P1-01
Theoretical calculations on proteins withfragment molecular orbital method
Yuji Mochizuki 1,2 Kaori Fukuzawa 2,3 Yoshio Okiyama 2,4
fullmoon@rikkyo.ac.jp fukuzawa.kaori@nihon-u.ac.jp okiyama.yoshio@riken.jp

Chiduru Watanabe 2,4 Teruki Honma 4 Shigenori Tanaka 5
Chiduru.watanabe@riken.jp honma@gsc.riken.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
School of Dentistry at Matsudo, Nihon University, 2-870-1 Sakaecho-Nishi, Matsudo,
Chiba, 271-8571, Japan.
RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku,
Yokohama, Kanagawa 230-0045, Japan.
Graduate School of System Informatics, Department of Computational Science, Kobe
University, 1-1 Rokkodai, Nada-ku, Kobe, Hyogo 657-8501, Japan

Keywords: FMO, parallel processing, K-computer, geometry optimization, electron density
The fragment molecular orbital (FMO) method has been one of the most successful techniques incalculating proteins in a fully quantum mechanical way based on efficient parallel computations [1].
The ABINIT-MP system is our original FMO program by which a wide variety of applications havebeen performed, to date [2]. In particular, the four-body corrected calculation (FMO4) [3] is available
only in ABINIT-MP, and the Cholesky decomposition (CD) for two-electron integrals [4] can be used
for acceleration. This poster will summarize the activities with ABINIT-MP including recent topics.
Recently, ABINIT-MP has become usable on the K-computer as a representative massively parallel
supercomputer. High execution efficiency with better than 10% was observed for real proteins with afew hundreds amino acid residues, through the DGEMM processing of CD-based MP2 and HF
calculations. It was found that an one-center restriction in CD (1C-CD) is effective in further reducing
the computational time. The use of cc-pVDZ basis has been made possible within acceptable cost.
Another new feature of ABINIT-MP has involved the MP2 geometry optimization [5] for crucial
ligand-binding regions of proteins. There are sizable differences in interaction energies between PDB
structures and MP2-optimized structures. Additionally, the electron densities can now be efficiently
generated at the MP2 level for the direct comparison with X-ray data.

[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] "Electron-correlated fragment-molecular-orbital calculations for biomolecular and nanosystems", S. Tanaka, Y. Mochizuki, Y. Komeiji, Y. Okiyama, K. Fukuzawa, Phys. Chem. Chem.
Phys., 16 (2014) 10310-10344.
[3] "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.
[4] "Acceleration of fragment molecular orbital calculations with Cholesky decomposition
approach", Y. Okiyama, T. Nakano, K. Yamashita, Y. Mochizuki, N. Taguchi, S. Tanaka, Chem.
Phys. Lett., 490 (2010) 84-89.
[5] "Partial geometry optimization with FMO-MP2 gradient: application to TrpCage", T.
Tsukamoto, Y. Mochizuki, N. Watanabe, K. Fukuzawa, T. Nakano, Chem. Phys. Lett., 535(2012) 157-162.

P1-02
FMO-based analysis on DsRed chromophore

1

2

3

4

Masataka Sakaguchi 1

Yuji Mochizuki 2,3

ms.sakaguchi@rikkyo.ac.jp

fullmoon@rikkyo.ac.jp

Chiduru Watanabe 3

Kaori Fukuzawa 3,4

chiduru.watanabe@riken.jp

fukuzwa.kaori@nihon-u.ac.jp

Laboratory of Mochizuki, Master’s Course, Chemistry Major, Graduate School of Science,
Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, 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
Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo
153-8505, Japan
School of Dentistry at Matsudo, Nihon University, 2-870-1 Sakaemachi-Nishi, Matsudo
271-8571, Japan.

Keywords: FMO, Fluorescence protein, Excitation energy, Protein modeling, Crystallization water
DsRed is a kind of red fluorescent protein (RFP), which was first isolated from Discosoma
coral；excitation energy and emission energy are 558 nm (2.22 eV) and 583 nm (2.13 eV),
respectively. [1].The crucial pigment of chromophore is the deprotonated p-hydroxybenzilideneimidazolinone moiety formed from three amino acid residues of Gln66-Tyr67-Gly68 [2]. There
were various theoretical studies for DsRed, so far, including ours [3,4]. However, the effects from bothwater molecules and configurations of neighboring residues in the chromophore have not yet been
examined enough. Therefore, we have performed a series of calculations based on the fragment
molecular orbital (FMO) method in these regards, as will be shown in the poster presentation.
As in Refs. [3,4], the “1ZGO” PDB structure of DsRed [5] was adopted in the present study, and
the ABINIT-MP program was used in all FMO calculations. It was found that the existence of two
water molecules with hydrogen-bonds to the pigment as well as the the conformation of Ser69 havecritical influences on the excitation energy in comparison with the experimental value. The energy
decomposition analysis [6] was performed to illuminate the importance of electrostatic interactionsin the choromophore.
[1] “Fluorescent proteins from nonbioluminescent Anthozoa species”, Matz M.V., Fradkov A.F.,
Labas Y.A. et al., Nature Biotech, 17 (1999) 969–973.
[2] “The structure of the chromophore within DsRed, a red fluorescent protein form coral”, Gross
L.A., Baird G.S., Hoffman R.C., Baldridge K.K., Tsien R.Y., Proc. Natl. Acad. Sci. USA, 97(2000) 11990-11995.
[3] "Fragment molecular orbital calculations on red fluorescent protein (DsRed)", Y. Mochizuki, T.
Nakano, S. Amari, T. Ishikawa, K. Tanaka, M. Sakurai, and S. Tanaka, Chem. Phys. Lett., 433(2007) 360-367.
[4] "Fragment molecular orbital calculations on red fluorescent proteins (DsRed and mFruits)", N.
Taguchi, Y. Mochizuki, T. Nakano, S. Amari, K. Fukuzawa, T. Ishikawa, M. Sakurai, and S.
Tanaka, J. Phys. Chem. B, 113 (2009) 1153-1161.
[5] “Crystallographic structures of Discosoma red fluorescent protein with immature and maturechromophores”, J. L. Tubbs, J. A. Tainer, and E. D. Getzoff., Biochem., 44 (2005) 9833-9840.
[6] “Pair Interaction Energy Decomposition Analysis”, D. G. Fedorov, K. Kitaura, J. Comp. Chem.
28 (2007) 222.

P1-03
Efficient Implementation of 3D-RISM Theory to the FMO
Method, and Its Applications
Norio Yoshida 1

noriwo@chem.kyushu-univ.jp

1

Department of Chemistry, Graduate University of Sciences, Kyushu University , 6-10-1
Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Keywords: FMO, 3D-RISM, Electronic structure calculation, Ligand docking
An efficient implementation of the three-dimensional reference interaction site model(3D-RISM) theory to the fragment molecular orbital (FMO) method was proposed. The method isreferred to as FMO/3D-RISM method. The method allows us to treat an electronic structure ofwhole part of macromolecules, such as protein, as well as a solvent distribution around the solute
macromolecules.
The electrostatic potential should be calculated on grid point in the three- dimensional real
space witch is used in 3D-RISM theory. Therefore, the reducing the computational cost to
calculate the electrostatic potential is most serious concern. In this article, we propose a procedure
to save the computational cost for calculating the electrostatic potential in the framework of FMO
method. The strategy of this procedure is to evaluate the electrostatic potential and the solvated
Fock matrix in different ways, depending on the distance between solute and solvent. In thevicinity of solute molecule, the electrostatic potential is evaluated directly by integrating themolecular orbitals of monomer fragments of solute molecule, whereas that is described as thesuperposition of multipole interactions by using Taylor expansion of the electronic distribution ofmonomer fragment.
The results are compared with those from the other methods. We apply the FMO/3D-RISM
method to investigate the selective binding of cellulose by cellulose binding module (CBM).
[1] Yoshida, N., Efficient implementation of the three-dimensional reference interaction site model
method in the fragment molecular orbital method, Journal of Chemical Physics,
140:210118(1-13), 2014.

P1-04
Fragment Based Drug Discovery (FBDD) vs Molecular
Evolution: Shared Strategy for the Induction of
Substrate/Target Selectivity
Osamu Ichihara 1
osamu.ichihara@schrodinger.com

	
  	
 

Shigeru Matsuoka 2
Daisuke Matsuoka 2

roy.kimura@schrodinger.com

Shigeru Sugiyama 2

matsuokas11@chem.sci.osaka-u.ac.jp

dmatuoka@chem.sci.osaka-u.ac.jp

S. Roy Kimura 1

	
  	
 

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

Michio Murata 2
murata@chem.sci.osaka-u.ac.jp

1

Schrödinger K. K., 17F Marunouchi Trust Tower North, 1-8-1 Marunouchi
Chiyoda-ku, Tokyo 100-0005, Japan
2
JST ERATO, Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama,
Toyonaka, Osaka 560-0043, Japan

Keywords: FBDD, FABP, Hydrophobic effect, Hydration Thermodynamics
Previously, we computationally investigate the effects of water molecules on the binding
energetics of over 20 fragment hits and their corresponding optimized lead compounds. [1] Theanalysis revealed that fragment hits tend to displace water molecules with notably unfavorable
excess entropies relative to those displaced by the newly added moieties of the lead compound
during the course of FBDD. The degree of difficulty in displacing hydrations sites by “design”
increases as the excess entropy of the hydration site increase. Thus, the water molecules in thebiding site can be classified into “difficult-to-replace” and “easy-to-replace” waters according totheir excess entropies. There is a clear trend that the “difficult” waters are displaced by thefragment hits and the leads eventually evolved from them by displacing the “easy” watermolecules.
Mitochondrial β-oxidation of long chain fatty acids (LCFAs) is a key metabolic pathway forenergy generation in the skeletal and heart muscle. Transporters and carrier proteins, including
fatty acid-binding proteins (FABPs), are required for the efficient cytosolic delivery of highlyhydrophobic LCFAs. However, it remains unclear how such proteins recognize LCFA’s flexible
alkyl chains of different length (c10 – c18) that do not exhibit defined structure or noticeable
electrostatic interactions. Recently, we suggested that the two clusters of water molecules found in
the FA binding pocket might play a crucial role in the recognition of substrate FAs by FABPs. [2]Our investigation of the thermodynamic properties of the two water clusters revealed their
distinctively different thermodynamic profiles. [2] Cluster 1 contains a well-defined hydrogenbonding network of well-ordered, “difficult”, water molecules with highly unfavorable excess
entropies. On the other hand, Cluster 2 consists of relatively disordered, “easy”, water molecules.
FABP’s selectivity towards the substrate FAs can be explained by considering the preferential
displacement of “easy” Cluster 2 water molecules. The entropy of the binding site water molecules
plays the key role in these two seemingly unrelated processes.
[1] Ichihara O., Shimada Y., Yoshidome D., The importance of hydration thermodynamics infragment-to-lead optimization, ChemMedChem, in press.
[2] Matsuoka, S., Sugiyama S., Matsuoka D., Hirose M., Sébastien L., Ano H., Hara T., Ichihara
O., Kimura S. R., Murakami S., Ishida H., Mizohata E., Inoue T., Murata M., Heart fatty
acid-binding protein performs a fuzzy search for “fuel” fatty acids, to be published.

P1-05Prediction of Protein-ligand Binding Affinities Using
Molecular Simulations (II)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 evaluated protein-ligand binding affinities
using 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.

P1-06
Toward precise determination of binding free energy
from molecular dynamics simulation
Teruhisa S. Komatsu 1

Noriaki Okimoto1

teruhisa.komatsu@riken.jp

okimoto@gsc.riken.jp

Makoto Taiji1

taiji@riken.jp

1

QBiC, RIKEN, 1-6-5 Minatojima-minami, Chuo-ku, Kobe, Hyogo 650-0047, Japan

Keywords: molecular dynamics simulation, protein-ligand binding affinity
In order to develop a new drug design or to improve the tentative drugs for the target receptor, theprecise estimation of the binding free energies for the various ligands is essential. Aiming at the application
to drug improvements, rather high precision ( around the order of thermal energy k BT=2.5kJ/mol ) for thebinding free energies should be established. Since atomistic (classical mechanical) forcefields optimized
for organic molecules have been developed[1-2], and methods to calculate binding free energies bymolecular dynamics simulations are proposed[3-7], we can smoothly set to work on the various casestudies. Unfortunately, however, it is not always easy to obtain successful results. Comparing thesimulated results with experimental one, we often disappointed the poor correlation among them. Thus it
seems that the blind use of the result from the molecular dynamics simulation is not yet guaranteed. Whatis a reason for such discrepancy between numerical and experimental results? Maybe there are many
possibilities, e. g. incorrectness of the force fields, incorrectness of classical mechanical description,
limited length of simulation time scale, and so on. Here we will focus on checking how precisely we cancalculate the free energies for a given force field. For this purpose, we will compare different protocols forthe calculation of the free energies. Such research could be a inevitable fundamental step before entering
many other issues.
[1] S. J. Weiner, P. A. Kollman, D. T. Nguyen, and D.A. Case, An All Atom Force Field for Simulations ofProteins and Nucleic Acids. J. Comp. Chem. 7, 230-252 (1986).
[2] J.W. Ponder and D.A. Case. Force fields for protein simulations. Adv. Prot. Chem. 66, 27-85 (2003).
[3] Y. Deng and B. Roux, Computations of Standard Binding Free Energies with Molecular Dynamics
Simulations, J. Phys. Chem. B 113, 2234–2246 (2009).
[4 ] J. Wang, Y. Deng, and B. Roux, Absolute Binding Free Energy Calculations Using Molecular
Dynamics Simulations with Restraining Potentials, Biophys. J. 91, 2798-2814 (2006).
[5] H. J. Woo and B. Roux, Calculation of absolute protein–ligand binding free energy from computer
simulations, PNAS 102, 6825-6830 (2005).
[6] D. L. Mobley, J. D. Chodera, and K. A. Dill, Confine-and-Release Method: Obtaining Correct Binding
Free Energies in the Presence of Protein Conformational Change, J. Chem. Theory Comput. 3,
1231-1235 (2007).
[7] H. Fujitani, Y. Tanida, and A. Matsuura, Massively parallel computation of absolute binding free energy
with well-equilibrated states, Phys. Rev. E 79, 021914:1-12 (2009).

