagatsuta-cho, Midori-ku,
Yokohama-shi, Kanagawa 226-8501, Japan
7
Department of Electrical Engineering and Bioscience, WASEDA University, 3-4-1 Okubo
Shinjuku-ku, Tokyo Japan
8
Department of Control and Systems Engineering, Tokyo Institute of Technology, 4259
Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan
9
Center for Biological Resources and Informatics, Tokyo Institute of Technology,4259
Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan

Keywords: Synthetic biology, iGEM, genetic circuit
International Genetically Engineered Machine competition ( iGEM) is an international synthetic
biology competition in which undergraduate teams from around the world compete one another. The
participating teams design and construct the original genetic circuits by assembling various gene parts
inside living cells during summer. The teams perform the presentation about their work on the
Jamboree held at MIT. Our Tokyo Tech iGEM team has been participating iGEM since 2006 andgetting bright results such as the acquisition of gold medal and track prizes.
In this year, we proposed to create E. coli that mimics some of the characteristics of ‘ninja’
warrior-spies. A ninja must receive and pass on correct information at all times. A mistake wil l be fatal.
We have designed an artificial genetic circuit that avoids crosstalk between two signals used inbacterial cell-cell communication. Ninjas are also known for their star-shaped ‘shuriken’ throwing
knives. Our E. coli ninja has a similar weapon, an M13 phage which is released to infect other E. coli,
injecting plasmid DNA into them. Moreover, ninjas must harmonize with the natural environment, sothat their relationship with nature is very important. Plant hormones help plants to grow efficiently, andwe attempted to construct a circuit that synthesizes two plant hormones depending on the soil
environment.

C-5-41
Construction and functional analysis of DNA origami base
DNA-RNAP hybrid nanomachine
Takeya Masubuchi 1

Hisashi Tadakuma 1

kk126450@mgs.u-tokyo.ac.jp

tadakuma@k.u-tokyo.ac.jp

Masayuki Endo 2
endo@kuchem.kyoto-u.ac.jp

Yoshie Harada 2
harada.yoshie.4r@kyoto-u.ac.jp

1
2

Hiroshi Sugiyama 2
hs@kuchem.kyoto-u.ac.jp

Takuya Ueda 1

ueda@k.u-tokyo.ac.jp

Graduate School of Frontier Science, The University of Tokyo, 5-1-5
Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan
Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University,
Yoshida-Ushinomiyacho, Sakyo-Ku, Kyoto 606-8302, Japan

Keywords: Molecular robotics, DNA nanostructure, RNA polymerase, gene expression
In the cell, gene expression is highly controlled. To create biologically inspired nanoscale
device enabling the control of gene expression, we made h ybrid nanomachine (T7-tile)
using DNA origami tile as the skeletal structure and T7 RNA polymerase (T7 -RNAP) as thefunctional module (Miyazono et al., EMBO J , 2010). T7-tile hybrid allowed us to evaluate
the effects of intermolecular distance of enzyme (T 7-RNAP) and substrate (target genecontaining T7 promoter). We will show our recent achievements.

[1] Miyazono Y., Hayashi M., Karagiannis P., Harada Y., Tadakuma H. Strain through the necklinker ensures processive runs: a DNA-kinesin hybrid nanomachine study. EMBO J. 29:93-106.
2010.

C-5-42

Heat-induced Morphological Transformation ofSupramolecular Nanostructures
Masato Ikeda 1,2
m_ikeda@gifu-u.ac.jp
12

Department of Chemistry and Biomolecular Science, Gifu University, 1-1
Yanagido, Gifu, 501-1193, Japan
United Graduate School of Drug Discovery and Medical Information Science, Gifu University,
1-1 Yanagido, Gifu, 501-1194, Japan

Keywords: Supramolecular nanostructure, Stimuli-response, HydrogelSelf-assembly is utilized increasingly in many disciplines and is one of the few practical strategy forcreating nanostructure.
Supramolecular hydrogel is a macroscopic material consisting ofself-assembled nanostructures (3D network of mostly 1D nanofibers). The supramolecular hydrogel
has recently attracted much attention because of their mild fabrication process, biocompatibility, anddynamic functions such as stimuli-responsivenessTo install stimuli-responsive functions in the supramolecular nanostructures and hydrogels,modulation of noncovalent interactions among component self-assembling molecules has been widely
employed. By contrast, rearrangement of covalent bonds in the component molecules has not yet
been explored to a significant extent. We believe that incorporation of a chemical reaction site into
the component molecules may open up a new avenue for exploring unprecedented stimuli-responsive
supramolecular materials, which is impossible to realize only by modulation of noncovalentinteractions.
Here we present heat-responsive supramolecular hydrogels on the basis ofself-assembling molecules bearing a chemical reaction site.
We designed a bolaamphiphile as a self-assembling precursor molecule of a hydrogelator as shown in
Figure; the bolaamphiphile is constructed from a hydrogelator bearing a dienophile (maleimide) and awater-soluble diene (furan) via Diels-Alder (DA) reaction. We demonstrated that heating facilitatedmolecular conversion from the bolaamphiphile to the hydrogelator by means of a retro-DA reaction,
which triggered a drastic morphological transformation from 2D nanosheets to a 3D network of 1D
nanofibers, giving rise to a unique heat-set supramolecular hydrogel [1] . Heat stimuli required in thepresent system (60 ºC, 5 h) would be too harsh for bio-applications, whereas the present rational
molecular design could allow us to decrease or tune the heat stimuli by using the otherdiene-dienophile pair [2] and to extend to the other structural motif such as vesicles and nanotubes.

[1] Ikeda, M., Ochi, R., Kurita, Y.-s., Pochan, D. J., Hamachi, I., Chemistry –A European Journal,
215:403-410, 1990.
[2] Ochi, R., Nishida, T., Ikeda, M., Hamachi, I., to be submitted.

C-5-43
Towards an Automatic Recognition of DNA Nanostructureson AFM images
Yuexing Han 1

Akito Hara 1

Akinori Kuzuya 23

hanyuexing@gmail.com

hara.a.ac@m.titech.ac.jp

kuzuya@kansai-u.ac.jp

Ryosuke Watanabe 2

Yuuichi Ohya 2

Akihiko Konagaya 1

k947410@kansai-u.ac.jp

yohya@kansai-u.ac.jp

kona@dis.titech.ac.jp

1

Department of Computational Intelligence and Systems Science, Tokyo Institute ofTechnology, 4259-J3-25 Nagatsuta, Midori, Yokohama, 226-8502, Japan.
2