P1-07
How does water in the binding site affect intermolecularinteraction between a protein and a ligand?
Hiroyuki Sato 1
shryk@jp.fujitsu.com

1

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

Fujitsu Laboratories Ltd., 10-1 Morinosato-Wakamiya, Atsugi 243-0197, Japan

Keywords: Intermolecular interaction, Binding affinity, Water, Polarization, FMO
We investigated the effects of water in the binding site on interaction energy between a protein
and a ligand from the viewpoint of enthalpic bridging.
We selected human coagulation factor Xa and some ligands (RRP, RTR, RDR, RRR) as the test
system of this study[1]. Three of these ligands (RRP, RTR, RDR) contain chlorobenzothiophene,
which is placed inside of the binding site, while RRR contains chlorothiophene at thecorresponding position. Firstly, we calculated interaction energy between each ligand and factor
Xa using Fragment Molecular Orbital (FMO) method[2]. Considering the polarization effectcaused by protein-ligand binding, the calculated interaction energy and the experimental binding
affinity show a correlation except for the ligand RRR. If the displacement of water in the bindingsite[3] explains the exception, entropically unfavorable water should exists in the vicinity ofchlorobenzothiophene; however, it does not exist in the vicinity of chlorobenzothiophene but
chlorothiophene[4], so the displacement of water does not explain the relatively high affinity of
RRR. Therefore, we focused on the role of the water to build a weak bridge between RRR andfactor Xa. We calculated interaction energy between RRR and the water in the vicinity ofchlorothiophene in addition to the one between RRR and factor Xa. The total interaction energy
and experimental binding affinity show an excellent correlation (R 2 = 0.95). This result indicatesthat the specific water molecules in the binding site have a role to enforce a protein–ligand binding
affinity by bridging the space between a ligand and a protein.
[1] Maignan, S., et al., V. J. Med. Chem. 2003, 46, 685-690.
[2] Kitaura, K., et al., M., Chem. Phys. Lett. 1999, 313, 701-706.
[3] Young, T., et al., Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 808-813.
[4] Abel, R., et al., J. Am. Chem. Soc. 2008, 130, 2817-2831.

P1-08Binding Mode Analysis of Protein Kinase CK2
Inhibitors with a Purine Scaffold
Atsushi Sakurai 1
1333420019n@kindai.ac.jp

Shinya Nakamura 1

nakas@phar.kindai.ac.jp

Seiya Taniguchi 11111610161z@kindai.ac.jp

Naoya Nagata 2

Masakuni Kurono 2

Katsumi Murata 2

nagata@pharm.kyoto-u.ac.jp

kurono@pharm.kyoto-u.ac.jp

murata@pharm.kyoto-u.ac.jp

Kazuo Kitaura 2

Akira Hirasawa 2

Gozoh Tsujimoto 2

kkitaura@pharm.kyoto-u.ac.jp

akira_h@pharm.kyoto-u.ac.jp

gtsuji@pharm.kyoto-u.ac.jp

Isao Nakanishi 1,2
isayan@phar.kindai.ac.jp
12Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-osaka 557-8502, Japan.
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Keywords: Protein kinase CK2, docking study, MM-PBSA, AMBER11
Protein kinase CK2 (CK2) is a serine/threonine kinase that employs either ATP or GTP as acoenzyme. CK2 is a potential target protein for trea ting a wide variety of tumours andglomerulonephritis, since it is involved in regulation of growth, proliferation, and the survival ofcell. [1] Several compounds with a purine scaffold were discovered through a virtual screening(Figure1). Binding modes of these compounds were elucidated by a docking study and binding
energies were calculated using the molecular mechanics Poisson-Boltzmann surface area
(MM-PBSA) method. The docking study was performed by using MOE2013 and binding energies
were calculated using the MMPBSA.py script of AMBER11. The predicted binding modes showedthat the polar groups such as nitro group, carboxamide group, and carbonyl group of purine
scaffold might interact with Lys68 or Val116 of CK2. The calculated binding energies are wellcorrelated with the experimental inhibitory activities (Table1).

Compound1
Compound2
Compound3
Figure1. Compounds discovered through virtual screening
Table1. Binding energies calculated by the MM-PBSA method (kcal/mol)
CompoundG bind
E vdW
E ele
G PB
G non-ele
1
-48.6
-49.4
-8.9
13.6
-3.8
2
-48.8
-48.4
-8.9
12.2
-3.4
3
-45.4
-45.8
-5.8
9.6
-3.6

IC 50 (M)
6.1
3.3
19.6

[1] Yamada, M.; Katsuma, S.; Adachi, T.; Hirasawa, A.; Shiojima, S.; Kadowaki, T.; Okuno, Y.;
Koshimizu, TA.; Fujii, S.; Sekiya, Y.; Miyamoto, Y.; Tamura, M.; Yumura, W.; Nihei, H.;
Kobayashi, M.; Tsujimoto, G. Inhibition of protein kinase CK2 prevents the progression ofglomerulonephritis. Proc. Natl. Acad. Sci. USA. 2005, 102, 7736-7741.

P1-09Biomolecular Computational Simulations and Organic
Synthesis: Prediction of the Enantioselectivity of
Lipase-Catalyzed Biotransformations
Yoichiro Yagi 1,2

Takatomo Kimura 3

yagi@sp.ous.ac.jp

kimura@konankako.co.jp

Makoto Kamezawa 3
makoto.kamezawa@konankako.co.jp

Yoshinobu Naoshima 1,2
naoshima@sp.ous.ac.jp
12

3

Research 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
Konan Chemical Industry Co., Ltd., 5-12 Nakagawa-cho, Takatsuki-shi, Osaka
569-0066, Japan

Keywords: MD calculation, FMO calculation, organic synthesis, lipase, enantioselectivity
Lipase-catalyzed biotransformatios have recently been utilized as a useful method in the synthesisof enantiomerically pure and biologically active organic compounds [1]. We have found in the
recent literature that computational simulations have become gradually useful as a research method in
the fields of organic chemistry and bioscience. The present effort involves a continuation of ourinterest in biomolecular computational simulations toward predicting the enantioselectivity ofenzyme lipases in organic synthesis, such as Burkholderia cepacia lipase (BCL) and Candida
antarctica lipase typeB (CALB) [2,3].
BCL- and CALB-substrate esters complexes in the presence of TIP3P water molecules were
subjected to MD calculations over a period of 2000ps with ABMER11, and the C-O interatomic
distance (R C-O ) between the carbonyl carbon of esters and the oxyge n of the active site amino acidresidue (BCL: SER87, CALB: SER105) side chain OH in each lipase complex was examined
during the MD trajectory. After MD calculations, surrounding water molecules were removed andfor the resulting lipase complexes FMO calculations with ABINIT-MP/BioStation program werecarried out at FMO2-MP2/6-31G level.
MD calculations show that R C-O for the fast reacting enantiomer of substrate esters remains
roughly unchanged during the computation, while R C-O for the slow reacting enantiomer of esters
increases with the elapsed time. In addition, FMO computations indicate that for esters with highenantioselectivity, each fast reacting enantiomer shows strong interactions with some particularamino acid residues including a definite amino acid residue in each lipase, that is, HIS286 in BCL
and THR40 in CALB, whereas for esters with low enantioselectivity, both ( R)- and (S)-enantiomers
interact with identical amino acid residues including HIS286 and THR40. We can safely say that
HIS286 in BCL and THR40 in CALB play an important role in the chiral recognition ofenantiomers using each lipase. Biomolecular computational simulations enable us to predict and tounderstand the reactivity and the enantioselectivity of lipases in organic synthesis.
[1] Naoshima, Y., Kamezawa, M., Kimura, T., Okimoto, F., Watanabe, M., Tachibana, H., andOhtani, T., Enantioselectivity and reactivity of Candida and Pseudomonas sp. lipases for synthetic
substrates: Biocatalytic synthesis of chiral building blocks with alkanol skeletons, Recent Res. Devel.
Org. & Bioorg. Chem., 4:1-16, 2001.
[2] Moriya, Y., Yagi, Y., Imagawa, A., Kimura, T., Kamezawa, M., Naoshima, Y., Large-Scale
Biomolecular Computations toward the Prediction of Burkholderia cepacia Lipase Enantioselectivity,
The 2011 Joint Conference of CBI & JSBi, Poster list/Proceeding , Kobe, CBI-P1-8, 2011.
[3] Yagi, Y., Tanaka, T., Imagawa, A., Moriya, Y., Mori, Y., Kimura, T., Kamezawa, M., andNaoshima, Y., Large-Scale Biomolecular Chemical Computations toward the Prediction of
Burkholderia cepacia Lipase Enantioselectivity, J. Adv. Simulat. Sci. Eng., accepted.

P1-10Analysis of the SLP-76 ligand recognition mechanismof Mona/gads by molecular dynamics simulation
Yoshiyuki Uemura 1
uemura-yoshiyuki-td@ynu.jp

1

Kazuyoshi Ueda 1
k-ueda@ynu.ac.jp

Graduate school of Engineering Yokohama National University, 79-5
Tokiwadai, Hodogaya-ku, Kanagawa 240-8501, Japan

Keywords: Molecular Dynamics simulation, Ligand docking,Intracellular signal transduction is an important mechanism for the growth and the differentiationof the immuno competent cell (T cell, etc.) in vivo. [1] Mona/gads and SLP-76 are known as themember proteins of such signal transduction pathway in cell. Meanwhile, SH3 is a domain which is
known as one of the most abundant protein interaction modules in nature a nd can binds Pro-rich
peptide strongly. Mona/gads also includes a SH3 domain in its structure. However, SH3 domain in
Mona/gads does not recognize the Pro-rich peptide, instead, it recognizes the RxxK motif included
in SLP-76. [2] The mechanism of these different binding, nature has not been cleared yet. In thiswork, we tried to elucidate the binding mode and recognition mechanism between Mona/gads and
SLP-76 complex using the molecular dynamics simulation method.
We performed 10 ns MD simulation on the system of Mona/gads and SLP-76 complex. From the
trajectories, we calculated the interaction energy between them to elucidate the modes of thebinding. Molecular dynamics calculation was performed using t he package of GROMACS. The
force field of protein is AMBER03, and TIP3P model was used for water. The peptide sequence of
PAPSIDRSTKPPL is used as a binding motif instead of whole SLP-76 peptide. The results showthat the interaction between SLP-76 and Mona/gads was found to be divided into two parts; One is
the part where the coulombic electrostatic interaction works mainly, and the other is the part wherethe van der Waals interaction works mainly. The ratio of these interactions is about 2:1. On theother hand, the residues of Pro1,Ser4,Asp6,Ser8,Pro12 of Mona/gads were found not to involved in
the interaction.
We next prepared some mutants of SLP-76, which is substituted one of the residues of SLP-76 toAla, to investigate the importance of the specific residue. The calculations were also performed
with 10ns MD simulation. The results show that the hydrophobic residues in SLP-76 affect theinteraction between Mona/gads and SLP-76. On the other hand, charged groups in SLP-76 affectthe interaction through the conformation change. In other words, coulomb interactions of thecharged residues play an important role to maintain the conformation of SLP -76.
[1] Andre E.Nel, J. Allergy. Clin. Immunol. 109, 763 (2002) .
[2] Maria Harkiolaki, Marc Lewitzky, Robert J.C.Gilbert , E.Yvonne Jones, Roland P.Bourette, Guy
Mouchiroud, Holger Sondermann, Ismail Moarefi and Stephan M.Feller.
The EMBO Journal. 22, 2571-2582 (2003)

P1-11
Structural Recognition of Local Anesthetic Enantiomer sin human Kv1.5 Channel Pore
Tomoyoshi Seto 1
tseto@belle.shiga-med.ac.jp

1

Department of Anesthesiology, Shiga University of Medical Science,
Seta Tsukinowa-cho, Otsu 520-2192, Japan

Keywords: Molecular Recognition, human Kv1.5, Local anesthetics, Enantiomer
Lethal arrhythmia and cardiac arrest are known as local anesthetic toxicity. Less toxic withenantio-selective ropivacaine and levobupivacaine are used in clinical practice. From animal
experiment, cardiac toxicity increases ropivacaine (ropi) < (S)-bupivacaine (bupi) < bupi <(R)-bupi order [1]. Inhibition of potassium channel has some contribution to cardiac toxicity [2].
We choose here human Kv1.5 voltage-gated channel as a potassium channel model. (R)- and(S)-bupi bind to hKv1.5. (Kd=4.1, 27.3 uM, respectively) , and (R)-bupi inhibits Kv1.5 current
strongly [3]. I, V and A mutants of T507, L510S and V514S mutants do not have enantiomericdifference of the inhibition [3]. T507, L510, V514 residues at the bottom of filter region in porecontribute to enantiomeric difference of bupi inhibition. This study investigates enantiomeric local
anesthetic binding to hKv1.5 channel using docking simulation, and surveys mechanisms ofmolecular recognition of R and S local anesthetic.
Method: The structure of the Kv1.2 channel from the Protein Data Bank (PDB:A79, 2.9 Å
resolution) [ 4 ] was used for homology modelling. ASEDock2013.11.14 program [ 5] (Ryoka
Systems Inc., Tokyo) of the MOE platform was used. Dissociated local anesthetic (LA’s), lidocaine(lido+), mepivacaine (mepi+), enantiomer of ropi+ and bupi+ were docked Kv1.5 channel.
Results: (1) LA’s bound around center of four T479 residues in pore region. (2) Binding energies(Udock kcal/mol) were (S)-bupi+(#59:-13.55) > (S)-ropi+(#35:-14.13) > mepi+(#1:-15.46) >(R)- ropi+(#3:-16.18) > (R)-bupi+(#1:-17.29) > lido+(#1:-17.97). ( R>S ) (3) Interactions of T479,
hydrophobic residue, T507, L510 and V514 with LA’s in pore were found. (4) Molecular recognition ofLA’s was determined by fitting of three groups around chiral nitrogen atom of amide LA’s to biding site.
R-body hydrophobic interactions of butyl- and propyl- to binding site augment to binding force andKv1.5 inhibition.
[1] Ohmura, S. et. al., Systemic toxicity and resuscitation in bupivacaine -, levobupivacaine-, orropivacaine-infused rats, Anesthesia & Analgesia, 93:743-748, 2001.
[2] Olschewski, A. et al., Effect of bupivacaine on ATP-dependent potassium channels in rat
cardiomyocytes, British Journal Anaesthesia, 82:435-438, 1990.
[3] Franqueza L., et al., Molecular determinants of stereoselective bupivacaine block of hKv1.5
channels. Circulation Research, 81:1053-1064(1997).
[4] Long, S.B., Cambell, E.B. and Mackinnon, R., Crystal Structure of a mammalianvoltage-dependent Shaker family K+ channel. , Science, 309:897-903, 2005.
[5] Goto, J., Kataoka, R., Muta, H., Hirayama, N., ASEDock -docking based on alpha spheres andexcluded volumes. Journal of Chemical Information and Modeling, 48:583-590, 2008.

P1-12Analysis of Barnase-Barstar Interactions
with Interfacial Mutations, based on 3D-RISM Method
Yasuomi Kiyota 1
kiyotay@pharm.kitasato-u.ac.jp

Narumi Sai 1
pl10017@st2.pharm.kitasato-u.ac.jp

Mayuko Takeda-Shitaka 1
shitakam@pharm.kitasato-u.ac.jp

1

School of Pharmacy, Kitasato University, 5-9-1 Shirokane Minato-ku, Tokyo 108-8641, Japan

Keywords: Protein-Protein interaction, 3D-RISM method, Barnase-barstar complex
Protein-protein interactions (PPIs) have a key role in numerous biological processes and are
deeply concerned with the many diseases. For understanding the binding mechanism of PPIs andmodulating these interactions by small molecules including water, quantitative information likebinding energy is required. Toward the computational methods for quantitative analysis to predict
the binding affinity of PPIs, we applied the 3D-RISM method [1], which is the statisticalmechanics theory for liquid, to barnase-barstar complex. The 3D-RISM method is a powerful tool
for studying solvation thermodynamics and properties of a solvation structure successfully. The
barnase-barstar complex is one of the widely used PPI systems for investigating the protein-protein
binding affinity, because they bind fast and with high affinity without drastic induced fit.
Additionally, it was found that interfacial water molecule s significantly contributed to the binding
free energy.
In this study, we first comprehensively generated
the 3D-structure models of 65 barnase-barstar
Water molecule in
X-ray structurecomplexes. We second calculated the binding freeenergy from these structures to compare withexperimental values [2,3]. The desolvation energies
on binding of each unit are obtained by 3D-RISM
method and other energies (non-bonding andrelaxation) are obtained by molecular mechanics(Amber99). As an instance, the distribution
Distributionfunction of water at interface of wild-type complex
Function of
was shown in Figure 1. As shown in this figure, thewaterwater molecules are concerned with the binding for
Figure 1. 3D distribution function ofcomplex. The distribution function reproduced thewater between the in interfaces oflocation of water molecule in X-ray structurewild-type complex.
including number of molecule.
References:[1] Kovalenko, A., Hirata, F., J. Chem. Phys., 110:10095−10112, 1999.
[2] Frisch, C., Schreiber, G., Johnson, C. M., and Fersht, A. R., J. Mol. Biol., 267:696-706, 1997.
[3] Schreiber, G., and Fersht, A. R., J. Mol. Biol., 248:478-486, 1995.

P1-13Development and cellular application of novel
streptavidin-binding small molecules
Takuya Terai 1
t-terai@mol.f.u-tokyo.ac.jp

Yasuteru Urano 1,2
uranokun@m.u-tokyo.ac.jp

1

2

3

Moe Kohno 1
2470493421@mail.ecc.u-tokyo.ac.jp

Tetsuo Nagano 3
tlong@mol.f.u-tokyo.ac.jp

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

Keywords: Protein-small molecule interaction, High-throughput screening, Fluorescence imaging
The interaction between streptavidin (protein from Streptmyces avidinii) and biotin (small
molecule ligand) is known as the strongest non-covalent interaction that exists in nature (K d = 10 -13
M) [1]. Streptavidin-biotin system is used for many applications in biotechnology, such as affinity
column chromatography (isolation), solid-phase sensing device (immobilization) or histochemical
imaging (detection). However, for some applications, disadvantages of this system have been
pointed out such as almost irreversible binding under physiological conditions. To disrupt a
streptavidin-biotin complex, it is often necessary to denature the streptavidin under very harshconditions, which destroys functions of many biomolecules. Therefore, in this research, we
screened a large chemical library to search for novel chemical scaffolds that mildly bind to (anddissociate from) streptavidin, then aimed to apply the hit compounds to biological experiments.
First, we developed an assay system based on fluorescence polarization for the screening ofligands of egg avidin, which has identical tertiary structure with streptavidin. More than 160,000
compounds were screened, and we selected 773 compounds as primary hits. These compounds
were subsequently screened for streptavidin, dose-dependently assayed, and analyzed by SPR(Surface Plasmon Resonance). As expected, several biotin analogs were detected among the hits,
but we excluded these compounds. Finally, 6 compounds were selected as novel ligand scaffolds
for streptavidin which exhibited fully reversible binding to the protein (K d = 10 -4 ~10 -6 M). We thenproceeded to characterize the binding mode of the compound that showed the strongest affinity indetail. Based on information from X-ray crystal structure and derivative synthesis, the amide
moiety in the 6-membered ring was most likely to be essential for the binding. Moreover, weapplied the compound to provide reversibility to the recently-reported protein transport system
inside cells [2]. When biotin was added to the system, protein transport occurred but it wasirreversible because of its high affinity. In contrast, when our novel compound was used, the target
protein moved back to the original positions after washout of the compound. We expect that thisstrategy may be a general method to control protein localization reversibly at a given time.
[1] Green, N.M., Avidin, Advances in Protein Chemistry, 29:85-133, 1975.
[2] Boncompain, G., Divoux, S., Gareil, N, de Forges, H., Lescure, A., Latreche, L., Mercanti, V.,
Jollivet, F., Raposo, G., Perez, F., Synchronization of secretory protein traffic in populations ofcells, Nature Methods, 9:493-498, 2012.

P1-14
Molecular dynamics simulations of ATP or ADP boundform of SR Ca 2+ -ATPase using CHARMM force fieldwith updated polyphosphate parameters
Yasuaki Komuro 1,2,3
ykomuro@riken.jp

Chigusa Kobayashi 3

ckobayashi@riken.jp

1

2

3

4

5

	
  	
 

Suyong Re 2

suyongre@riken.jp

Eiro Muneyuki 1

emuneyuk@phys.chuo-u.ac.jp

Yuji Sugita 2,3,4,5

sugita@riken.jp

Graduate School of Science and Engineering, Chuo University, 1-13-27, Kasuga,
Bunkyo-ku, Tokyo 112-8551, JapanRIKEN Theoretical Molecular Science Laboratory, 2-1, Hirosawa, Wako-shi, Saitama
351-0198, JapanRIKEN Advanced Institute for Computational Science, International Medical Device
Alliance (IMDA) 6F, 1-6-5 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047,
Japan
RIKEN Quantitative Biology Center, International Medical Device Alliance (IMDA) 6F,
1-6-5 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, JapanRIKEN iTHES, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan.

Keywords: sarcoplasmic reticulum Ca 2+ -ATPase, force-field optimization, ATP-Mg 2+ interactions
Sarcoplasmic reticulum (SR) Ca 2+ -ATPase transports two Ca 2+ from the cytoplasm into the SR
lumen utilizing ATP hydrolysis. It is fundamental to know how ATP/ADP can bind stably in thecytoplasmic domains of the ATPase. We performed all-atom molecular dynamics (MD)simulations of the ATPase in the ATP/ADP bound forms [1, 2] with explicit solvent and a DOPC
lipid bilayer. We observed the phosphates structure in the bound ATP changed rapidly into theextended form using the original CHARMM force field. After some modifications of the force
field following the CHARMM protocol [3, 4], we could simulate the protein stably [5]. We
examine the essential interactions between ATP bound with Mg 2+ and the ATPase and discuss therelationship between the simulation results and experimental data.
[1]
Toyoshima, C.; Mizutani, T. Crystal structure of the calcium pump with a bound ATP
analogue. Nature 2004, 430, 529-535.
[2]
Sørensen, T. L.-M.; Møller, J. V.; Nissen, P. Phosphoryl transfer and calcium ion
occlusion in the calcium pump. Science 2004, 304, 1672-1675.
[3]
Pavelites, J. J.; Gao, J.; Bash, P. A.; Mackerell, A. D. A molecular mechanics force field
for NAD + NADH, and the pyrophosphate groups of nucleotides. J. Comput. Chem. 1997, 18,
221-239.
[4]
Foloppe, N.; MacKerell, J. All-atom empirical force field for nucleic acids: I. Parameter
optimization based on small molecule and condensed phase macromolecular target data. J. Comput.
Chem. 2000, 21, 86-104.
[5]
Komuro, Y.; Re, S.; Kobayashi, C.; Muneyuki, E.; and Sugita, Y. CHARMM Force Fields
with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of
Ca 2+ -ATPase. J. Chem. Theory Comput . 2014, 10, 4133-4142.

P1-15
Proposal for novel inhibitors to acetyl- andbutyryl-cholinesterases by ab initio molecular simulations
Takeru Murakawa 1 , Tomoya Suzuki 1 , Yuki Matsushita 1 , Mahmud Tareq Hassan Khan 2
and Noriyuki Kurita 1
kurita@cs.tut.ac.jp

1

Depart. Computer Science & Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan

2

Holmboevegen 3B, 9010 Tromsø, Norway

Keywords: Protein-ligand docking; molecular mechanics; molecular orbital calculation;
inhibitor; acetylcholine
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) play important roles in thehydrolyzing mechanism of neurotransmitter acetylcholine (ACh). Recently, AChE and BChE haveattracted much attention because of the possibility that they may contribute to ACh hydrolysis in
the brain of Alzheimer’s patients [1]. Since an adequate amount of ACh is contained in intact
brains, the hydrolysis of some ACh causes no serious problem in living organisms. In contrast, thebrain of a person diagnosed with Alzheimer ’s disease usually has a considerable shortage of ACh,so that the transmission mechanism of information between nerve cells is damaged. It is thus
expected that if the hydrolysis reaction of ACh by AChE and BChE can be inhibited in the brain of
Alzheimer’s patients, the amount of ACh in the synapse will not be decreased so significantly andthe transmission mechanism will become smoother. Many types of ligands have been produced forinhibiting the hydrolysis reaction by AChE.
In the previous study [2], we investigated the specific interactions between AChE and several
carbamates and proposed some potent inhibitors for AChE, using molecular simulations based onclassical molecular mechanics (MM) and ab initio fragment molecular orbital (FMO) methods.
In the present study, we investigate the specific interactions between BChE and some types ofligands, whose biochemical properties were investigated by our previous experiments [ 3], using
the same molecular simulations. Based on the results simulated for AChE and BChE, we attempted
to propose novel ligands, which have strong binding affinity to both AChE and BChE. The detailsof the results will be shown in the conference.
[1] H. Joachim, K. Cornelia, G.D. Ellen, L. Oksana, H.G. Nigel, K. Jochen, Excessive hippocampal
acetylcholine levels in acetylcholinesterase-deﬁcient mice are moderated by butyrylcholinesterase activity, J. Neurochem. 100 (2007) 1421–1429.
[2] T. Okada, T. Murakawa, M.T.H. Khan, K. Komers, M. Kovářová, N. Kurita, Ab initio fragment
molecular orbital calculations on specific interactions between acetylcholinesterase and its
carbamate inhibitors, Current Topics in Med. Chem., (2014) in press.
[3] M.T.K. Khan, Putative molecular interactions involving naturally occurring steroidal alkaloids
from sarcococca hookeriana against acetyl- and butyryl-cholinesterase, Curr. Bioinfo. 8 (2013)
416–428.

P1-16
Assessment of Protein-ligand Binding Energy Calculation
Using Fragment Molecular Orbital Method
Takao Otsuka 1

Noriaki Okiomoto 1

totsuka@riken.jp

okimoto@gsc.riken.jp

Makoto Taiji 1
taiji@riken.jp

1

RIKEN Quant itative Biology Center (QBiC), 1-6-5 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan

Keywords: electronic structure calculation, fragment molecular orbital method, protein-ligand
binding energy
Computational prediction of protein-ligand binding affinities is so important for rational drug
design. Especially computational methods using molecular docking and/or molecular dynamics
simulation are central methods for evaluation of protein-ligand binding affinity, and are expected
to improve the efficiency of the drug development in the near future [1].
Recently first-principles (or ab initio) electronic structure calculations are also used in this field
to evaluate accurate protein-ligand interactions. However, the electronic structure calculations areoften not available for the large molecular system because of its high computational costs. One of
the most popular calculation methods of electronic structure for large biomolecular system is thefragment molecular orbital (FMO) method proposed by Kitaura and co-workers [2]. Nowadays
many studies on protein-ligand interactions by FMO method have been reported [3].
In this study we report further study on how accurately the binding energy of the protein-ligand
systems by using FMO method. We tested the binding energies with some computational
conditions for FMO method and have validated the computational conditions for molecular design
toward the practical use of the drug development [4].

[1] Gilson, M. K., Zhou, H., Annu. Rev. Biophys. Biomol. Struct., 36, 21-42, 2007.
[2] Kitaura, K., Ikeo, E., Asada, T., Nakano, T., Uebayasi, M., Chem. Phys. Lett., 313, 701, 1999.
[3] For example, Tanaka, S., Mochizuki, Y., Komeiji, Y., Okiyama, Y., Fukuzawa, K., Phys. Chem.
Chem. Phys., 16, 10310-10344, 2014.
[4] Otsuka, T., Okimoto, N., Taiji, M., in preparation.

P1-17
Molecular Similarity Based on
Electronic Interaction Topology
Tatsuhiro Doi 1

141d8329@kumamoto-u.ac.jp 	
 1Manabu Sugimoto 1

sugimoto@kumamoto-u.ac.jp

Department of Applied chemistry and Biochemistry, Graduate School of Science andTechnology Kumamoto University, 2-39-1, Kurokami, Chuo-ku,
Kumamoto, 860-0862, Japan

Keywords: Electronic structure calculation, molecular similarity, molecular shape
Prediction of biological activity of compounds is essential for drug discovery. One of the promising
methods for this purpose is to investigate “structural similarity” among molecules because it isbelieved to be directly related to biological activity. [1] In this study, we would like to introduce anothertype of similarity which can reflect characteristics of intermolecular interactions exerted in biologicalsystems. Herein, we suggest a method defining a shape of a molecule, which should reflect topologicalfeatures of hydrogen-bond interactions.
In our approach, molecular shape is defined in two steps: one is geometry optimization of a target
compound, and another is the calculation of the interaction energy. The geometry of the target
molecule was optimized at the DFT(B3LYP)/6-31G** method. The three-dimensional gird forpotential-energy-surface (PES) mapping, the target compound is placed at the origin of the Cartesian
coordination. Then, grid points for proton are generated. At each grid point, the interaction energy
between the target molecule and the probe (proton) is evaluated by using the PM6 method. These
calculations were implemented with GAUSSIAN09.	
 For demonstration, we discuss about the application of the present method to investigate
topological similarity between glucose and psicose. The latter compound attracts recent interest
because of its biological activity for suppressing absorption of glucose in the small intestine. [2]The molecular shape of psicose was calculated as follows. Here
we focus on the ring-form compound of glucose and psicose called
β -D-glucopyranose andβ -D-psicopyranose, respectively. For thesestructural isomers, conformation search was carried out by using the
CONFLEX software in order to find stable conformers within energy
tolerance of 100 kcal/mol. Further selection of conformers was madeby referring to the Boltzmann distribution in the compound space.
Then the DFT method was applied to finally determine the moststable conformer. Its PES calculated using the PM6 method is shownin Fig. 1. It is seen that proton is energetically stabilized when it isclose to the oxygen atoms (oxygen lone pairs) of the target
compound. In the poster presentation, we will discuss about
quantitative molecular similarity on the basis of the molecular Fig.1 Molecular shape of β-D-psicopyranoseshapes obtained with the present method.
	 [1] Q. Du, R. Huang, Y. Wei, Z. Pang, L. Du, and K. Chou, Fragment-Based Quantitative
Structure-Activity Relationship (FB-QSAR) for Fragment-Based Drug Design, Journal ofComputational Chemistry, 30, 295-304(2008)[2] www.kagawa-u.ac.jp/ccip/images/2-02/ME-11-002.pdf

P1-18
Electronic-Structure Data Mining
For Molecular Property Analysis
Takayuki Kurogi

144d8313@st.kumamoto-u.ac.jp

Manabu Sugimoto

sugimoto@kumamoto-u.ac.jp

Department of Applied Chemistry and Biochemistry, Graduate School of Scienceand Technology, Kumamoto University, 2-39-1, Kurokami, Chuo-ku, Kumamoto
860-8555, Japan

Keywords: Electronic-structure calculation, Data mining, Structure-property relationship!Computational chemistry is one of the promising methods for drug discovery. Among a varietyof computational methods, the electronic-structure calculation is expected to be insightful forunderstanding structure-property relationships of a molecule, because both its structure andproperty are essentially determined by its electronic structure. In this study, we aim at developing
an analytical method for data mining which would be useful for molecular design and discovery in
Chemistry and Pharmaceutical Sciences. Herein we will also discuss its application to bio-related
compounds.
We have developed a computer program that automatically discovers a mathematicalcorrelation function between an electronic descriptor and a chemical property. Such a correlationis revealed through a variety of fittings of experimentally measured values for various electronic
factors such as orbital energy levels, ionization energies, reorganization energies, and so on. A
correlation between an experimental and theoretical parameters is automatically discovered by our
computer program in which versatile fitting functions are applied: they consist of polynomial
fittings, the Gaussian fitting, exponential functions, the>%?2<,4%?
Rayleigh distribution, sigmoid functions, the Box-Lucas model/01%+2+3(4,!"#$%"&5)+63%+function, and power law functions as shown in Fig. 1.
Throughout such fittings we can know a hidden mathematical@%0,A)6(*,$%&24'()**"(+,
relation which may be called “data mining”.
-.+#In this presentation, an application of the above
782,9(:42"#8,
computational method will be discussed for an organic pigment&"*;<"=)3%+
for bio-imaging. Herein we focus on a derivative of fluorescein
which is called 	  TokyoGreen (TG) [1]. We have tried to Fig. 1 Mathematical functions applied
discover an electronic factor that is well correlated with for data mining
experimental fluorescent quantum yield of TokyoGreen!"'$##
derivatives. For this purpose, we have investigated electronic!"'!##!"&$##
factors discussed above. After a large number of function!"&!##
fittings, it has been shown that the best fitting is obtained when!"%$##!"%!##
the orbital energy of the molecular orbital localized on the!"!$##
012(3455671
benzene moiety is chosen as a descriptor (Fig. 2). This!"!!##
computational finding is consistent with the conclusion(!"&%##(!"&'##(!"&$##(!"&*##(!"&)##
obtained by Urano et al.
!"#$%&'()*+",-(.(/&"%"++
[1] Urano, Y., Kamiya, M., Kanda, K., Ueno, T., Hirose, K.,
and Nagano, T., Evolution of Fluorescein as a Platform for
Finely Tunable Fluorescence Probes, Journal of theAmerican Chemical Society, 127:4888-4894, 2005.