Department of Chemistry and Material Engineering, Kansai University,

3-3-35, Yamate, Suita, Osaka, 564-8680, Japan.
3

PRESTO, JST

Keywords: DNA nanostructure, AFM image recognition, curvature scale space method,
convexity-concavity detection, open robot technology middleware
With the technology of Atomic Force Microscope (AFM), we can directly observe DNA
nanostructures in high resolution. An AFM image of DNA nanostructures contains a variety ofshapes including single DNA nanostructures, overlapped DNA nanostructures and a lot of noise
shapes. Automatic recognition and classification of DNA nanostructures are strongly demanded toreduce human efforts in AFM image analysis.
In this research, automatic-recognition of single DNA nanostructures in AFM images based on theinformation of their outer contours has been proved. The information about the outer contour of a
DNA nanostructure includes not only peak points in the curvature scale space (CSS) images, butalso the convexity-concavity of the outer contour. Our prototype system of DNA nanostructure
recognition demonstrates that the CSS information is effective for the classification of open andclosed forms of DNA origami pliers [1,2].
The prototype system is developed on robot technology middleware (OpenRTM-aist) [3] tointegrate multiple components written in different languages running on multiple operating
systems such as linux and windows. The prototype system currently consists of two components
named “GetNanoDNASize” and “RecognizeNanoDNA”. The former component is a preprocessing
system for picking DNA nanostructures in AFM image focusing on the lengths and areas of DNA
nanostructures. The latter component classifies DNA nanostructures based on CSS andconvexity-concavity information as well as the lengths and the areas of DNA nanostructures.
[1] A. Kuzuya, Y. Sakai, T. Yamazaki, Y. Xu, and M. Komiyama. Nanomechanical DNA origami'single-molecule beacons' directly imaged by atomic force microscopy. Nature Communications,Vol. 2, DOI: 10.1038/ncomms1452, 2011.
[2] T. Yamazaki, Y. Aiba, K. Yasuda, Y. Sakai, Y. Yamanaka, A. Kuzuya, Y. Ohya, and M.
Komiyama. Clear-cut observation of PNA invasion using nanomechanical DNA origami devices.
Chemical Communications, Vol. 48, Pp. 11361-11363, 2012.
[3] http://www.openrtm.org/

C-5-44
Simple Local-information-based Self-optimizing
Algorithms in Grid Networks

1

Fukuhito Ooshita 1

Shusuke Takatsu 1

f-oosita@ist.osaka-u.ac.jp

s-takatu@ist.osaka-u.ac.jp

Hirotsugu Kakugawa 1

Toshimitsu Masuzawa 1

kakugawa@ist.osaka-u.ac.jp

masuzawa@ist.osaka-u.ac.jp

Graduate school of information science and technology, Osaka University
Yamadaoka 1-5, Suita, Osaka 565-0871, Japan

Keywords: Distributed algorithm, Self-optimization, Grid network, Shortest path construction,
BFS tree construction
Recently molecular robotics, which uses molecules as robot components, has received a lot of
attention as a new paradigm for artifacts [1]. To realize molecular robots, it is necessary toassemble a large number of molecular devices in a self-organizing manner. That is, each moleculardevice operates autonomously based on local information (obtained with their sensors), but themolecular devices as a whole should work cooperatively as a robot. This behavior is similar to thatof large-scale distributed systems, where each computer operates based on local information but
the computers as a whole work cooperatively as a system.
For this reason, it is natural to apply distributed algorithms (i.e., algorithms for distributed
systems) to molecular robotics. Many distributed algorithms are proposed for many problems inliterature. However, since such distributed algorithms are designed for computers to achieve thehighest performance, the algorithms are complex and implementing them in molecular devices isdifficult. To apply distributed algorithms to molecular robotics, it is necessary to develop simple
distributed algorithms.
In this poster, we focus on grid networks, and introduce simple distributed algorithms for the
shortest path construction [2] and the BFS (bread-first-search) tree construction. The shortest pathis useful to transmit a signal from the source to the destination quickly, and the BFS tree is usefulto transmit a signal from the source to all devices quickly. The algorithm for the shortest path
construction (resp., the BFS tree construction) constructs the shortest path (resp., the BFS tree)
from any initial path (resp., tree) in a self-optimizing manner. Both algorithms are simple: thealgorithm for the shortest path construction uses only three rules and that for the BFS tree
construction uses only four rules. These rules locally update a path or a tree with preserving theconnectivity and can be executed asynchronously.
While the characteristics of the above distributed algorithms are suitable for molecular robots, it
still requires some researches to implement them in molecular robots because the capability ofcurrent molecular devices is limited. As future work, we intend establishing a theoretical model formolecular robotics and developing distributed algorithms on the model.
[1] Murata, S., Konagaya, A., Kobayashi, S., Saito, H., and Hagiya, M., Molecular robotics: A newparadigm for artifacts, New Generation Computing, 31, 27-45, 2013.
[2] Takatsu, S., Ooshita, F., Kakugawa, H., and Masuzawa, T., Zigzag: Local-information-based
self-optimizing routing in virtual grid networks, Proc. of the 33rd International Conference onDistributed Computing Systems, 2013.

C-5-45

A Study on pH-Responsive Photosensitizers for Potential
Applications in Molecular Robotics
Takuya Terai 1

Hiroki Ito 1

t-terai@mol.f.u-tokyo.ac.jp

6770313964@mail.ecc.u-tokyo.ac.jp

Tetsuo Nagano 2
tlong@mol.f.u-tokyo.ac.jp
1

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2
Open Innovation Center for Drug Discovery, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Keywords: Molecular robotics, Photosensitizer, Organic synthesis
Molecular robotics has emerged as a novel research area that aims to rationally design andimplement dynamic artificial systems consisting of self-assembled molecules [1]. In order torealize robot-like molecular systems which correctly respond to outer stimuli, several classes ofmolecular devices are necessary including sensors, actuators, and compartments. With respect tosensors, a number of fluorescent probes were developed in our laboratory in the last decade thatwere designed on the principle of photoinduced electron transfer (PeT) [2-3], Förster resonanceenergy transfer (FRET) [4], and so on. However, these fluorescent sensors are not suitable for use
in molecular robotics because it is difficult to convert their output signal (i.e. fluorescenceemission) to the next chemical reaction. Hence, we are focusing on the development of a novel
sensor system that is based on activatable photosensitizers [5].
As a proof of principle, we herein designed and synthesized a novel pH-responsive
photosensitizer using 2I-BODIPY [6]. In this presentation, we will present the photophysicalproperties of the compound and discuss future directions.
[1] Murata, S., Konagaya, A., Kobayashi, S., Saito, H., and Hagiya, M., Molecular Robotics: A
New Paradigm for Artifacts, New Generation Computing, 31:27-45, 2013.
[2] Terai, T., Urano, Y., Izumi, S., Kojima, H., and Nagano, T., A practical strategy to createnear-infrared luminescent probes: conversion from fluorescein-based sensors, Chem. Commun.,
48:2840-2842, 2012.
[3] Terai, T., Kikuchi, K., Urano, Y., Kojima, H., and Nagano, T., A long-lived luminescent probe
to sensitively detect arylamine N-acetyltransferase (NAT) activity of cells, Chem. Commun.,
48:2234-2236, 2012.
[4] Myochin, T., Hanaoka, K., Komatsu, T., Terai, T., and Nagano, T., Design Strategy for a
Near-Infrared Fluorescence Probe for Matrix Metalloproteinase Utilizing Highly Cell
Permeable Boron Dipyrromethene, J. Am. Chem. Soc., 134:13730-13737, 2012.
[5] Yogo, T., Urano, Y., Kamiya, M., Sano, K., and Nagano, T., Development of enzyme-activatedphotosensitizer based on intramolecular electron transfer, Bioorg. Med. Chem. Lett.,
20:4320-4323, 2010.
[6] Yogo, T., Urano, Y., Ishitsuka, Y., Maniwa, F., and Nagano, Y., Highly Efficient andPhotostable Photosensitizer Based on BODIPY Chromophore, J. Am. Chem. Soc.,
127:12162-12163, 2005.