Fig. 2 Computationally discovered
correlation
functionforthefluorescence quantum yield (φ) of
TGs. The horizontal axis wascomputationally suggested as thebenzene-localized orbital energy.

P1-19Classification of Bioisostere Based on Similarity
Evaluated by Electronic-Structure Calculation
Ryo Iwane

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

Manabu Sugimoto

sugimoto@kumamoto-u.ac.jp

Department of Applied Chemistry and Biochemistry,
Graduate School of Science and Technology, Kumamoto University
2-39-1, Kurokami, Chuo-ku, Kumamoto 860-8555, Japan

Keywords: Quantum chemistry, Electronic structure calculation, Molecular similarity
catechol type
Discovery of similar molecules is one of the
c1
c2
c3
c4
c5
c6
key processes in Pharmaceutical Sciences. It has
been believed that similar molecules should beester type
similar in their functionality. One of the interesting
e1
e2
e3
e4
e5
e6
ideas for drug discovery is “bioisosterism”. This isa concept useful for chemical-substructure
replacement. Its knowledge is indispensable in lead
e7
e8
e9
e10
e11
optimization.
Compounds
showingsimilarbiological activity are called “bioisosteres”. The
classification seems essentially empirical, so thatthiourea type
its a priori deviation seems challenging. This work
t1
t2
t3
t4
has been motivated by a simple question: canquantum chemical similarity be a good measure forbioisosterism ? Herein we apply our method ofquantum-chemical similarity [1] for classifying
Fig. 1. Chemical structures of molecules investigatedmolecules, which is expected comparable toin this work.
bioisosterism.
We have studied 21 molecules in Fig. 1. It includes bioisosteres of catechol, ester, and thiourea.
Theirelectronic
groundstatesandvibrational
statesareevaluatedusing
the
DFT(CAM-B3LYP)/6-31G(d,p) method. In order to obtain overall electronic spectrum, electronic
excited states are calculated using TDDFT(CAM-B3LYP)/6-31G(d,p) method. Throughout thesecalculations, we evaluate “electronic similarity”, “vibrational similarity”, “quantum chemicalsimilarity”, and structure-based similarities. Based on numerical values of these similarities, astatistical analysis method called cluster analysis is applied. The result is compared with thebioiosteres defined in Ref. [2] where correct answer rate (CAR) is evaluated.
Cluster analysis and CAR evaluation were performed in every possible combination among theabove-mentioned descriptors. Comparison shows that CAR is large when either of HOMO, Raman
spectrum, or molecular geometry is chosen as a descriptor: the CAR for the HOMO descriptor is62.5 %. A cluster analysis for the catechol bioisostere successfully includes c1, c2, c4, c5 and c6 given
in Fig. 1, while only c3 is excluded in the cluster. This reasonable agreement between oursimilarity-based classification and the empirical bioisostere is encouraging for further study. In theposter presentation, we will discuss about more extensive cluster analysis for other bioisosteres.

[1] Sugimoto, M. and Iwane, R., to be submitted.
[2] Silverman, R. B., The Organic Chemistry of Drug Design and Drug Action (2nd Ed.),
Academic Press, 2004
[3] Patani, G. A. and LaVoice, E. J., Bioisosterism: a rational approach in drug design, Chemical
Reviews, 96:3147-3176, 1996.

P1-20
Dynamics of the Large Progenitor Toxin Complex of
Clostridium Botulinum
Yosuke Kondo 1
j3a12701@ed.tus.ac.jp

Yeondae Kwon 1
yekwon@rs.noda.tus.ac.jp

Satoru Miyazaki 1
smiyazak@rs.noda.tus.ac.jp

1

Department of Medicinal and Life Science o f Pharmaceutical Sciences
Tokyo University of Science, Yamazaki 2641, Noda -shi, Chiba 278-8510, Japan

Keywords: Normal mode analysis, Molecular dynamics, Principal component analysis
Clostridium Botulinum produces one of the potent toxins called a botulinum neurotoxin and several
proteins called a non-toxic component. The non-toxic components consist of a non-toxic
non-hemagglutinin and a few hemagglutinins and can form various stoichiometric complexes withbotulinum neurotoxins. As the complex becomes larger, the oral toxicity tends to be stronger and oneof the complexes is referred to as a large progenitor toxin complex (L-PTC). Recently, thethree-dimensional structure of the L-PTC has been revealed by X-ray crystallography and electron
microscopy [1,2,3]. However, most of the dynamics of the L-PTC are unclear. Therefore, in this study,
to estimate the dynamics of the L-PTC, three analyses are conducted by constructing three types ofcomputational models, each of which has a different force between p articles, and then solving anequation of motion. The first analysis called normal mode analysis with anisotropic network model
considers a harmonic potential between particles and gives an analytical solution of the differential
equation [4]. The second and third analyses are molecular dynamics with united -atom andcoarse-grained models, respectively [5,6]. These analyses consider both harmonic and non-harmonic
potentials between particles and give a numerical solution of the differential equation. As the result,when we compared the three analyses after a principal component analysis was performed to each of
the trajectories of the molecular dynamics, a characteristic motion of the L-PTC was obtained. We arepresently considering associating the characteristic motion with the oral toxicity.

[1] Gu S., Rumpel S., Zhou J., Strotmeier J., Hans B., Kay P., Shoemaker C. B., Rummel A. andRongsheng J., Botulinum neurotoxin is shielded by NTNHA in an interlocked complex, Science,
335(6071):977-981, 2012.
[2] Benefield D. A., Dessain S. K., Shine N., Ohi M. D. and Lacy D. B., Molecular assembly of
botulinum neurotoxin progenitor complexes, Proceedings of the National Academy Science of
the United States of America, 110(14):5630-5635, 2013.
[3] Lee K., Gu S., Jin L., Le T. T. N., Cheng L. W., Stotmeier J., Kruel A. M., Yao G., Perry K.,
Rummel A. and Jin R., Structure of a biomodular botulinum neurotoxin complex provides
insights into its oral toxicity, PLoS Pathogens, 9(10):e1003690, 2013.
[4] Bakan A, Meireles L. M. and Bahar I., ProDy: protein dynamics inferred from theory andexperiments, Bioinformatics, 27(11):1575-1577, 2011.
[5] Pronk S., Páll S., Schulz R., Larsson P., Bjelkmar P., Apostolov R., Shirts M. R., Smith J. C.,
Kasson P. M., van der Spoel D., Hess B. and Lindahl E., GROMACS 4.5: a high-throughput andhighly parallel open source molecular simulation toolkit, Bioinformatics, 29(7):845-854, 2013.
[6] Monticelli L., Kandasamy S. K., Periole X., Larson R. G., Tieleman D. P. and Marrink S., The
MARTINI coarse-grained force field: extension to proteins, Journal of Chemical Theory andComputation, 4:819-834, 2008.

P1-21
Binding Model Construction of GPCRs
Specifically Activated by Lysophosphatidylserine
	
  	
  	
  	
  	
  	
  	
  	
  	
  Misa Sayama 1 	
  	
  	
  	
  	
 
4884487850@mail.ecc.u-tokyo.ac.jp

	
 

	
  	
  Tomohiko Ohwada 1
ohwada@mol.f.u-tokyo.ac.jp

Takatsugu Hirokawa 2
t-hirokawa@aist.go.jp

Sejin Jung 1 , Yuko Otani 1
1

2

Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi,
Koto-ku, Tokyo 135-0064, Japan

Keywords: G-protein-coupled receptor, Lysophosphatidylserine, Homology modeling, Ligand
docking
Recently, three orphan G-protein-coupled receptors, GPR34, P2Y10 and GPR174 have beenshown to recognize lysophosphatidylserine (lysoPS) as an endogenous ligand, a kind oflysophospholipids generated from plasma membrane lipid, phosphatidylserine (PS). 1, 2 These
receptors are suggested to control immune system, but their detailed biological roles are still
unknown. To design and synthesize active and/or subtype-selective agonists as useful tools to
study the functions of these receptors, we aim to explore how lysoPS and its analogues bind to thereceptors. In this study, we will propose plausible binding models, which are virtually consistent
with the experimental structure-activity relationships.
LysoPS consists of three parts, a polar head group (phosphoserine), a hydrophobic tail (fatty acid)and a glycerol linker connecting them. In present modeling, we focused on the effect of fatty acidmoiety such as structures of non-lipid surrogates. Homology models were constructed and active
compounds were successfully docked into the model. Stability of these binding models wasevaluated using molecular dynamics simulation. We found plausible hydrophobic pockets, which
are thought to accommodate the non-lipid fatty acid surrogates effectively.

fatty acid moiety
O

OH

HO

O
NH 2

P
O

O

O

O

potent agonists

OH

lysophosphatidylserine (lysoPS)

synthesis of
lysoPS analogues

ligand docking
to homology model

ligand binding model[1] Sugo, T., et al., Identification of a lysophosphatidylserine receptor on mast cells, Biochem.
Biophys. Res. Commn, 341:1078-1087, 2006.
[2] Inoue, A., et al., TGFα shedding assay: an accurate and versatile method for detecting GPCR
activation, Nature Methods, 9:1021-1029, 2012.

P1-22
Random Matrix Theory and Principal Component Analysis
of Protein-Ligand Interactions by Using 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, Correlation matrix, Variance-covariance matrix
We apply the random matrix theory [1,2,3] and the principal component analysis (PCA) to study
explicit and hidden interactions between protein and ligand. The time series data of motion of
atoms for proteins-ligand complex which is produced by all atom molecular dynamics simulation[4] with solvent is used.
The PCA is a multivariate technique that analyzes a data table in which observations aredescribed by several inter-correlated quantitative dependent variables. Its goal is to extract theimportant information from the table, to represent it as a set of new orthogonal variables called
principal components, and to display the pattern of similarity of the observations and of thevariables as points in maps.
In the PCA, the first several components are considered to be meaningful as the collective motionof protein and the higher order components are discarded in usual. It works well when the size ofthe collective domains is fairly big. When the size of the domains are small, for example, theprotein-ligand interaction, we should take into account the higher order PCA components andreduciton of the noise is essential. The Inverse Participation Ratio analysis in the random matrix
theory gives a criterion to distinguish the meaningful higher components from noise in PCA, whichenables us to reduce the noise. We also calculate the fundamental statistical quantities whichcharacterize the universality class in the random matrix theory. We find that the eigenvalue spacing
agree well with the Gaussian orthogonal ensembles. Our method is an attempt to improve andrefine the principal component analysis of protein domain [5]. As an example, we demonstrate ourmethod to PDBID:2B18 to study the protein-ligand 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, Phys. 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 networks 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 Bi ologicaland Chemical Systems. Chapter 12: edited by Qiang Cui & Ivet Bahar, , Chapman & Hall/CRC,
Boca Raton, FL, USA, 233-251, 2006, and references therin.

P1-23
Benchmark for new fragmentation breaking peptide
bonds
Kaori Fukuzawa 1,2,3

Masataka Sakaguchi 2

fukuzwa.kaori@nihon-u.ac.jp

ms.sakaguchi@rikkyo.ac.jp

Chiduru Watanabe 4
Chiduru.watanabe@riken.jp
1

2

3

4

Yuji Mochizuki 2,3
fullmoon@rikkyo.ac.jp

School of Dentistry at Matsudo, Nihon University, 2-870-1 Sakaecho-nishi, Matsudo,
Chiba 271-8571, 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
Institute of Industrial Science, Universit y of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo
153-8505, JapanRIKEN Center for Life Science Technologies, 1-72-22 Suehiro-cho, Tsurumi-ku,
Yokohama 230-0045, Japan.

Keywords: FMO method, fragmentation method, peptide bond, protein-ligand complex,
In the Fragment molecular orbital (FMO) method [1-3], the way of fragmentation directly
influence the accuracy of calculation. Intra-molecular fragmentation has been usually done at theposition of sp3 carbon as the bond detached atom (BDA). In such a manner, fragmentation ofprotein broke bonds between alpha carbon and carbonyl carbon of main chain, and therefore unitsof fragment and amino acid were different. Such inconsistency has caused misinterpretation of
the inter-fragment interaction energy (IFIE) when a carbonyl group of main chain was important
for residue-residue or residue-ligand interactions. Here, we examined benchmark calculations of
new fragmentation which breaks peptide bond to conform units of fragmentation to amino acidone.The systems used for this benchmark were two peptides (polyalanine; (Ala)n, n=1-10, andchignolin) and two protein-ligand complexes (estrogen receptor and influenza virus neuraminidasewith their ligand). Accuracy of fragmentation in total energy was acceptable by using many-body
expansion of FMO method (FMO3/FMO4) [4,5].
[1] Kitaura, K., Ikeo, E., Asada, et al., Fragment molecular orbital method: an approximate
computational method for large molecules, Chem. Phys. Lett. 313 (1999) 701-706.
[2] Fedorov, D. G., Nagata, T., & Kitaura, K. Exploring chemistry with the fragment molecularorbital method, Phys. Chem. Chem. Phys. 14 (2012) 7562-7577.
[3] Tanaka, S., Mochizuki, Y., Komeiji, et al., Electron-correlated fragment-molecular-orbital
calculations for biomolecular and nano systems. Phys. Chem. Chem. Phys., 16 (2014)
10310-10344.
[4] Nanano, T., Mochizuki,Y., Yamashita, et al., Development of the four-body corrected fragment
molecular orbital (FMO4) method. Chem. Phys. Lett. 523 (2012) 128–133.
[5] Watanabe, C., Fukuzawa, K., Okiyama Y., et al., Three- and four-body corrected fragment
molecular orbital calculations with a novel subdividin g fragmentation method applicable tostructure-based drug design, J. Mol. Graphics Modell., 41:31–42, 2013.
Acknowledgement: KF thanks to Dr. Tatsusada Yoshida at Tokushima University. A portion ofthis 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, andTechnology (MEXT)’s “Development and Use of Advanced, High -Performance, General-Purpose
Supercomputers Project” and SFR project at Rikkyo University.

P1-24
Prediction of activity cliff among Pim1 inhibitors
using FMO and MM-PBSA calculations
Chiduru Watanabe 1,2

Hirofumi Watanabe 1

Kaori Fukuzawa 2,3

Yoshio Okiyama 1

chiduru.watanabe@riken.jp hirowatanabe@ssbc.riken.jp fukuzwa.kaori@nihon-u.ac.jp yoshio.okiyama@riken.jp

Yuji Mochizuki 2,4Lorien J. Parker 1

fullmoon@rikkyo.ac.jp lorienjparker@gmail.com

1

2

3

4

Hitomi Yuki 1

Teruki Honma 1

yuki@gsc.riken.jp

honma@gsc.riken.jp

RIKEN Center for Life Science Technologies, 1-72-22 Suehiro-cho, Tsurumi-ku,
Yokohama 230-0045, Japan.
Institute of Industrial Science, The University of Tokyo, 4 -6-1 Komaba, Meguro-ku,
Tokyo 153-8505, Japan.
School of Dentistry at Matsudo, Nihon University, 2 -870-1 Sakaecho-nishi, Matsudo,
Chiba 271-8571, 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: FMO, MM-PBSA, Activity cliff, Serine/threonine kinase Pim1
We have predicted activities among serine/threonine kinase Pim1 inhibitors (compounds 1 to 6, asseen in figure) [1] based on fragment molecular orbital (FMO) method in combination withmolecular mechanics-Poisson Boltzmann surface area (MM-PBSA) scheme [2]. Nakano et al. [1]has reported that activities of five compounds including 7 -azaindole derivative (compounds 2 to 6)
are shown various values. The compound 2 is a potent inhibitor as well as 1 including indole ring,
however, compound 6 shows 200-fold decrease in potency by comparison with 1 and 2. This result
is a typical example of “activity cliff”, because the slight difference in structure among theinhibitors with only replacement of a carbon atom with a nitrogen one and its position.
Additionally, X-ray structures of complexes with compounds 1, 3, 5 and 6 are almost no change inconformation of pharmacophore, the origin of variation in inhibition remains enigmatic. Since
dramatic change in activity are caused by the difference of electronic state derived from thereplacement of atom, an approach by only MM calculations is not sufficient to predict theactivities and explain the origin. Hence, we examine the prediction of activity by using FMO and
MM-PBSA calculations.
In this study, we estimate a binding free energy G bind as a sum of interaction energy E int invacuo, deformation energy of ligand in its complex form from free state E def , and solvationenergy G solv . FMO calculations with many-body correction at FMO3-MP2/6-31G* level wereachieved for the complexes with the protein divided into main/side chain of amino acids and eachligand into two functional parts by using ABINIT-MP/BioStation Viewer [3]. The E int was here
estimated as a sum of inter-fragment interaction energy between Pim1 protein fragments and each
inhibitor. On the other hand, the E def and G solv were obtained from MM-PBSA calculations with
AMBER10. Moreover, we prepared three structures of the complex, which are X -ray crystal
structure and MM and QM/MM optimized structures based on template using complex between
Pim1 protein and compound 1, and verified theappropriateness of construction protocol. The G bindobtained from the FMO-PBSA calculation are highly
correlated with logIC 50 more than that of MM-PBSA.
In addition, the result of the QM/MM optimized
structure is preferable to that of the X-ray and the
MM optimized structures. This approach to prediction
Figure Serine/threonine kinase Pim1
activity will be helpful tool to rational drug design.
[1] Nakano, H., et al., Rational Evolution of a Novel Type of Potent and Selective Proviral Integration Site in Moloney Murine
Leukemia Virus Kinase 1 (PIM1) Inhibitor from a Sc reening-Hit Compound, J Med Chem., 55:5151-64, 2012.
[2] Mazanetz, M. P., Ichihara, O. et al., Prediction of cyclin -dependent kinase 2 inhibitor potency using the fragment molecularorbital method. J. Cheminformatics, 3:2, 2011.
[3] Watanabe, C., Fukuzawa, F., Okiyama Y., et al., Three- and four-body corrected fragment molecular orbital calculations with anovel subdividing fragmentation method applicable to structure -based drug design, J. Mol. Graphics Modell., 41:31–42, 2013.

P1-25
Study on QCLO method for canonical molecular orbital
calculation of cofactor-containing protein
Junya Yoshida 1

Toshiyuki Hirano 2

yosjunya@iis.u-tokyo.ac.jp

t-hirano@iis.u-tokyo.ac.jp

Fumitoshi Sato 1, 2
satofumi@iis.u-tokyo.ac.jp

12

Department of Mechanical Engineering, University of Tokyo, 7 -3-1, Hongo, Bunkyo-ku, Tokyo
113-8654, Japan
Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku,
Tokyo 153-8505, Japan

Keywords: All-electron calculation, CMO, QCLO, DFT, Glucose oxidase
All-electron calculation of protein is becoming an indispensable tool to analyze protein electronic
structure and accurate intermolecular interactions. However, convergence of SCF calculation isdifficult for canonical molecular orbital (CMO) method, especially density functional method, andthe effective initial guess creating method is required for the practical use.
We have developed QCLO method [1, 2] to make a precise initial guess of large systems . The
QCLO, quasi-canonical localized orbital (LO), has the character of both CMOs and LOs. An initial
guess of large system is created by gradually lengthening the QCLO of the subunit, which is called
frame molecules. Since it assumed dividing at C, sp 3 carbon, it was difficult to apply to protein
containing hetero molecules.
In this study, in order to remove the restriction, we have developed the new program which candivide by arbitrary subunits and create QCLO. The partial structure of the active center of Glucose
oxidase (GOD, Fig. 1) [3] was applied for a verification calculation. Since the several ionicfunctional groups exist around cofactor FAD the whole calculation is very difficult.
By the new QCLO method, CMO calculation of not only protein but various nano-molecules isattained. It is also applicable for the analyses of intra- and/or inter- molecular interactions between
the arbitrary subunits [2].

Fig.1 Structure of GOD (FAD is shown by CPK model)[1] Kashiwagi, H. et.al., Convergence process with quasi-canonical localized orbital in all-electron
SCF calculation on proteins, Molecular Physics, 101:1-2,81-86, 2003.
[2] Nishino-Uemura, N. et.al., Study of the quaicanonical localized orbital method based onprotein structures, J. Chem. Phys., 127:184106, 2007.
[3] Phaneeswara-Rao, K. et.al., Probing Oxygen Activation Sites in Two Flavoprotein Oxidases
Using Chloride as an Oxygen Surrogate, Biochemistry, 50:5521-5534,2011.

P1-26
Improvements of a method to predict interfaces for
GPCR oligomerization
Akira Saito 1 	  	
 14rmb06@ms.dendai.ac.jp

Wataru Nemoto 1	  	
  w.nemoto@mail.dendai.ac.jp

Hiroyuki Toh 2

toh-hiroyuki@aist.go.jp

12

Graduate School of Science and Engineering Tokyo Denki University, Ishizaka,
Hatoyma-machi, Hiki-gun, Saitama, 350-0394, Japan
Computational Biology Research Center (CBRC), National Institute Advanced Industrial
Science and Technology (AIST), Koto-ku, Tokyo, 135-0064, Japan

Keywords: G protein-coupled receptor, Oligomerization, Interface
G protein-coupled receptors (GPCRs) interact with each other to form homo- and/or
hetero-oligomers. Some GPCR oligomers are known to be associated with diseases. An accurate
prediction of the residues that interact upon oligomerization interface would further our
understanding of signal transduction and the diseases in which GPCRs are involved. We had
previously developed a method to predict interfaces for GPCR oligomerization. The method
requires both homologous sequences and a closely related structure of a target GPCR. Homologous
sequences are used to calculate conservation scores of residues. A template structure is used to
identify conserved residue clusters on the template structure as interfaces.
Recently, we modified the method as follows. Firstly, the source code of the method was
rewritten in Java, which made the execution speed faster than that of the previous version.
Secondly, a template can be selected from more than one hundred GPCR structures, which havebeen registered in Protein Data Bank in the past few years. Thirdly, a user can predict theinterfaces of a GPCR structure model without inputting homologous sequences. These two
modifications improved the usability of the method. The prediction server is available at
http://grip.b.dendai.ac.jp.

P1-27
Complexity of Moving Robots on a Graphwith Group Constraints
Hirotaka Ono
hirotaka@econ.kyushu-u.ac.jp

Department of Economic Engineering, Kyushu Universit y, Japan
6-19-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan

Keywords: Computational Complexity, Reconfiguration
Given a graph G and a set of k robots, we consider the scenario of moving the robots on somevertices to some other vertices under a certain group constraint, such as independent set constraint,
dominating set constraint, clique constraint, and so on. As an example, suppose that we are given
two independent sets Fb and Fr of a graph such that |Fb|=|Fr|=k. and imagine that robots arelocated on Ib. We call an independent set of size k a formation. The problem that we consider here
is to determine whether we can change the initial formation Fb to the target formation Fr by
moving a robot along an edge one by one with keeping the independent set (or dominating set,clique). The problem for independent set is shown to be PSPACE-complete even for planar graphs,
and also for bounded treewidth graphs, by using the results in [1], In the poster presentation, we
review some known results and new results for several group constraints.
[1] Hearn, R.A., Demaine, E.D.: PSPACE-completeness of sliding-block puzzles and otherproblems through the nondeterministic constraint logic model of computation. TheoreticalComputer Science 343, pp. 72–96 (2005)

P2-01
Identification of novel potential antibiotics against
Mycobacterium using pharmacophore-based in silico
screening targeting thymidine monophosphate kinase
Yuji Koseki 1 , Hironori Kanetaka 1 , H é l è n e M u n i e r -L e h m a n n 2 , 3 , and Shunsuke Aoki 1,4,**Corresponding author aokis@bio.kyutech.ac.jp
1

2

3
4

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
Institut Pasteur, Unité de Chimie et Biocatalyse, Dé partement de Biologie Structurale
et Chimie, 28 rue du Dr Roux, F-75015 Paris, France
CNRS, UMR3523, F-75015 Paris, France
Biomedical Informatics R&D Center, Kyushu Institute of Technology, 680-4 Kawazu,
Iizuka-shi, Fukuoka 820-8502, Japan

Keywords: Antibiotics, in silico screening, Mycobacterium, Pharmacophore model, Thymidine
monophosphate kinase (TMPK),
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 concern [1]. Advances in the identification of novelanti-TB drugs have been progressed against several biological pathways, such as mycobacterialcell wall synthesis, energy production, and DNA/RNA synthesis [2]. The Mycobacterium
tuberculosis thymidine monophosphate kinase (mtTMPK) is a potential enzymatic target for thetreatment of TB. In fact, several research groups have reported that the successful development ofinhibitors of mtTMPK with potential antibacterial activity against Mycobacterium strains [3].
In this study, we attempted to identify novel chemical compounds specifically targeting the
mtTMPK. We performed pharmacophore-based in silico screening [1,4,5] using mtTMPK crystal
structure data (PDB ID: 1MRN) and the virtual chemical library including 461,383 chemicals. We
then evaluated the antibacterial effects of the candidate chemicals, and we found one hit (KTP3),
which was able to inhibit the growth of model mycobacteria M. smegmatis. We also performed
similarity analysis to identify four additional chemicals (KTPS1-KTPS4) with similar structures tothe active chemical from ChemBridge web-based database. The most potent inhibitor (KTPS1)
does not have any toxic effects on model intestinal bacteria (E. coli BL21 and JM109 strains) andseveral mammalian cells (MDCK, SH-SY5Y, THP-1, HL-60, and K562 cells). Moreover, we also
confirmed that these chemicals directly inhibit the enzymatic activity of mtTMPK.
In conclusion, the structural and biological assays data regarding these novel chemicals are likely
to be useful for the development of novel antibiotics for the treatment of TB. Furthermore, ourscreening methodology presented in this study could contribute to the further identification ofnovel hit chemicals for other candidate medicinal drugs.
References[1] Y. Koseki et al., Curr. Top. Med. Chem. 2014 (14), 176-188.
[2] B. Villemagne et al., Eur. J. Med. Chem. 2012 (51), 1-16.
[3] S. V. Calenbergh, et al., Curr. Top. Med. Chem. 2012 (12), 694-705.
[4] Y. Koseki et al., Eur. J. Med. Chem . 2013 (60), 333-339.
[5] T. Kinjo, et al., J. Chem. Inf. Model. 2013 (53), 1200-1212.

P2-02
Ligand Based Pharmacophore Modeling and In silico
Screening of Protein Tyrosine Phosphatase 1B Inhibitors for
Identification of Novel Antidiabetic Lead Compounds
Shabana Bibi1
dazzling.grin@gmail.com
1, 2

Katsumi Sakata2
ksakata@maebashi-it.ac.jp

Department of Environment and Life Engineering, Maebashi Institute of Technology, 460-1
Kamisadori-machi, Maebashi, Gunma 371-0816, Japan

Keywords: Diabetes mellitus; PTP1B inhibitors; Pharmacophore mapping; Hydrophobic domain.
Diabetes is a metabolic disorder of the pancreas, in which the pancreas loses its functionality to produce
insulin hormone properly in the body [1, 2]. It has been effecting to the middle-aged and elderly, while
there is still lack of efficient drug with improved efficacy and tolerability against this disease. The
purpose of this study is to evaluate therapeutic applications of drug like compounds as antidiabetic
activity through computational studies. Data set consist of reported antidiabetic compounds belongs tobenzylidene-2, 4-thiazolidinedione derivatives; after substitutions on the phenyl ring at the ortho or para
positions of the thiazolidinedione (TZD) group of compounds were synthesized as PTP1B inhibitors withbiological activity (IC50) [3]. PTP1B is a negative regulator of the insulin signaling pathway and is mostpromising potential therapeutic target for discovery of antidiabetic drugs [4-6]. A ligand based
pharmacophore model has been mapped for 59 new antidiabetic compounds with significance for thedevelopment of new drugs by using Ligand Scout software and distance estimation using effectual
software visual molecular dynamic (VMD). The best pharmacophore model in terms of predictive value
consisted of two features like two hydrogen bond acceptors (HBA) and one hydrophobic domain (HYD).
The derived pharmacophore models were then filtered using Lipinski’s rule of five criteria. Therefore theresults obtained in this study hope that the model generated will be helpful in identification of structurally
diverse novel and potentially active lead compounds with improved activity against diabetes.
[1] Patel, D.K., Kumar, R., Prasad, S.K., Sairam, K., Hemalatha S, Anti-diabetic and in vitro antioxidant
potential of Hybanthus enneaspermus (Linn) F. Muell in streptozotocin-induced diabetic rats. Asian
Pacific Journal of Tropical Biomedicine, 1(4):316-22, 2011.
[2] Warjeet Singh L, Traditional medicinal plants of Manipur as anti-diabetics, Journal of Medicinal
Plants Research, 5(5): 677-87, 2011.
[3] Bhattarai, B.B., Kafle, B., Hwang, J.S., Khadka, D., Lee, S.M., Kang, J.S., Ham, S.W., Han, I.O.,
Park, H., Cho, H., Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic andantiobesity effects. Medicinal Chemistry Letters, 19: 6161–6165, 2009.
[4] Ferreira, C.V., Justo, G.Z., Souza, A.C.S., Queiroz, K.C.S., Zambuzzia, W.F., Aoyama, H.,
Peppelenbosch, M.P., Natural compounds as a source of protein tyrosine phosphatase inhibitors:
Application to the rational design of small-molecule derivatives. Biochimie 88,1859-1873, 2006.
[5] Vintonyak, V.V., Waldmann, H., Rauh, D., Using small molecules to target protein phosphatases. Bioorganic Medicinal chemistry, 19: 2145-2155, 2011.
[6] Jain, A.K., Vaidya, A., Recent developments and biological activities of thiazolidinone derivatives: A
review. Bioorganic & Medicinal Chemistry, 11 (20), 378-3395, 2012.