C-5-46
Acceleration of DNA strand exchange reaction by cationic
comb-type copolymers
Naohiko Shimada 1

Wei Song 2

nshimada@bio.titech.ac.jp

songwei@ms.ifoc.kyushu-u.ac.jp

Atsushi Maruyama 1
amaruyama@bio.titech.ac.jp

1

School and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa Pref. 226-8501, Japan2
Departments in Graduate School of Engineering, Kyushu University,744, Motooka, Nishi-ku,
Fukuoka, 819-0395, Japan

Keywords: molecular robotics, cationic polymers, DNA strand exchange reaction
DNA strand exchange reaction (SER) between double-stranded DNA(dsDNA) and its
complementary single-stranded DNA(ssDNA) is a key reaction in biotechnological applications(1)There are many reports describing DNA nanomachines triggered by the SER. Highly programed
SER has been exploited to build DNA logic gate (2). In these application, the dsDNAs generally
have sticky end as known as toehold in order to control rate of the SER because of very slow rateof SER between dsDNA having blunt end and ssDNA. In nature, recombination proteins promote
the SER even in DNA having blunt end. However, the applications of the proteins to DNA
biotechnology based on the SER are limited by the instability and resource ineffectiveness of theproteins. An artificial agent showing the activity of the proteins has been desired for theapplication of the DNA biotechnology including molecular robotics.
We have previously demonstrated that cationic comb type copolymers consisting of polycation
backbone and abundant hydrophilic graft chain influence kinetics and thermodynamics of nucleic
acid hybrid under physiologically relevant condition. For example, poly( L -lysine)-graft-dextran(PLL-g-Dex) copolymer significantly accelerates DNA hybridization over 200-folds(3).
Moreover, the copolymer markedly accelerated the SER by 4-5 orders of magnitude (4), whilestabilizing dsDNA DNA (5). However, the stabilization of dsDNA is considered to be principally
unfavorable for acceleration of the SER, because partial dissociation of dsDNA is required for an
initial step of SER. In this study, we focused on urea which is a well-known destabilizing agent
for dsDNA, we prepared cationic comb type copolymers partially modified with urea (ureido)groups, poly(allylamine-co-allylurea)-graft-dextran. The copolymers showed higher acceleration
effect of SER than unmodified copolymer.
[1]Zhang, D.Y. and Seelig, G. (2011) Dynamic DNA nanotechnology using strand-displacement
reactions. Nat Chem, 3, 103-113.
[2]Qian, L. and Winfree, E. (2011) Scaling Up Digital Circuit Computation with DNA Strand
Displacement Cascades. Science, 332, 1196-1201.
[3]Wu, L., Shimada, N., Kano, A. and Maruyama, A. (2008) Poly(l-lysine)-graft-dextran
copolymer accelerates DNA hybridization by two orders. Soft Matter., 4, 744-747.
[4]Kim, W.J., Akaike, T. and Maruyama, A. (2002) DNA Strand Exchange Stimulated by
Spontaneous Complex Formation with Cationic Comb-Type Copolymer. Journal of the American
Chemical Society, 124, 12676-12677.
[5]Maruyama, A., Katoh, M., Ishihara, T. and Akaike, T. (1997) Comb-Type Polycations
Effectively Stabilize DNA Triplex. Bioconjugate Chemistry, 8, 3-6.

C-5-47

A study on controller structure of biochemical reaction
networks
Takashi Nakakuki 1 	  	  	  Jun-ichi Imura 2nakakuki@ces.kyutech.ac.jp
12

imura@mei.titech.ac.jp

Department of Systems Design and Informatics, Faculty of Computer Science and Systems
Engineering, Kyushu Institute of Technology, 680-4, Kawazu, Iizuka, Fukuoka, 820-8502,
Japan
Department of Mechanical and Environmental Informatics, Graduate School of Information
Science and Engineering, Tokyo Institute of Technology, 2-12-1-W8-1, O-Okayama,
Meguro-ku, Tokyo, 152-8552, Japan

Keywords: controller structure, frequency response analysis, biochemical reaction networks
Many mathematical models of intracellular signal transduction systems have been developed
toward model-based analysis [1]. However, since the models are typically complex and nonlinear,
it is still a difficult task to analyze them [2]. In this poster, we address a fundamental study onwhat characteristics the biochemical reaction networks have if the system is considered to be acontroller.

[1] Nakakuki, T., et al., Ligand-specific c-Fos expression emerges from the spatiotemporal controlof ErbB network dynamics, 141, 5, 884-896, CELL, 2010.
[2] Nakakuki, T. et al., Methods for the Analysis of Intracellular Signal Transduction Systems (In J.
Wu, Ed., Biomedical Engineering and Cognitive Neuroscience for Healthcare: Interdisciplinary
Applications), Medical Infor-mation Science Reference (an imprint of IGI Global), 2012.

C-5-48
Toward Spatial and Temporal Gel-Sol Transition of
Hydrogel Driven by DNA Hybridization Reaction
Ibuki Kawamata 1,2
ibuki@is.s.u-tokyo.ac.jp

Satoshi Murata 1
murata@molbot.mech.tohoku.ac.jp

Masami Hagiya 2
hagiya@is.s.u-tokyo.ac.jp

1
2

Tohoku University, 6-6-01, Aoba Aramaki-Aza, Aoba-ku, Sendai, Miyagi, JapanThe University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan

Keywords: Molecular robotics, DNA hydrogel, Polyacrylamide gel, DNA computing,
One of the goals of molecular robotics is synthesizing a hydrogel whose state transition can be driven
by DNA devices [1]. One possible demonstration of the hydrogel is a system where gel state gradually
propagates in one direction. For example, gel is gradually formed in a capillary from one end, wheresome signal is put, to the other end. We propose a candidate implementation of such hydrogel anddiscuss preliminary experimental results.
Gel-sol transition of hydrogel has potential applications such as drug release, chemical sensor, andmicro fluidics. Recently, various gel-sol transitions of DNA-functional hydrogel have been
demonstrated, which make use of DNA hybridization reactions [2]. One of the hydrogels is made ofpolyacrylamide that is cross-linked by complementary DNA strands, which can solate by adding an
additional DNA strand that displaces the linker DNA [3]. Synthesizing this kind of DNA gel is very
costly because high concentration of DNA is required for gelation. However, another hydrogel fully
made of DNA using enzymatic reaction does not require high concentration of DNA because
polymerization begins with small concentration of primer DNA [4].
We show the spatial and temporal solation of DNA cross-linked hydrogel, which is achieved by the
diffusion of displacement strand. To propagate a gel state in a sol solution, we need to implement amechanism that only the sol near the boundary of a gel turns into a new gel. Our idea to achieve suchmechanism is releasing the primer of polymerization by the product of the polymerization. Morecomplex control of gel-sol transition may be possible in the future by combining with DNA statetransition devices such as logic gates.
[1] Murata, S., Konagaya, A., Kobayashi, S., Saito, H., and Hagiya, M., Molecular Robotics: A New
Paradigm for Artifacts, New Generation Computing, 31:27-45, 2013.
[2] Liu, J., Oligonucleotide-functionalized hydrogels as stimuli responsive materials and biosensors,
Soft Matter, 7:6757-6767, 2011.
[3] Lin, D. C., Yurke, B., Langrana, N. A., Mechanical Properties of a Reversible, DNA-Crosslinked
Polyacrylamide Hydrogel, Journal of Biomechanical Engineering, 126:104--110, 2004
[4] Lee, J. B., Peng, S., Yang, D., Roh, Y. H., Funabashi, H., Park, N., Rice, E. J., Chen, L., Long, R.
Wu, M., Luo, D., A mechanical metamaterial made from a DNA hydrogel, Nature Nanotechnology,
7:816--820, 2012