P2-03
Predicting drug efficacy and bias for GPCRs
by molecular dynamics simulation

Osamu Ichikawa 1

Akihiko Arakawa 1

Kazuto Yamaza ki 1

osamu-ichikawa@ds-pharma.co.jp

akihiko-arakawa@ds-pharma.co.jp

kazuto-yamazaki@ds-pharma.co.jp

Kazushi Fujimoto 2

Hideyuki Fujii 2

Atsushi Yamada 2

Susumu Okazaki 2

fujimoto@ogmail.apchem.nagoya-u.ac.jp fujii.hideyuki@a.mbox.nagoya-u.ac.jp ayamada@apchem.nagoya-u.ac.jp okazaki@apchem.nagoya-u.ac.jp

1

Dainippon Sumitomo Pharma Co. Ltd., Suita, Osaka, Japan 3-1-98 Kasugade-naka,
Konohana-ku, Osaka 553-0022, Japan2
Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603,
Japan

Keywords: molecular dynamics, drug discovery, agonistic activity, biased signaling, GPCR
The strength of signal transduction at a target protein induced by a drug is closely associated withthe benefits and side effects of the drug. Especially, partial agonist activity and β-arrestin-biased
agonist activity for G-protein coupled receptors (GPCRs) are key issues in drug discovery forcentral nervous system disorder 1 . However, it is challenging problem to design the GPCR drugs
with appropriate efficacy and bias, because the structural mechanism underlying the differences inthe signaling levels is still not clear. Here, we utilized molecular dynamics simulation tocharacterize the differences of the dynamic fluctuations that associate with agonistic activation in
the various drug 2 –β2-adrenergic receptor (β2AR) complexes. Our computational analyses
revealed that the drug efficacy and bias correlated with the rms fluctuation and the cross-correlated
fluctuation at the sub-microsecond time scale in the transmembrane region, which compose theligand-binding site and the G-protein-binding site. These findings show that the simulation of the
dynamic fluctuations enables the prediction of the efficacy and the bias for drug candidates.

[1] Kahsai, A. W., Xiao, K., Rajagopal, S., Ahn, S., Shukla A. K., Sun J., Oas T. G. and Lefkowitz R. J.,
Multiple ligand-specific conformations of the b2-adrenergic receptor, Nature Chemical Biology,
7:692–700, 2011.
[2] Rajagopal S., Ahn S., Rominger D. H., Gowen-MacDonald W., Lam C. M., DeWire S. M., Violin J.
D. and Lefkowitz R. J., Quantifying Ligand Bias at Seven-Transmembrane Receptors, Mol Pharmacol,
80:367–377, 2011.

P2-04
Identification of ‘dynamic hotspots’ for lead discovery using
MD simulation and spatiotemporal cluster analysis
Akihiko Arakawa 1

Osamu Ichikawa 1

Kazuto Yamazaki 1

akihiko-arakawa@ds-pharma.co.jp

osamu-ichikawa@ds-pharma.co.jp

kazuto-yamazaki@ds-pharma.co.jp

Kazushi Fujimoto 2

Masaki Okada 2

Atsushi Yamada 2

fujimoto@ogmail.apchem.nagoya -u.ac.jp

masaki.okada@apchem.nagoya-u.ac.jp

ayamada@apchem.nagoya-u.ac.jp

Susumu Okazaki 2
okazaki@apchem.nagoya-u.ac.jp

12

Sumitomo Dainippon Pharma Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku,
Osaka 554-0022, Japan
Department of Applied Chemistry, Nagoya University, Furo -cho, Chikusa-ku,
Nagoya, Aichi 464-8603, Japan

Keywords: in silico drug discovery, dynamic hotspots, molecular dynamics simulation,
spatiotemporal cluster analysis, induced-fit binding
Lead compounds with high potency and selectivity often take advantage of induced-fit binding,which implies that design of lead compounds in consideration of induced-fit binding enhances
their potency and selectivity. On the other hand, driving forces of ligand-protein binding arecharacterized as hotspots, which contribute to the binding free energy. If we can identify hotspots
for target protein, they are helpful for design of lead compounds 1 . We therefore establish a method
for identification of hotspots involving induced -fit binding, ‘dynamic hotspots’, using molecular
dynamics (MD) simulation and spatiotemporal cluster analysis of the MD trajectory.
As a test case, we attempted to find dynamic hotspots of the dimeric BTB domains of BCL-6,
which interact with several transcriptional regulators such as SMRT and BCOR. At first, we
performed 1 μsec MD simulation of BCL-6 in the presence of multiple organic solvents. From theentire MD trajectory, spatiotemporal clusters of organic solvents with long residential time insubstrate-binding region were obtained by cluster analysis. Then, we classified the solvent cluster,
according to their 3D localizations, their pharmacophores, and the contemporary structural statesof BCL-6, and identified the several groups of solvent clusters as dynamic hotspots . Finally, we
confirmed that the identified dynamic hotspots corresponded to free energy maps calculated basedon the MD trajectory and the SMRT and BCOR peptides in the reported crystal structures.
Our method is similar with the X-ray crystallographic method in aqueous solution of organic
solvents (MSCS) 2 and the MD simulation-based method with the probability distributions oforganic solvents (SILCS) 3 . However, these methods reveal only static or time-averaged hotspots,
and are unable to detect hotspots specific to the particular structural state of the protein. Our new
method overcomes these problems and is expected to guide the design of lead compounds withhigh potency and selectivity.
[1] Landon, M.R., et al., Identification of Hot Spots within Druggable Binding Regions by
Computational Solvent Mapping of Proteins, Journal of Medicinal Chemistry, 50:1231-1240,
2007.
[2] Mattos, C., et al., Multiple Solvent Crystal Structures: Probing Binding Sites, Plasticity andHydration, Journal of Molecular Biology, 357:1471-1482, 2006.
[3] Yu, W., et al., Site-Identification by Ligand Competitive Saturation (SILCS) assisted
pharmacophore modeling, Journal of computer-aided molecular design, 28:491-507, 2014.

P2-05In silico cardiac safety assessment of drug effects using
heart simulator
Jun-ichi Okada 1
okada@sml.k.u-tokyo.ac.jp

Takashi Yoshinaga 2
t2-yoshinaga@hhc.eisai.co.jp

Junko Kurokawa 3
junkokuro.bip@mri.tmd.ac.jp

Tetsushi Furukawa 3
t_furukawa.bip@mri.tmd.ac.jp

Seiryo Sugiura 1
sugiura@k.u-tokyo.ac.jp

1

2
3

Takumi Washio 1
washio@sml.k.u-tokyo.ac.jp

Kohei Sawada 2
k-sawada@hhc.eisai.co.jp

Toshiaki Hisada 1
hisada@mech.t.u-tokyo.ac.jp

Graduate School of Frontier Sciences, University of Tokyo, 178-4-4 Wakashiba, Kashiwa,
Chiba 277-8563, Japan
Global CV Assessment, Eisai Co., Ltd., Ibaragi, Japan
Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical
and Dental University, Tokyo, Japan

Keywords: Heart simulator, Multiple ion current assay , Cardiotoxicity testing

To save time and cost for drug discovery, a paradigm shift in cardiac safety assessment isrequired. Here, we present the results of a novel assay system using heart simulation
technologies coupled with the multi-ion current assays. Our heart simulator, UT-Heart [1],is a finite-element-based model of human heart to each element of which molecular models
of electrophysiology and excitation-contraction coupling process are implemented. For thedetailed analysis of ECG, we coupled the heart model with the finite element model ofhuman torso and solved the bidomain equations with the novel solving technique [2],[3].
The effect of the drugs under various plasma concentrations was reproduced in UT-Heartby changing the parameters of each channel model according to the experimentallyobtained dose-inhibition relations. We show that our system combining in vitro and in
silico technologies can predict drug-induced arrhythmogenic risk reliably and efficiently.
[1]Sugiura, S. et al. Multi-scale simulations of cardiac electrophysiology and mechanics using the
University of Tokyo heart simulator. Prog Biophys Mol Biol 110, 380-389 (2012).
[2]Okada, J. et al. Transmural and apicobasal gradients in repolarization contribute to T-wave
genesis in human surface ECG. Am J Physiol 301, H200–208 (2011).
[3]Washio, T., Okada, J. & Hisada, T. A parallel multilevel technique for solving the bidomain
equation on a human heart with Purkinje fibers and a torso model. SIAM Review 52, 717–743(2010).

P2-06
Construction of the Protein 3D Fragment Library System
based on Glycine Neighboring Environment
Ryohsuke Itoh

itoh @ mbi.cs.tut.ac.jp

Hiroaki Kato

kato @ cs.tut.ac.jp

Department of Computer Science and Engineering, Toyohashi University of Technology,
1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580 Japan

Keywords: glycine neighboring environment, fragment library, similarity search, motif pattern
PROSITE, Folylpolyglutamate synthase
With the rapidly increasing number of proteins of which three -dimensional (3D) structures are
known, the structure database is one of the key elements in many attempts being made to derive theknowledge of structure-function relationships of proteins. It is known that a glycine residue
appears in various positions such as a turn domain and has important roles in protein structure [1].
In the present work, the authors have constructed the 3D fragment library which focused on theglycine neighboring environment. It has also applied to the structural feature analysis using
known motif patterns.
We have focused on a peptide fragment which contained the target glycine residue and its
first/second neighboring residues in N-, C-terminal sides. The fragment is characterized withstructural information such as secondary structure assignment [2], dihedral angles (φ,ψ), and a
distance matrix for alpha-carbon atoms. Two types of library have been constructed for the
purpose of structural feature analysis: (1) full dataset consists of 3,283,178 glycine-targeted 3D
fragments taken from 87,090 proteins in PDB 2013, and (2) the representative dataset consists of
34,009 fragments from 2,616 proteins.
The library system provides us a fragment condition search using PDBID, sequence pattern,
secondary structure, and/or dihedral angle information. The sequence pattern search with regularexpressions, e.g., T-[NK]-G-K-x which is a subsequence of folylpolyglutamate synthase motif(PS01011) taken from PROSITE database [3], is available in our system. In addition, we havealso implemented structure similarity fragment search based on dihedral angle or distance matrix
in the present work. Folylpolyglutamate synthase motif has rich glycine residues and could playa role in the catalytical activity [4]. For this subsequence pattern, 1,656 fragments were found in
the full dataset library, and then structure similarity search was carried out with the query of the
known motif 3D fragment (1JBVA 47-51). As the result, similar fragments close to the substratebinding sites were identified in several proteins. The result shows the potential applicability of
our approach for 3D structural data mining of proteins.

References:[1] H.Kato, et al., J. Comput. Chem. Jpn., 4, 33-42 (2005).
[2] W.Kabsch, C.Sander, Biopolymers, 22, 2577–2637 (1983).
[3] C.J.A.Sigrist, et al., Nucleic Acids Res., 41, 344-347 (2013).
[4] B.Shane, et al., Adv. Exp. Med. Biol., 338, 629-634 (1993).

P2-07
Discovery of InhA inhibitors with anti-mycobacterial activity
through matched molecular pair approach
Hironori Kanetaka 1

Yuji Koseki 1

n673207h@bio.kyutech.ac.jpm791201y@bio.kyutech.ac.jp

Shunsuke Aoki 1,2
aokis@bio.kyutech.ac.jp

12

Department of Bioscience and Bioinformatics, Graduate School of Computer Science andSystems Engineering, Kyushu Institute of Technology, 680 -4 Kawazu, Iizuka-shi, Fukuoka
820-8502, Japan
Biomedical Informatics Research and Development Center ( BMIRC), Kyushu Institute ofTechnology, 680-4 Kawazu, Iizuka-shi, Fukuoka 820-8502, Japan

Keywords: Antibiotics, Enoyl-acyl-carrier protein reductase (InhA), Fragment library, in silico
structure based drug screening, Matched molecular pair (MMP), Mycobacterium
Tuberculosis (TB) is global chronic infection and there are lots of health concerns represented byemergence of multidrug-resistant TB (MDR-TB) or co-infection with human immunodeficiency
virus (HIV). System of bacterial cell wall synthesis is one of potential targets, and isoniazid (INH)
which has inhibitory effect against mycobacterium Inh A is a major drug in TB treatment [1,2].
Lately, in the research and development of medicinal drugs, the information of bioisosteric andnonbioisosteric transformation is useful for medicinal chemists to modify lead compounds. Of
such approaches, matched molecular pair (MMP) analysis can describe the relation of substructure
and biological property, and the analysis leads to comprehension of chemical pharmacology andprotein-ligand interactions [3].
In this study, by combination of the MMP and docking simulation approaches , we tried to
discover novel InhA inhibitors alternative to a compound (KES4) we previously identified [4].
461,384 compounds in the virtual chemical library were cut at all exocyclic single bonds, andsingle-, double-, triple-cut [5] unique MMPs were generated. After that, 10 candidate compounds
associated with KES4 were identified through the docking simulation with M. tuberculosis InhA
structure. We then evaluated the antibacterial effects of these compounds on growth of
mycobacteria M. smegmatis as a model bacteria of M. tuberculosis, and all of 10 compounds
showed inhibitory effects on growth of M. smegmatis. In addition to this, 6 of 10 compounds havelower experimental IC 50 value than 50 μM and do not have any toxicity against enterobacteria (E.
coli BL21 and JM 109) and mammalian cells (MDCK and SH-SY5Y cells).
We found various potential anti tuberculosis agents with exchanged molecular substituents fromthe prototype compound (KES4) through in silico approaches, and these structural andexperimental data exhibited reasonable correlations. Moreover, protein-ligand interaction data inthis study have potential to improve chemical and biological properties in TB drug design.
[1] Dhar, N, et al., Proc. Natl Acad. Sci. USA 107:12275-12280, 2010.
[2] Izumizono, Y., et al., Eur. J. Med. Chem. 46:1849-1856, 2011.
[3] Antonio, L., et al., Med. Chem. Commun., 5:64-67, 2014.
[4] Kinjo, T., et al., J. Chem. Inf. Model., 53:1200-1212 2013.
[5] Hussain, J., et al., J. Chem. Inf. Model., 50:339-348, 2010.

P2-08Predicting Molecular Mechanisms of Action
Associated with Binary Endpoints
Josep Prous, jr 1
jprous@prousresearch.com

Antoni Valencia 1
avalencia@prousresearch.com

Marcel.li Carbo 1
mcarbo@prousresearch.com

1

Prous Institute for Biomedical Research, Rambla Catalunya 135, 08008 Barcelona, Spain

Keywords: Safety and efficacy profiling, in silico drug discovery, chemoinformatics, brain
cancer
The in silico identification of mechanisms of action related to drug candidates can help elucidat e
therapeutic targets and discover off-target effects, providing information to better understand theunderlying pathways involved in the efficacy and adverse effects of drugs. Additionally, predicting
the mechanism of action of chemicals is of utmost impo rtance to determine their environmental
fate, and to predict the effects of long term exposure.
To address these needs, we have used Prous Institute’s computational platform SYMMETRY to
train a model that predicts around 650 mechanisms of action based on a training set extracted fromliterature and patent analysis. The training set contains approximately 1.5 million
Structure-Activity Relationships (SAR) with a ratio of ~1.6 SAR per training set structure, and it iscontinuously updated. The prediction quality of the Mechanism of Action (MoA) model has beenassessed in a 10% hold-out external validation yielding an average recall of 93%.
One interesting application of SYMMETRY’s MoA model is to predict the mechanisms of action
associated with a binary endpoint and using that information to find differentially expressedmechanisms, i.e. mechanisms associated with positives but unrelated to negatives for a given
endpoint. A methodology is proposed to find the relevant molecular mechanisms of action
associated with a binary training set, and avoid spurious MoA relationships.
As a case study, a binary data set of brain cancer cell line SNB -78 tumor growth inhibitors has
been analyzed and MoAs related to this endpoint have been highlighted. The resulting MoAs
associated with the anticancer activity in the SNB -78 cell line are compared with those describedin the literature.
[1] Valencia, A., Prous, J., Mora, O., Nakissa, S., Valerio, L.G., A novel QSAR model of
Salmonella mutagenicity and its application in the safety assessment of drug impurities, Toxicol.
Appl. Pharmacology, 273, 427-434, 2013
[2] Preusser, M., de Ribaupierre, S., Wöhrer, A., Erridge, S.C., Hegi, M., Weller, M., Stupp, R. ,Current concepts and management of glioblastoma, Ann. Neurol., 70, 9-21, 2011[3] Moffat, J.G., Rudolph, J., Bailey D., Phenotypic screening in cancer drug discovery - past,present and future, Nature Rev. Drug Discovery, 13(8):588-602, 2014[4] Kishi, Y., Okudaira, S., Tanaka, S., Hama, K., Shida, D., Kitayama, J., Yamori, T., Aoki, J.,
Fijimaki, T., Arai, H., Autotaxin is overexpressed in glioblastoma multiforme and contributes tocell motility of glioblastoma by converting lysophosphatidylcholine to lysophosphatidic Acid, J.
Biol. Chem., 281,17492-17500, 2006

P2-09
OEDocking
Package.

3.1:

Pose

2
3

in

One

Hideyuki Sato, PhD 1

Gregory Warren, PhD 2

Mark McGann, PhD 3

Brian Kelley, PhD 3mcgann@eyesopen.com

kelley@eyesopen.com

hsato@eyesopen.com

1

Prediction

Awesomegreg@eyesopen.com

OpenEye Japan K.K., AIOS Toranomon #904, 1-6-12 Nishi-Shimbashi, Minato-ku, Tokyo
105-0003, Japan
OpenEye Scientific Software Inc., 9 B is b e e C t, S u ite D , Santa Fe, NM 87508, USA
OpenEye Scientific Software Inc., 2 2 2 3 r d S t, S u ite 3 2 1 0 , C a m b rid g e , M A 0 2 1 4 2 , U S A

Keywords: Ligand docking, Pose Prediction
The history of molecular docking has been to generate a single algorithm that can doeverything. This strategy ignores that the two problems where docking has been successfully
applied to (posing prediction and virtual screening) are very different problems. The strategy at
OpenEye is to develop applications that are optimized to produce the best performance for a single
task. We present the first of these applications, POSIT for posing, and show that the new
application is best class for generating pose predictions for lead optimization.

P2-10Computational Studies for Drug Design
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, 1-6-5, Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan

Keywords: Molecular Modeling, Drug Design, Molecular Screening

In early stage of drug development process, “seed” compounds as drug candidate aredetected from huge chemical compound library by experimental and computational
screenings. Recently, computational screening using molecular docking plays animportant role in this process. In general, molecular docking treats protein and ligand
as flexible and rigid molecules and predicts binding pose and affinity of a ligand to atarget protein. In living cell, proteins are obviously not static objects andconformational changes are key elements in ligand binding. Therefore, it is essential tointroduce protein flexibility into molecular docking for detection of various seed
compounds.
In this work, we performed molecular simulations of some proteins to understand the
key protein-ligand interactions and the importance of protein flexibility to moleculardocking. Based on these results, we will extend the information of protein flexibility to
the drug design.

P2-11Development of a novel anti trypanosoma drug
Ryunosuke Yoshino 1

Nobuaki Yasuo 2

Yohsuke Hagiwara 3

yoshino.r.aa@m.titech.ac.jp

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

yohsuke.hagiwara@astellas.com

Kazuki Ohno 4

Ichiji Namatame 3

Masaya Orita 3

kazuki.ono@astellas.com

ichiji.namatame@astellas.com

masaya.orita@astellas.com

Masakazu Sekijima 1,2
sekijima@gsic.titech.ac.jp

1

2

3

Global Scientific Information and Computing Center, Tokyo Institute of Technol ogy, Meguro,
Tokyo 152-8550, Japan
Department of Computer Science, Graduate School of Information Science and Engineering ,Tokyo Institute of Technology, Meguro, Tokyo 152 -8550, Japan
Drug Discovery Research, Astellas Pharma Inc, Tsukuba, Ibaraki 305 -8585, Japan

Keywords: Trypanosoma, Spermidine synthase, Molecular dynamics, Protein-Ligand docking,Allosteric inhibitor
Chagas’ disease, African trypanosomiasis, and leishmaniasis are some examples of tropicalneglected diseases (NTDs) caused by the parasite Trypanosoma [1-2]. While nifurtimox andbenznidazole are currently available for treatment of Chagas' disease, they have major drawbacks,
such as side effects and their insufficient effectiveness in the chronic stage [3-4].
In order to discover a new anti-trypanosoma drug, this study focused on spermidine synthase(SRM) as target protein. SRM is one of the proteins involved in the spermine synthesis pathway,
and is believed to be a promising target protein of trypanocidal agents.
While docking simulation is carried out exclusively for drug discovery, a protein can assume
conformation states different from the one determined by X -ray crystallography due to thermal
fluctuation. Therefore, we performed molecular dynamics (MD) simulation for SRM as target
protein (PDBID: 3WBC) using Desmond [5]. Docking simulation was performed on the open
structure obtained from MD simulation and approximately 4.8 million drug-like compounds using
Glide [6]. We successfully obtained active compounds with IC 50 values of the 10 M order against
SRM.
[1] Lockman J.W., Hamilton A.D., (2005) Recent developments in the identification ofchemotherapeutics for Chagas disease. Curr. Med. Chem. 12: 945-959.
[2] Hayley E.M., et al., (2014) Neglected Zoonotic Diseases—The Long and Winding Road to
Advocacy, PLOS Neglected Tropical Diseases, 8(6): e2800.
[3] Van den Bossche H (1978) Chemotherapy for parasi tic infections. Nature 626-630.
[4] Linares G.E.G., Ravaschino E.L., Rodriguez J.B., (2006) Progresses in the field of drug design
to combat tropical protozoan parasitic diseases. Curr. Med. Chem. 13: 335-360.
[5] Bowers KJ, Chow E, et al., (2006) Scalable algorithms for molecular dynamics simulations oncommodity clusters. In: Proceedings of the 2006 ACM/IEEE Conference on supercomputing , 84.
Tampa, FL[6] Richar A.F., Jay L.B., et al., (2004) Glide: A New Approach for Rapid, Accurate Docking andScoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem., 47: 1739 -1749.

P2-12Estimate of Pharmacophore for Anti-Trypanosome Drug
Development Using Fragment Molecular Orbital Method
Nobuaki Yasuo 1

Ryunosuke Yoshino 2

Inaoka Daniel Ken 3

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

yoshino.r.aa@m.titech.ac.jp

danielken@m.u-tokyo.ac.jp

Yohsuke Hagiwara 4

Kazuki Ohno 4yohsuke.hagiwara@astellas.com

kazuki.ono@astellas.com

Kiyoshi Kita 3
kitak@m.u-tokyo.ac.jp

1

2

3
4

Masaya Orita 4
masaya.orita@astellas.com

Masakazu Sekijima 1,2
sekijima@gsic.titech.ac.jp

Department of Computer Science, Graduate School of Information Science and Engineering
Tokyo Tech, Tokyo, Japan
Global Scientific Information and Computing Center, Tokyo Institute of Technology, Meguro,
Tokyo 152-8550, Japan
Graduate School of Medicine, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan
Chemistry Research Labs, Drug Discovery Research, Astellas Pharma Inc, Tsukuba, Ibaraki
305-8585, Japan

Keywords: Chagas disease, Dihydroorotate dehydrogenase, Pharmacophore, Fragment molecularorbitalChagas disease is caused by the parasite Trypanosoma cruzi (T.cruzi) and is one of the neglected
tropical diseases. This disease affects people from approximately 20 countries, particularly thoseliving in the southern United States and Latin America [1-3]. Currently, nifurtimox andbenznidazole are available for treatment of Chagas disease. However, there are serious problems
including adverse effects and limited effectiveness during the chronic phase of this disease [4-5].
In order to develop a novel anti-trypanosoma drug, we analyzed interaction energy fordihydroorotate dehydrogenase (DHODH) as target protein using fragment molecular orbital (FMO)
method [6].
As a result, FMO calculation revealed that polar amino acids of DHODH and the cofactor flavinmononucleotide (FMN) interact with the orotate derivative by hydrogen bond and π-stacking. Thus,
effective inhibitors of TcDHODH require hydrogen bond donor, acceptor and aromatic cycle.
Based on these results, we estimated pharmacophore model that was effective for development ofanti-trypanosoma drug.
[1] Schmunis GA, Yadon ZE (2010) Chagas disease: a Latin American health problem becoming aworld health problem. Acta Trop 115: 14-21.
[2] Roellig DM, Brown EL, Barnabe C, Tibayrenc M, Steurer FJ, et al. (2008) Molecular typing of
Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14: 1123–1125.
[3] Marcet PL, Duffy T, Cardinal MV, Burgos JM, Lauricella MA, et al. (2006) PCR-based
screening and lineage identification of Trypanosoma cruzi directly from faecal samples oftriatomine bugs from northwestern Argentina. Parasitology 132: 57–65.
[4] Van den Bossche H (1978) Chemotherapy for parasitic infections. Nature 626-630.
[5] Linares GEG, Ravaschino EL, Rodriguez JB (2006) Progresses in the field of drug design tocombat tropical protozoan parasitic diseases. Curr Med Chem 13: 335-360.
[6] Kitaura K, Sawai T, Asadaa T, Nakanob T, Uebayasi M (1999) Pair interaction molecularorbital method: an approximate computational method for molecular interactions. Chem Phys
Lett 312: 319-324.

P2-13
Statistical analysis of hydrogen-bond preferences between
chemical fragment and amino acid in Protein Data Bank

1

Tomohiro Sato 1

Teruki Honma 1

tomohiro@gsc.riken.jp

honma@gsc.riken.jp

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

Keywords: Electronic structure calculation, Ligand docking, Protein modeling
In a drug-discovery process, hit to lead optimization is a complicated task to achieve multiple
optimization of sufficient potency, physical properties, ADME profile, and toxicity. To assist theprocess, many chemoinformatics approaches for bioisosteric transformation [1] in order to change
physiochemical properties retaining potency against target protein are reported. Previously, suchbioisosteric transformations were derived from examples of successful chemicaltransformations in public and/or in house screening data . Compared to such data mining fromscreening data, the number of data mining efforts for lead optimization from 3D structural
information has been limited. In this study, we focused on the hydrogen-bonds between small
molecules and receptor proteins, and tried to derive bioisosteric transformation fromprotein-ligand complex structures in Protein Data Bank (PDB),
At first, hydrogen bonding pairs of small molecules and amino acids were collected. Then, thefrequencies of the interacting chemical fragment and each amino acid were analyzed. To clarify thedifferences between amino acids, we focused on the hydrogen bonds with side chain atoms in thisstudy. Using this data, each chemical fragment was characterized the frequencies of observedhydrogen bonds with each of the amino acids. Using Kohonen Self-Organizing Map [3], thefragments were categorized into 5×5 map according to their preferences on amino-acids as theirhydrogen-bond partners. The results proposed the possible bioisosteric transformations by whichsimilar hydrogen bond preferences to the original compound can be expected.
[1] Sheridan, R. P. The most common chemical replacements in drug-like compounds. J. Chem. Inf.
Comput. Sci., 42, 103–8, 2002
[2] Kenny, P. W.; Sadowski, J. Structure Modification in Chemical Databases. In
Chemoinformatics in Drug Discovery, Wiley-VCH, 2005
[3] Kohonen,T. Self-Organizing Maps, Springer Series in Information Sciences, 2001

Figure 1. SOM analysis of chemical fragments by their H -bond preferences on amino acids

P2-14
A New Virtual Screening Method for Identifying Novel
Active Compounds Based on Protein Promiscuity
Kato Koya 1

Okuno Tatsuya 2kato@tbp.cse.nagoya-u.ac.jp

 tatsuya.med@gmail.com

Chikenji George 1

chikenji@tbp.cse.nagoya-u.ac.jp
12

Dept. of Computational Science and Engineering, Graduate School ofEngineering, Nagoya University
Nagoya University Graduate School of Medicine

Keywords: virtual screening, protein promiscuity, ligand based virtual screening, scaffold hopping,in silico screening,
We propose a new virtual screening method for identifying novel compounds that acts against atarget protein of interest. The method performs three-dimensional structural comparison to "theprotein structure based multiple template", using a geometric hashing method, to rank database
compounds. Here, the protein structure based multiple template is consists of a number of known
active ligands that bind to the target protein or its close homologs and is obtained by protein
structure superposition. This approach enables us to simultaneously exploit the diversity anduniversality of protein-ligand interactions for a target protein, and thus it may be suitable forfinding novel active compounds.
We evaluated its performance on several protein targets from the directory of useful decoys(DUD) and compared it to other popular screening programs. The results indicate that theperformance of our approach is comparable to that of one of the most famous screening program
Glide. We also show that our method can identify active compounds that are not similar to any ofthe ligands used in the multiple template. These results suggest that our approach is useful for
identifying novel compounds that acts against a target protein.