C-5-49

Science Communication in DNA nano-engineering
Satoru Yoshizawa 1
yoshizawa@molbot.mech.tohoku.ac.jp

	
  	
 

Kei Fujiwara 1
fujiwara@molbot.mech.tohoku.ac.jp

Shin-ichiro M. Nomura 1

Satoshi Murata 1

nomura@molbot.mech.tohoku.ac.jp

murata@molbot.mech.tohoku.ac.jp

1

Graduate school of Engineering, Tohoku University

Keywords: Molecular Robotics, DNA nano-engineering, learning process, science communication
DNA nano-engineering is a new field of technology that gives the foundation of
Molecular Robotics. Most of people know the word “DNA”, however, for them DNA is regarded asa molecule that contains genetic information. In order to understand the concept of DNA
nano-engineering, structural and functional aspects of DNA as a nano-material is crucially
important. Understanding properties of DNA is usually difficult for layman because it requires
many background knowledge in biology, chemistry and physics. The purpose of this research is todevelop an aid that helps general people understand the properties of DNA. We need to make theunderstanding process as interesting as we can, to let learners self-motivated. As a consequence, itwill increase the number of people interested in DNA nano-engineering.
As a first step of this attempt, we have made a movie to explain basic principles of DNA
nano-engineering. We also have conducted experiments with visitors who came to our open
laboratory. By using questionnaire we measured the level of understanding and interest. Someanalysis of the result will be reported in the poster. As the next step, we adopt gamification method[1] to improve the effectiveness of the learning process.
[1] Jane MacGonical, “Reality Is Broken : Why Games Make Us Better and How They Can Change
the World”, Penguin Books (2011)

C-5-50
Spatio-temporal change of BZ gel

1
2

Hiroyuki Mayama 1

Yusuke Hara 2

mayama@asahikawa-med.ac.jp

y-hara@aist.go.jp

Department of Chemistry, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi,
Aasahikawa, Hokkaido 078-8510, Japan

Nanosystem Research Institute (NRI), National Institute of Advanced Industrial Scienceand Technology (AIST), Central 5-2, 1-1-1 Higashi, Tsukuba 305-8565, Japan

Keywords: Volume phase-transition of gel, the Belousov-Zhabotinsky reaction, Non-equilibrium
conditions
The Belovsov-Zhabotinsky reaction is well-known as one of the oscillatory chemical reactions tounderstand rhythmic phenomena under non-equilibrium conditions including living matter andliving cell. On the other hand, living body is made from various biopolymers. In particular, motor
proteins (myosin, kinesin) and their scaffolds (actin, microtubule) generate motion under thehydrolysis of adenosine triphosphate (ATP), a kind of non-equilibrium conditions. Here, we
present a theoretical scenario for the BZ gel, which is a gel containing the BZ reaction field, basedon free-energy arguments of volume-phase transition of cross-linked polymer gel and theOregonator (2 parameter version), a simple model of the BZ reaction.
The essence is that polymer chain elongates in good solvent, while it collapses in poor solvent,where the BZ reaction switches the solvent quality (good or poor solvents) spontaneously.
Addition to this, we roughly assume that the volume of polymer gel reflect the size of a sub-chain,
which is the short polymer chain between cross-linking points. Free energy of the gel, F gel , can be
described as [1]1 / 2 2(1)
Fgel ~ (3 2 N sub ) 2   2   C *  6 / N sub  ln1   /( N sub )
where N sub is the length of a sub-chain,  is the normalized size of a polymer chain (  ~ 0; collapse,
 ~1; elongate), -C * is the solvent quality (smaller: good solvent, larger: poo solvent), the aspectratio of a sub-chain  . The BZ reaction changes - C * , which is obtained from the Oregonator. The
Oregonator is(2)(3)
where x, y, z are the concentrations of chemical species, and 1 - z = - C * although we skip the
explanation of the parameters. From the calculations of the Oregonator, we obtain oscillating - C * .
We thus succeed to describe the oscillatory volume change of the BZ gel. Based on this scenario,
we succeeded to explain other experimental results of polymer system under the BZ reaction.
[1] Mayama, H., Nakai, T., Takushi, E., Tsujii, K., and Yoshikawa, K., Marked differences involume phase-transitions between gel and single molecule in DNA, J. Chem. Phys., 127:034901,
2007.

C-5-51

Construction of Giant Vesicle Containing Microspheres
at High Volume Fraction and Its Transformation

1

2

Yuno Natsume 1

Taro Toyota 2

natsumey@fc.jwu.ac.jp

cttoyota@mail.ecc.u-tokyo.ac.jp

Faculty of Science, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo
112-8681, Japan
Department of Basic Science, Graduate School of Arts and Sciences, The Universityof Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan

Keywords: Giant Vesicle, Microspheres, Phospholipid, Fluorescence microscopy
Giant vesicles (GVs), which are closed lipid bilayer compartments with cellular size, have muchdrawn attention as a container of cell model. In the cytoplasm, biological macromolecules occupy
a sizeable fraction of the total volume of the media (typically 20–30 vol%), and the effects of this“crowded” state are important for their functions. 1 Crowding significantly influences two
physicochemical features of the macromolecules: (i) thermodynamics stabilization to increases thefree volume of the macromolecules, and (ii) changes in the mobility of the macromolecules. For
the purpose of quantitatively understanding these features, GVs containing microspheres serve as
valuable experimental model systems. 2
Here our purpose is the preparation of GV containing microspheres at a variety of volume
fractions and the clarification of the crowding effect for the GV transformation. Because thewater-in-oil emulsion centrifugation method 4 is fast and easy for encapsulation of soft matterparticles inside of GVs, we utilized it herein to encapsulate 1-m microspheres with volume
fractions in the range of 0–45 vol%. 5 GVs containing polymers or colloidal particles exhibit aunique transformation from an oblate (or prolate) spheroid to linked spheres after external stimuli
such as osmolality change or electrical stimulation. 3,6 These findings are thought to be typicalexamples of the crowding effect for GVs concerning about the thermodynamics of the membranebending energy and the entropic action of encapsulated particles. Here we found that GVs
encapsulating 1-m microspheres with a specific volume fraction transform from sphere topolygonal structure transiently after osmolality change. The fluorescence microscopy observation
on GV membrane proved that this transient state associated with the tubular membrane protrusion
apart from the GV. As far as we know, the polygonal structure of GVs is a characteristic shape only
if GV contains polymerizable proteins such as actin or microtubules. 7 Although the mechanism of
the transient polygonal structure of the GV is still veiled, the current finding can provide us a clue
to clarify the crowding effect for the GV in a non-equilibrium state.
[1] Minton, A. P. and Ellis, R. J., Nature, 425: 27-28, 2003.
[2] Dinsmore, A. D., Wong, D. T., Nelson, P., and Yodh, A. G., Physical Review Letters, 80:
409-412, 1998.
[3] Natsume, Y., Pravaz, O., Yoshida, H., and Imai, M., Soft Matter, 6: 5359-5366, 2010.
[4] Pautot, S., Frisken, B. J., and Weitz, D.A., Langmuir, 19: 2870-2879, 2003.
[5] Natsume, Y., and Toyota, T., Chemistry Letters, 42: 295-297, 2013.
[6] Terasawa, H., Nishimura, K., Suzuki, H., Matsuura, T., and Yomo, T., Proceedings of the
National Academy of Sciences of the U. S. A., 109: 5942-5947, 2012.
[7] Miyata, H., and Hotani, H., Proceedings of the National Academy of Sciences of the U. S. A.,
89: 11547-11551, 1992.

C-5-52

Reversible Gel-Sol Transition of DNA Gel
Daisuke Kandatsu1

Shin-ichiro M. Nomura 1

kandatsu@molbot.mech.tohoku.ac.jp

nomura@molbot.mech.tohoku.ac.jp

Satoshi Murata 1
murata@molbot.mech.tohoku.ac.jp

1

Graduate School of Engineering, Tohoku University,
6-6-01 Aobayama, Sendai, 980-8579 Japan

Keywords: Molecular Robotics, DNA Gel, Photo-cross linker
DNA gel have been attracted much attention for its programmable functionality. We propose a
DNA gel made of DNA motifs with designed base sequences. The motif is a cross shaped DNA
junction with a self-complementary sticky end and a photo-linking artificial base at each arm.
Under controlled temperature with UV irradiation, we have been succeeded in making thesol-gel state transition in repetitive manner. The physical properties of DNA gel such as theswelling degree and diffusion coefficient is measured in both sol or gel state of the DNA. Thisdata will be used to design slime-type molecular robot.
[1] Soong Ho Um, Jong Bum Lee, Nokyoung Park, Sang Yeon Kwon, Christopher C. Umbachand Dan Luo, Enzyme-catalysed assembly of DNA hydrogel . Nature Materials 5, 797-801(2006)[2] Yongzheng Xing, Enjun Cheng, Yang Yang,Ping Chen, Tao Zhang,Yawei Sun, Zhongqiang
Yang, Dongsheng Liu, Self-Assembled DNA Hydrogels with Designable Thermal andEnzymatic Responsiveness. Advanced Materials 23, 1117–1121 (2011)[3] Yoshinaga Yoshimura and Kenzo Fujimoto, Ultrafast Reversible Photo-Cross-Linking
Reaction: Toward in Situ DNA Manipulation. Org. Lett., 10 (15), 3227–3230 (2008)[4] Satoshi Murata, Akihiko Konagaya, Satoshi Kobayashi, Hirohide Saito, Masami Hagiya,
Molecular Robotics: A New Pardigm for Artifacts, New Generation Computing, 31, 27-45(2013)

C-5-53
Smart Lenses Created withTransparent Shape Memory Gels
Jin Gong 1

Takahiro Hazama 2

Masanori Arai 2

jingong@yz.yamagata-u.ac.jp

hzmtaka@gmail.com

taylor.made1golf@gmail.com

Shota Murata 2

Yosuke Watanabe 1

Masato Makino 1

niwasann0420@gmail.com

yosuke.watanabe.y.watana@gmail.com

mstmkn@gmail.com

M. Hasnat Kabir 1

Hidemitsu Furukawa 1

hasnatkabir@yz.yamagata-u.ac.jp

furukawa@yz.yamagata-u.ac.jp

1,2Soft &Wet matter Engineering Laboratory (SWEL), Department of Mechanical Systems Engineering,
Graduate School of Science and Engineering, Yamagata University,
4-3-16 Jonan, Yonezawa, Yamagata, Japan, 992-8510

Keywords: Gel, Shape memory, Transparent, Lens, Medical, Health and care
Gel is a dispersion system consisting of a solid three-dimensional network as the continuous
phase and a liquid (dispersing medium) within a solid as the discontinuous phase. Gel exhibits noflow and behave like solids when in the steady state due to the three-dimensional cross-linked
internal network structure, which may result from chemical bonds (chemical gels) or physicalbonds (physical gels), as well as crystallites or other junctions. Virtually any fluid can be used as
an extender including water (hydrogels), organic solvent (organogel), and air (aerogel). Since 2001,
high-strength gels like topological gel,nanocomposite hydrogel, tetra-PEG gel,double-network gel were developed [1-3]. In
our group, one novel hydrogel [4,5] isdeveloped. The hydrogel has the property ofshape memory, and is great transparent andflexible (Fig. 1). We named it TF-SMG. In thispaper, we report the development of smart
lenses (Fig. 2) with this excellent gel materialof TF-SMG.
Fig.1 TF-SMG
Fig. 2 Smart lens
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas"Molecular Robotics"(No. 24104005) of The Ministry of Education, Culture, Sports, Science, andTechnology, Japan.
This study was also partly supported by the following funding agencies: The New Energy andIndustrial Technology Development Organization (NEDO) of Japan, (Project ID: 09A25003a),
Japan Society for the Promotion of Science (JSPS) (Project No.: 22350097, etc.), Green Network
of Excellence (GRENE) (Green TriboNet), A-STEP (AS2421731K), Grant program from theYonezawa city in 2012, the start-up grant from Yamagata University in 2012, and Grant-in-Aid forYoung Scientists (B) (25810123).
[1] Okumura, Y., and Ito, K., Advanced Materials, 13(7): 485-487, 2001.
[2] Haraguchi, K., and Takeshita, T., Advanced Materials, 14(16): 1120-1124, 2002.
[3] Gong, J. P., Katsuyama, Y., Kurokawa, T., Osada, Y., Advanced Materials, 15(14): 1155-1158, 2003.
[4] Amano, Y., Hidema, R., Gong, J., Furukawa, H., Chemisty Letter, 41: 1029-1031, 2012.
[5] Harada, S., Hidema, R., Gong, J., Furukawa, H., Chemisty Letter, 41: 1047-1049, 2012.

C-5-54
Chain-Reactive Molecular Releasing System
BIOMOD2013 Team Sendai 1

biomod2013@molbot.mech.tohoku.ac.jp

Kei Fujiwara 2
fujiwara@molbot.mech.tohoku.ac.jp

Shinichiro M. Nomura 2

nomura@molbot.mech.tohoku.ac.jp

Satoshi Murata 2

murata@molbot.mech.tohoku.ac.jp

1
2

BIOMOD2013 Team Sendai, Tohoku University, 6-6-1 Aobayama, Sendai, Japan
Graduate School of Engineering, Tohoku University, 6-6-1 Aobayama, Sendai, Japan

Keywords: International Biomolecular Design Competition (BIOMOD), Molecular releasingsystem, liposome, DNA nanostructure
BIOMOD is a design competition where undergraduate students compete to master controlof biomolecules on the nanometer scale [1]. Students execute projects during the summer
and then gather in Boston in November to present their work and win awards.
This year, Team Sendai [2] aims at creating a novel chain-reactive molecular releasingsystem by using liposome as a container. In the proposed system, chain reactive collapse of
liposomes will be triggered by absorption of DNA nanostructure onto the surface ofliposome. By designing appropriate DNA-liposome interaction mechanism, we try to realize
quantitative control of drug release over time.

[1] BIOMOD2013 Website
http://biomod.net/[2] BIOMOD2013 Team Sendai Wiki
http://openwetware.org/wiki/Biomod/2013/Sendai

C-5-55

Dynamic biomolecular computing system for artificial
genetic network
Takashi Nukada

Koh-ichiroh Shohda

nukada @genta.c.u-tokyo.ac.jp

shohda@genta.c.u-tokyo.ac.jp

Akira Suyama
suyama@dna.c.u-tokyo.ac.jp

Department of Life Sciences and Institute of Physics, University of Tokyo, 3-8-1
Komaba, Meguro-ku, Tokyo 153-8902, Japan

Keywords: DNA computing, Logic gate, Genetic network
RTRACS (Reverse-transcription and TRanscription-based Autonomous Computing System) is a
modular biomolecular computing system composed of DNA, RNA and enzymes, whose mechanism
is based on retroviral replication [1, 2]. The modularity of RTRACS allows various simple modules
to be combined together by using output RNA of a module as input for successive modu les tocreate systems capable of sophisticated computational operations. When the output RNA of themodule is assigned to mRNA, RTRACS is available for artificial genetic network with cell-freeprotein synthesis.
RTRACS can basically perform computation to process time-dependent input data by employing
modules capable of dynamically changing output RNA depending on input RNA. However, logic
gate modules previously developed [2, 3] do not operate dynamically because output RNA
production continues indefinitely once the logical operation is complete. Here, we report a new
AND gate module that can operate dynamically in response to input RNA data. The dynamic
operation was achieved by two additional degradation reactions, one for the newly synthesized
DNA strands and the other for the remaining input and output RNA strands. These degradation
reactions return the AND gate module to the original state when the input RNA strands are nolonger present. Since the degradation reactions are effective for all kinds of RTRACS modules, thepresent method can be applied to other RTRACS modules to make them succeed in operating
dynamically.
[1] N. Nitta and A. Suyama., Autonomous biomolecular computer modeled after retroviral
replication. Lect.Notes Comput. Sc., 2943, 203-212 (2004).
[2] M. Takinoue, D. Kiga, K. Shohda, and A. Suyama., Experiments and simulation models of abasic computation element of an autonomous molecular computing system. Phys. Rev. E,
78:041921 (2008).
[3] A. Kan, K. Shohda, and A. Suyama., A DNA-based molecular logic gate capable of a broadclass of logical operations. Lect. Notes Comput. Sc., 7433, 86–97 (2012).

C-5-56
Towards Persistent Molecular Computers for
Molecular Robots
Masami Hagiya 1

Ibuki Kawamata 1

hagiya@is.s.u-tokyo.ac.jp

ibuki@is.s.u-tokyo.ac.jp

Nathanael Aubert 1
nathanael.aubert@gmail.com

1

Department of Computer Science, Graduate School of Information Science and Technology
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Keywords: Molecular computing, DNA computing, Molecular robotics
Although a large number of models for molecular computers have been proposed a nd many of them
have actually been implemented typically by using DNA molecules with or without modifications,most of those models are restricted in the sense that computational resources are consumed by each
instance of computation and cannot be reused i teratively. Models based on strand displacement ofDNA, such as that of seesaw gates by Qian et al. [1], are not exceptions . On the other hand, theidea of molecular robots is now commonly accepted [2], and computers for molecular robots are
expected to be reusable and sustainable. The model of dynamical systems by Rond elez et al. is oneof few such models [3]. We envision that molecular computers for molecular robots should havethe following properties. They 1) receive input from the environment, 2) adapt to the environment
by solving some optimization (or learning) problems, and finally 3) save the result of computation(solutions to the problems) for handling the next input from the environment.
In this ongoing and preliminary work, we take the k -SAT problem as an optimization problem
whose clauses model input from the environment. The molecular computer solves the problem, andre-solves it when its clauses are modified or new ones are added. As in the work by Sakamoto et al.
[4], each clause of an instance of the k-SAT problem is represented by a computational unit. While
one literal is chosen from a clause in advance in [4], a computational unit in this work takes one of
the literals in its corresponding clause as a state, and changes the state for sol ving or re-solving the
problem.
In this poster, we propose and compare three molecular implementation s of the above abstractscheme. 1) Implementation by a molecular complex whose conforma tional changes realize
progression of state transitions by alternating irradiation of two kinds of light [5]. 2)
Implementation by polymerase extension and digestion by nicking enzymes, which can beconsidered as an extension of DWPCR [6]. 3) Implementation by a dynamical system of Montagne
et al. [3].
[1] Qian et al.: Neural network computation with DNA strand displacement cascades, Nature
475:368, 2011.
[2] Molecular Robotics: http://www.molecular-robotics.org/en/[3] Montagne et al.: Programming an in vitro DNA oscillator using a molecular networking
strategy, Molecular Systems Biology 7:466, 2011.
[4] Sakamoto et al.: Molecular computation by DNA hairpin formation, Science 288:1223, 2000.
[5] Nishioka et al.: A photon-fueled DNA nanodevice that contains two different photoswitches,
Angewandte Chemie International Edition, 51:1165, 2012.
[6] Rose et al.: Displacement Whiplash PCR: Optimized Architecture and Experimental Validation,
Lecture Notes in Computer Science 4287:393, 2006.

C-5-57

A Computing Model for Biochemical Reactions
Fumiya Okubo 1
f.okubo@akane.waseda.jp

1

2

Takashi Yokomori 2	  	 yokomori@waseda.jp

Graduate School of Education, Waseda University,
1-6-1 Nishiwaseda, Shinjuku-ku, Tokyo 169-8050, Japan
Department of Mathematics, Faculty of Education and Integrated Arts and Sciences,
Waseda University, 1-6-1 Nishiwaseda, Shinjuku-ku, Tokyo 169-8050, Japan

Keywords: Models of biochemical reactions, Reaction automata, Turing computability
Recent developments on the theory of reaction automata ([4,5,6]) are surveyed. In these years,
Ehrenfeucht and Rozenberg have introduced a formal model, called reaction systems ([2]), forinvestigating interactions between biochemical reactions. Reaction systems provide a formal
framework suited for investigating at an abstract level the way of biochemical functioning.
Meanwhile, the notion of a multiset has frequently appeared and been investigated in many areasof computer science and related fields ([1,7]) because of its usefulness. In fact, identifying a
volume of compound chemicals as a multiset makes it possible that a multiset rewriting provides anatural way to express chemical reactions. Combining two notions of a reaction system and of amultiset rewriting, we have introduced reaction automata as computing devices for accepting string
languages ([4]), in order to model and analyze the behaviors of biochemical reactions in thecomputational framework.
The notion of reaction automata is an extension of reaction systems in which reaction rules aredefined by triples consisting of reactants, inhibitors, and products. A computation process of areaction automaton is performed in such a way that each time receiving one symbol from an input
string, a reaction automaton changes its memory (multiset) by applying reaction rules to themultiset. Since reaction automata are a computing model based on multiset rewriting that accepts
string languages, their modeling capability for chemical reactions can be investigated in terms ofthe theory of computation ([3]).
It is shown that reaction automata are computationally Turing universal. Further, Computational
complexity issues for some classes of reaction automata are explored in comparison to language
classes in the Chomsky hierarchy.
[1] C.Calude, Gh. Paun, G. Rozenberg and A. Salomaa (Eds.), Multiset Processing, LNCS 2235, Springer,
2001.
[2] A. Ehrenfeucht, G. Rozenberg, Reaction systems, Fundamenta Informaticae vol.75, pp.263-280, 2007.
[3] J.E. Hopcroft, T. Motwani, J.D. Ullman, Introduction to automata theory, language and computation 2nd ed, Addison-Wesley, 2003.
[4] F. Okubo, On the Computational Power of Reaction Automata Working in Sequential Manner, 4thWorkshop on Non-Classical Models for Automata and Applications, book@ocg.at series 290, pp.149-164,Österreichische Computer Gesellschaft, 2012.
[5] F. Okubo, S. Kobayashi, T. Yokomori, Reaction Automata, Theoretical Computer Science, 429,
pp.247-257, 2012.
[6] F. Okubo, S. Kobayashi, T. Yokomori, On the Properties of Language Classes Defined by Bounded
Reaction Automata, Theoretical Computer Science, 454, pp.206-221, 2012.
[7] Y. Suzuki, Y. Fujiwara, J. Takabayashi, H. Tanaka, Artificial Life Applications of a Class of P Systems,in [1], pp.299-346, 2001.

C-5-58i

Progress in the Enumeration Approach to Computing
Equilibrium of Interacting Nucleic Acid Strands
Satoshi Kobayashi 1
Kobayashi.satoshi@uec.ac.jp

1

Dept. of Communication Engineering and Informatics, Univ. of Electro-Communications,
1-5-1, Chofugaoka, Chofu, Tokyo 182-8585, JAPAN

Keywords: Nucleic Acid Strand Interaction, Equilibrium, Molecular Robotics, DNA Computing
DNA is one of the most promising material for constructing logical circuits, memory, intelligent
devices equipped in molecular robots([1]). The problem of designing nucleic acid strands to avoid
unwanted structures is difficult since they can form tremendously many number of structures andthus we need much of computation time for the analysis. Nucleic acid strand interaction analysis atequilibrium has been tackled by Dirks, et al.([2]),who have nicely extended Mackaskill's partition
function computation algorithm for a single RNA molecule ([3]) to the case of multiple strands,
and furthermore succeeded in computing the equilibrium state of interacting RNA molecules byusing convex programming after computing partition functions of all strand complexes. The author
introduced a new general approach to computing chemical equilibrium based on graph theory andoptimization theory([4][5]). Distinguished feature of the method was that it only used convex
programming but not dynamic programming. Essential point of the method based on the idea of
enumerating all the structures in a reaction system by using a graph. This method was applied also
to nucleic acid strands interaction, where we considered only linear secondary structures([6]). In
this poster presentation, we will present further progress of this approach to the analysis ofinteracting nucleic acid strands.
[1] S. Murata, A. Konagaya, S. Kobayashi, H. Saito, M. Hagiya, Molecular Robotics: A New
Paradigm for Artifacts, New Generation Computing, 31 (2013), 27-45, Ohmsha, Ltd. andSpringer.
[2] R.M. Dirks and J.S. Bois and J.M. Schaeffer and E. Winfree and N.A. Pierce, Thermodynamic
Analysis of Interacting Nucleic Acid Strands, SIAM Review, 49, 65-88, 2007.
[3] J.S. McCaskill, The Equilibrium Partition Function and Base Pair Binding Probabilities for
RNA Secondary Structure, Biopolymers, 29, 1105-1119, 1990.
[4] S. Kobayashi, A New Approach to Computing Equilibrium State of Combinatorial
Hybridization Reaction Systems, in Proceedings of Workshop on Computing andCommunications from Biological Systems: Theory and Applications, CD-ROM, paper2376,
2007.
[5] S. Kobayashi, Enumeration approach to computing chemical equilibria, Theoretical Computer
Science, 499, 51-88, 2013.
[6] Satoshi Kobayashi and Takaya Kawakami, Enumeration Approach to the Analysis ofInteracting Nucleic Acid Strands, in Biomolecular Information Processing --- From Logic
Systems to Smart Sensors and Actulators, Chap.12, pp.225-244, 2012.

C-5-59

Modeling and Simulation of self oscillating gel- toward a molecular gel robot

1

Hiroshi Morita 1

Yusuke Hara 1

h.morita@aist.go.jp

y-hara@aist.go.jp

Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology（AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8568, Japan

Keywords: dissipative particle dynamics method, OCTA, self-oscillating gel, meso-scale
simulation
The self-oscillating gel is one of the interesting applications of the gel with theswelling-shrinking dynamics. The self-oscillating gel is the gel which pulsates
spontaneously itself using the dynamics of BZ reaction inside the gel, and it is now
developing as materials for actuators or soft robots. To use self-oscillating gel as thesematerials, we need much precise design of functions and properties, and encountermuch more difficulties. For the purpose of the precise designing of the self-oscillatinggel, we make the model for self-oscillating gel, and performing these simulations.
In the dynamics of self-oscillating gel, swelling and shrinking dynamics occur
repeatedly, and in these dynamics the solvents and the polymers moves inside the gel.
To model the self-oscillating gel, we must treat both dynamics of solvents and polymers.
In this study, the model of self-oscillating gel based on dissipative particle dynamics(DPD) [1] method is constructed on OCTA system[2], which is the multi-scale
simulation system for soft materials in the meso-scale. The swelling-shrinking
dynamics was represented by changing the interaction parameter between polymer andsolvent particles. In the simulations, the frequency of the changing the interaction
parameter can be controlled, and with changing its frequency, the structure and the
dynamics of gel can be observed. In the slower frequency case, gel can take almost
equilibrium structure at each time and the change of size of gel in theswelling-shrinking cycle is much larger, however in the faster case, the movement ofsolvents and polymers cannot follows along the change of the interaction parameterand the delay of phase of the swelling-shrinking dynamics occurs. Noted that our model
follows the previous theoretical model of gel, stress-diffusion coupling model [3], which
can be applicable to the many kinds of dynamics of gel widely.
Our model is also applied to the peristaltic motion of gel. This is one of the models ofworm or soft molecular robot. Our simulations indicate that our worm-like gel moves
with pulsating. Our model indicates that our gel model is swimming in the solvent
using the power of the BZ reaction. Detail of our simulation results and some movies
will be shown in the session.
References[1] R.D. Groot and P.B. Warren, J. Chem. Phys. 107, 4423 (1997)[2] http://octa.jp[3] T.Yamaue, T. Taniguchi, M. Doi, AIP Conf., Proc. 519, 584 (2000)

C-5-60

Numerical studies of protein-induced shape changes ofliposomes
Tamiki Umeda
umeda@maritime.kobe-u.ac.jp

Graduate School of Maritime Sciences, Kobe University, 5-1-1, Fukae-Minami
Higashinada-ku, Kobe 658-0022 Japan

Keywords: Lipid bilayer membranes, cytoskeleton, numerical calculations, molecular robotics
Liposomes are closed vesicles formed by lipid bilayer membranes. They have been well studied
as simplified models of biological membranes, and are also considered as a possible component formolecular robots. When cytoskeletal proteins, such as actin and tubulin, are polymerized insideliposomes, elongating cytoskeletal filaments push the membrane and deform it. As a result,liposomes change their morphology into a lemon shape, flattened shape, spoon shape, or a shapehaving long membrane projections. Other proteins, such as talin, induce large holes in themembrane, and liposomes become cup-like shapes. We have analyzed these phenomena using amembrane elasticity model, and showed that the numerical calculations reproduce most of theseshape changes. Only a few piconewtons of cytoskeletal forces are needed for large membrane
deformations, but actual liposomal morphologies are determined also by the membrane properties,
arrangement of cytoskeletal filaments, and pressure difference between inside and outside of themembrane. We expect that the methods used in the calculations may also be useful for designing
the molecular robots.

C-5-61

Robustness Criteria of Hybridizationof double-stranded DNA Sequences
Hirotaka Ono
hirotaka@econ.kyushu-u.ac.jp

Faculty of Economics, Kyushu University, Japan
6-19-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Keywords: Electronic structure calculation, Ligand docking, Protein modeling
Predicting the behavior of DNA molecules in vitro is a fundamental issue on DNA computing andrelated areas, but is also known to be quite difficult. Computational simulation is one of the most
reasonable approaches for the prediction, which enables us to circumvent money/time-consuming
biochemical experiments, and several simulation models have been proposed indeed. However,simulation approaches are still time-consuming in some scenario; for example, in order to design
DNA sequences that hybridize in some preferable way, standard local search algorithms need torefer thousands of simulation results, which is actually impossible to complete it in reasonable
computational time. In this paper, we propose simple criteria that are useful to roughly predict the
behavior of DNA sequence hybridization. By using this criteria, we can determine whether a given
DNA sequence s and its complement sequence s’ form the perfect double stranded structures
quickly, or slowly. The proposed criteria do not require computational simulations and can beeasily computed. We conducted computational experiments that simulate hybridization of
double-stranded DNA sequences under a probabilistic simulation model proposed in [1], whose
validity is shown via comparison with biochemical experiments. The simulation results show thatthe proposed criteria are promising to predict the hybridization speed of double-stranded DNA
sequences.
[1] Masashi Shiozaki, Hirotaka Ono, Kunihiko Sadakane, Masafumi Yamashita: A Probabilistic
Model of the DNA Conformational Change. Proceedings of DNA 2006, Lecture Notes in
Computer Science, 4287 274-285, 2006.

C-5-62

Recapitulation of the hepatic function using
in vitro liver model from murine ES/iPS cells

1

Miho Tamai 1

Yoh-ichi Tagawa 1

tamai.m.aa@m.titech.ac.jp

ytagawa@bio.titech.ac.jp

Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology,
Tokyo Institute of Technology, B-51, 4259 Nagatsuta, Midori−ku, Yokohama, Kanagawa
226−8501, Japan

Keywords: Embryonic stem cell, Liver, Metabolism, Cell polarity, Urea cycle
Polygonal and multipolar hepatocytes in the liver are surrounded by sinusoids, bile canaliculus,
and adjacent hepatocytes. It is only in the context of hepatic tissue architecture that hepatocytes
can express their specific and multiple functions. Hepatocyte polarity exerts a major influence on
the physiology of the cell. Recently, we established a unique system of in vitro liver model derived
from murine ES/iPS cells, i.e., IVL mES/iPS [1, 2]. The IVL mES/iPS , consisting of not only hepatocytes,
but also endothelial networks, together with cardiac mesoderm differentiation, was induced after
the embryoid body formation.
To confirm cellular polarities of the IVL mES/iPS , first, dichlorofluorescein diacetate (CDFDA) was
added into the IVL mES/iPS . In liver, CDFDA is incorporated into hepatocytes via OATP2 which
expresses at apical side, and afterward CDFDA was hydrolyzed by cytoplasmic esterase to green
fluorescent CDF, and which is excreted to bile canaliculus via MRP2. CDF was observed to beaccumulated at the boundary of the cells in the IVL mES/iPS , but not in primary hepatocyte culture.
Second, we tried to activate urea cycle by addition of L-ornithine in the IVL mES/iPS or liver
perfusion system. Urea production increased and ammonia decreased in a dose-dependent mannerwith respect to the amount of L-ornithine both in the IVL mES/iPS and the liver perfusion system, but
not in primary hepatocyte culture [3].
Here, we demonstrated that architectural and functional properties in the IVL mES/iPS were quite
similar to those in the liver perfusion system, but different from those in the culture of primary
hepatocytes. The IVL mES/iPS has great promise to be useful for drug metabolism andpharmacokinetics in liver as an alternative to animal experiments.
[1] Ogawa, S., Tagawa, Y., Kamiyoshi, A., Suzuki, A., Nakayama, J., Hashikura, Y., Miyagawa, S.,
Crucial roles of mesodermal cell lineages in a murine embryonic stem cell-derived in vitro liverorganogenesis system, Stem Cells, 23:903-913, 2005.
[2] Tamai, M., Yamashita, A., Tagawa, Y., Mitochondrial development of the in vitro hepaticorganogenesis model with simultaneous cardiac mesoderm differentiation from murine induced
pluripotent stem cells, Journal of Bioscience and Bioengineering, 112:495-500, 2011.
[3] Tamai, M., Aoki, M., Nishimura, A., Morishita, K., Tagawa, Y., In vitro recapitulation of theurea cycle using murine embryonic stem cell-derived in vitro liver model, Amino Acids, in press.

C-5-63

A Design Method of Artificial Genetic Circuits on
Effective Search of These Logical Structure s
Manabu Sugii1 1

Adrien Fauré 2manabu@yamaguchi-u.ac.jp

afaure@yamaguchi-u.ac.jp

Hiroshi Matsuno3 2
matsuno@sci.yamaguchi-u.ac.jp

12Media and Information Technology Center, Yamaguchi University, 2 -16-1 Tokiwadai, Ube-shi,
Yamaguchi 755-8611, Japan
Graduate School of Science and Engineering, Yamaguchi University, 1677 -1 Yoshida,
Yamaguchi-shi, Yamaguchi 753-8512, Japan

Keywords: Synthetic biology, Artificial genetic circuit, Logical modeling, Dynamic modelingIn the context of synthetic biology, artificial genetic circuits are designed in the following way:after setting a biological target phenomenon to be investigated, reaction para meter estimations
among related molecules are conducted based on the dynamic analyses with mathematical models,
finally, a system of biological reactions is developed with these molecules in vivo or in vitro. It is
desired to develop an effective method to select the suitable circuits for realizing the target
phenomenon, because not a few models are possible as candidates for the target phenomenon.
We propose a new procedure to effectively design a mathematical model by the following two
steps. The first step is the process creating possible structures of the mathematical by a logical
technique. The second step is the process creating dynamic models in two ways; a system ofdifferential equations for the analysis of dynamics in the model and a model of hybrid functional
Petri net as a common platform for knowledge sharing between biologists and computer scientists.

[1] Gardner, T.S., Cantor, C.R., Collins, J.J., Construction of genetic toggle switch in Escherichia
coli, Nature 403:339-342, 2000.
[2] Fauré, A., Thieffry, D., Logical modeling of cell cycle control in eukaryotes: a comparative
study, Molecular Biosystems, 5:1769-81, 2009.
[3] Cell Illustrator: http://www.cellillustrator.com/homeC-5-64

Identification of Key Regulators in Glycogen Utilization in
E. coli Based on the Simulations from a Hybrid Functional
Petri Net Model
Zhongyuan Tian 1

Adrien Fauré 1

Hirotada Mori 2

Hiroshi Matsuno 1
hmatsuno@yamaguchi-u.ac.jp

12

Graduate School of Science and Engineering, Yamaguchi University, 1677 -1 Yoshida,
Yamaguchi-shi, Yamaguchi, 753-8512, Japan
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916 -5
Takayama, Ikoma, Nara 630-0101, Japan

Keywords: metabolic pathway; glycogen; hybrid functional Petri net; PTS; HPr and EIIA Glc
proteins; gene regulation
Background: Glycogen and glucose are two sugar sources available during the lag phase of E. coli,
but the mechanism that regulates their utilizatio n is still unclear.
Methods: Attempting to unveil the relationship between glucose and glycogen, we propose an
integrated hybrid functional Petri net (HFPN) model including glycolysis, PTS, glycogenmetabolic pathway, and their internal regulatory systems .
Results and Conclusions: By comparing known biological results to this model, basic necessary
regulatory mechanism for utilizing glucose and glycogen were identified as a feedback circuit inwhich HPr and EIIAGlc play key roles. Based on this regulatory HFPN model, we discuss theprocess of glycogen utilization in E. coli in the context of a systematic underst anding ofcarbohydrate metabolism.