P2-15
Quantum-Chemistry-Based Chemoinformatics System for
Molecular Discovery: Database, Search Engines, and More
Manabu Sugimoto 1,2,3

sugimoto@kumamoto-u.ac.jp

12
3

Department of Applied Chemistry and Biochemistry, Graduate School of Science andTechnology, Kumamoto University, 2-39-1, Kurokami, Chuo-ku, Kumamoto
860-8555, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science andTechnology Agency(JST), 4-1-8,Honcho, Kawaguchi, Saitama, 332-0012, Japan
Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585,
Japan

Keywords: Electronic structure, Database, Data mining, Text mining, Molecular discovery,
Molecular design
We propose a chemoinformatics system for molecular discovery on the basis of electronic-structure
calculations. In this presentation, we will provide the overview of our project consisting ofdevelopments of electronic-structure database, search engines for quantum chemical similarities
reflecting electronic spectra and interaction topology, data-mining tools for “electronic
structure-property” relationships, and knowledge search based on text-mining. Each of the research
topics will be separately given in the other three presentations in this Meeting from our group. See theposter presentations by Iwane and Sugimoto, Doi and Sugimoto, and Kurogi and Sugimoto.
By combining these developments, we aim at constructing an informatics system by which users
can learn, study, design, and discover functional molecules according to their individual needs. As ademonstration, we will discuss about our application of our system to predict tyrosinase inhibitors.
Some parts of the system will be open to public. Therefore we would like to discuss with theaudience about what characteristics the system should have for enhancing applicability, reliability, andefficiency for molecular discovery with a particular focus on drug discovery.
This work is a result of collaborations with Naoki Yoshida, Toshihiro Ideo, Ryo Iwane, Takayuki
Kurogi, Tatsuhiro Doi, Hideyuki Nagaya, Erika Makiyama, Mizuki Iida, and Kimitaka Kuroiwa ofKumamoto University. Their contributions are gratefully acknowledged. A part of the present work isfinancially supported by Grant-in-Aid for Scientific Research (KAKENHI) on Innovative Areas(#2601) by The Ministry of Education, Culture, Sports, Science and Technology(MEXT).

P2-16
Quantitative prediction of binding affinity fortheophylline-RNA using alchemical transformation method
Yoshiaki Tanida
tanida.yoshiaki@jp.fujitsu.com

Azuma Matsuura

matsuura.azuma@jp.fujitsu.com

Fujitsu Laboratories Ltd., Atsugi, Kanagawa 243 -0197, Japan

Keywords: drug design, free energy, alchemical transformation
Recent advances in computing and communication power provide us the realization of thecomputer-aided rational drug discovery and design using high performance computing (HPC)
system. Alchemical free energy calculation is nowadays well understood and widely accepted tocompute the quantitative estimation of free energy change between two different equilibrium states,e.g., from the unbound state to the bound state.
In this work, the standard binding free energy of theophylline-RNA complex (PDB code 1O15[1]) is calculated using alchemical transformation method with an application of a harmonic
restraint. The amber14sb force field and general amber force field (GAFF) are used for describing
potential function of RNA and for that of theophylline molecule, respectively. To restrict theconformation sampling of theophylline molecule to a finite volume within the binding site, we use
the harmonic potentials restraining the orientation of the molecule relative to RNA [2]. The longrange dispersion correction EXP-LR is also added [3]. The multistate Bennett’s acceptance ratio(MBAR) is employed in evaluating free energies [4]. Our obtained value is -8.8 kcal/mol fortheophylline-RNA complex, which agrees well with the experimental one (-8.9 kcal/mol).
We believe that this methodology leads to a standard approach of drug development in HPC
system.

[1] G. M. Clore and J. Kuszewski, J. Am. Chem. Soc., 125, 1518 (2003).
[2] S. Boresch, F. Tettinger, F. Leitgeb and M. Karplus, J. Phys. Chem. B, 107, 9535 (2003).
[3] M. R. Shirts, D. L. Mobley, J. D. Chodera and V. S. Pande , J. Phys. Chem. B, 111, 13052(2007).
[4] M. R. Shirts and J. D. Chodera, J. Chem. Phys., 129, 124105 (2008).

P3-01
Approach to Meta-Analysis of Microarray Datasets
Reveals Muscle Remodeling-related Drug Targets andBiomarkers in Duchenne Muscular Dystrophy
Kosuke Negishi 1

Maria Shkrob 2

k.negishi@elsevier.com

m.shkrob@elsevier.com

Nikolai Daraselia 2

Ekaterina Kotelnikova 3

N.Daraselia@elsevier.com

ekotelnikova@gmail.com

Mikhail A. Pyatnitskiy 4
mpyat@bioinformatics.ru
1
2
3

4

Elsevier Japan K.K., 1-9-15 Higashi Azabu, Minato-ku, Tokyo 106-0044, Japan
Elsevier, 5635 Fishers Lane, Suite 510, Rockville, MD, 20852, USA
Institute Biomedical Research August Pi Sunyer (IDIBAPS) - Hospital Clinic of Barcelona,
Spain/Personal Biomedicine ZAO, Russia/Kharkevich Institute for Information Transmission
Problems, Russian Academy of Sciences, Russia.
Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Russia

Keywords: Pathway analysis, Drug target, Biomarker, Systems biology, Omics data analysis

We suggested a computational approach for drug target and biomarkers discovery that relies on theaggregation of individual gene expression profiling experiments and the use of a Sub-Network
Enrichment Analysis (SNEA) algorithm and a literature-extracted network of biological relations
to find key regulators of differentially expressed genes (1). We applied this approach to publicly
available human muscle gene expression studies of Duchenne muscular dystrophy (DMD). Our
analysis revealed possible regulators of transcriptional changes (e.g. AMPK, TORC2, PPARGC1A)
within a skeletal muscle remodeling pathway. Some of the regulators had not been associated with
Duchenne Muscular Dystrophy previously. Our principal findings were later confirmed byindependent experiments.

[1] Ekaterina Kotelnikova, Maria A. Shkrob, Mikhail A. Pyatnitskiy, Alessandra Ferlini and
Nikolai Daraselia, Novel Approach to Meta-Analysis of Microarray Datasets Reveals Muscle
Remodeling-related Drug Targets and Biomarkers in Duchenne Muscular Dystrophy, PLoS
Comput Biol. Feb 2012; 8(2): e1002365.

P3-02
Identifying Potential Indications for Drug Repositioning
Using Text Mining Analyses
Yuki Sato 1
y.sato@elsevier.com

Anton Yuryev 2

Matthew Clark 3

a.yuryev@elsevier.com

m.clark@elsevier.com

George Jiang 2
g.jiang@elsevier.com

1
2
3

Elsevier, 1-9-15 Higashi-Azabu, Minato-ku, Tokyo 106-0044, Japan
Elsevier, 5635 Fishers Lane Suite 510, Rockville, MD 20850, USA
Elsevier, 1600 John F Kennedy Boulevard Suite 1800, Philadelphia, PA 20850, USA

Keywords: Humira, Adalimumab, Drug repositioning, Text mining, Bioinformatics
There is increasing pressure on pharmaceutical companies to continue producing new therapies
while still returning shareholder investment. While research and development continue to lookfor discovery of new compounds and targets, pharmaceutical companies are also looking to
supplement their drug discovery pipelines with drug repositioning of their existing drug portfolio.
This is beneficial to maximize the value of their compound portfolio, particularly for compounds
that may have limited patent protection left.
Text mining analysis can be used to help in drug repositioning efforts in several ways. Text
mining is much better than keyword searching as text mining allows for searching with particularsemantic relationships, e.g. finding only the items where adalimumab inhibited disease. Doing so
reveals true positive results that contain semantically relevant information, whereas keyword
searching may lead to very high noise and false positives as it relies on document levelco-occurrence in which keywords could occur but are not semantically related. Furthermore, text
mining analyses can be automated thereby allowing higher throughput analyses of content andoverall project team efficiency. We validate the methodology of using text mining analyses toassist with drug repositioning by finding additional indications from the literature for adalimumab,
a well-known TNF inhibiting drug.
Text mining analyses were performed on several content sources (biomedical literature, clinical
trials, and patents) to identify all relevant documents in which adalimumab was used as aneffective treatment condition, and also those in which other anti-TNF drugs were used. Different
disease conditions, compound information, and supporting reference information were thenextracted. Our data show 820 diseases, syndromes, physiological effects and toxicities reportedwith up-regulation of TNF-alpha activity. Data analyses confirm the top approved indications ofadalimumab and also suggest other indications that may be of interest for potential drug
repositioning. This information could be used to guide additional research and development
efforts.

P3-03What factors influenced the estimation of HIV-1
CRF07_BC evolutionary history in China
Asiya Hapaer1,3Fengrong Ren1

0430bio@tmd.ac.jp

Yoshihito Niimura

ren@bioinfo.tmd.ac.jp
2

aniimura@mail.ecc.u-Tokyo.ac.jp

Hiroshi Tanaka1
tanaka@bioinfo.tmd.ac.jp

1

Department of Bioinformatics, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo,
Tokyo 113-8510, Japan
2
Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo
113-8657, Japan
3
Center for Disease Control and Prevention, Xinjiang Uyghur Autonomous Region, China

Keywords: HIV-1, Virus origin, Evolutionary Analysis
It has been known that subtypes B’ and C from Thailand and India, respectively, wereintroduced into China in the late 1980s and then the two subtypes became a recombined
subtype CRF07_BC. There is a controversy on evolutionary history of CRF07_BC (HIV-1)
in China by two studies, and both of the studies used Bayesian Evolutionary Analysis by
Sampling Trees (Beast) program. The first paper published in 2011 suggested that
CRF07_BC probably spread form Xinjiang, whereas another paper published in 2012
demonstrated that the virus originated in Yunnan province. Because the two studies useddifferent viral samples and also analysed the data in different styles, there is a great concernthat these differences might have affected the results. Therefore, we conducted acomprehensive analysis to clarify this issue.
We retrieved the viral samples from more than one public database 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 (15 provinces), more sampling time points (1996-2011) and the longer genome
regions of viral genes. In addition, the most recent version of BEAST was used in this study.
The results showed that: (1) outgroup can affect the estimation of viral origin, (2) nosignificant difference between recombinant and non-recombinant genome analyses and (3)sampling bias and early samples impact on the estimation of viral origin.

[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 ofsequences from 12 provinces over a decade. PLoS One, 7(12): p. e52373, 2012.

P3-04
The regulatory mechanisms of adipocyte and osteoblast
differentiation revealed by the integrated transcriptome
analysis
Yosuke Mizuno 1
mizuno@saitama-med.ac.jp

Yutaka Nakachi 1,2
nakachiy@saitama-med.ac.jp

Yasushi Okazaki 1,2
okazaki@saitama-med.ac.jp

1

Division of Functional Genomics & Systems Medicine, Research Center for Genomic Medicine,
Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan
2
Division of Translational Research, RCGM, Saitama Medical University, 1397-1 Yamane,
Hidaka, Saitama 350-1241, Japan

Keywords: Transcriptome, Osteoblast, Adipocyte, Differentiation
Mesenchymal stem cells can differentiate into various cell types. Particularly the regulatory
mechanisms of the balance for differentiation to adipocyte and osteoblast are closely linked to thepathogenesis of metabolic syndrome and osteoporosis. We have displayed the gene regulatory
networks for osteoblastogenesis and adipogenesis of mesenchymal cells, and several genes andmicroRNAs (miRNAs) were identified as regulators for those differentiation. Meanwhile, it has
been reported that expression of several RNA variants in which such as their transcription startingsite or exon usage are different can be occurred from a gene locus, and they are functionallydiscriminated among body tissues and cell types. In addition, it is revealed that various types ofnoncoding RNAs (ncRNAs) as well as miRNAs are involved in most of biological function.
Therefore we aim to identify the RNA variants and ncRNAs that are involved in regulation of
osteoblastogenesis and adipogenesis, and perform detailed RNA expression analysis using total
RNAs which are obtained from the time-course samples during osteoblastogenesis andadipogenesis. Differentiation of the cells into adipocyte and osteoblast were confirmed by theincreased expression of marker genes for each differentiation, then differential expression of eachexon and ncRNAs are comprehensively analyzed using exon array, miRNA array andRNA-sequencing by next generation sequencer. As a result, we could detect specific increasing of
PPARγ2 expression, which is a variant of PPARγ transcription factor gene and has crucial function
for adipogenesis using exon array, suggesting that this tool is effective for identifying thefunctional RNA variants for adipogenesis and osteoblastogenesis. In this analysis, we could detectvarious RNA variants and ncRNAs that are differentially expressed during both types ofdifferentiation. We estimate that specific RNA variants which regulate differentiation are involvedin those differentially expressed RNA variants, and currently the experiment for their validation
are underway.
[1] Mizuno, Y., et al., miR-125b inhibits osteoblastic differentiation by down-regulation of cellproliferation, Biochem. Biophys. Res. Commun, 368:267–272, 2008.
[2] Mizuno Y, et al., miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b,
FEBS Letters, 583:2263-2268, 2009.
[3] Tokuzawa, Y., et al., Id4, a new candidate gene for senile osteoporosis, acts as a molecularswitch promoting osteoblast differentiation, PLoS Genet, 6:e1001019, 2010.

P3-05
A novel role of sphingosine-1-phosphate receptor inproliferation of breast cancer stem cells
Naoya Hirata 1

n-hirata@nihs.go.jp

Masaaki Kurihara 2

masaaki@nihs.go.jp

1

2

Shigeru Yamada 1

Takuji Shoda 2

shyamada@nihs.go.jp

tsho@nihs.go.jp

Yuko Sekino 1

Yasunari Kanda 2

yukos@nihs.go.jp

kanda@nihs.go.jp

Division of Pharmacology, National Institute of Health Science, 1-18-1
Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Division of Organic Chemistry, National Institute of Health Science, 1-18-1
Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

Keywords: sphingosine-1-phosphate, S1PR3, antagonist, cancer stem cells
Growing evidence suggests that breast cancer is originated from breast cancer stem cells(CSCs), which are defined by their ability for self-renewal and high tumorigenicity. Althoughtargeting CSCs is believed to be a more effective approach for therapy, the regulation of CSCs at
the molecular level has not been fully understood. We have previously reported that CSCs can beisolated from human breast cancer MCF-7 cells by aldehyde dehydrogenase (ALDH)
activity-based flow cytometry analysis. In the present study, we investigated a receptor that
regulates proliferation of CSCs by the ALDH assay system.
Using microarray screening in ALDH-positive cell population of MCF-7 cell, we found that
S1PR3, a receptor for a lipid mediator sphingosine-1-phosphate (S1P), was highly expressed in the
ALDH-positive cell population. Stimulation with S1P increased the proportion of ALDH-positive
cell population in a dose-dependent manner. The effect of S1P was inhibited by s selective S1PR3
antagonist, suggesting that S1P increases breast CSCs via S1PR3.
S1P is produced by sphingosine kinase (SphK) from sphingosine. Overexpression of SphK1
increased the ALDH-positive cell population, whereas overexpression of SphK2 did not. S1PR3
antagonist inhibited SphK1-induced increase in the ALDH-positive cell population. These datasuggest that S1P, which is produced by SphK1, stimulates S1PR3 and leads to increase breast
CSCs.
We next performed nude mouse xenograft assay using breast CSCs.
SphK1-overexpressing ALDH-positive cell population exhibited high tumorigenicity,
compared with vector-overexpressing ALDH-positive cell population. Knockdown of S1PR3
inhibited the tumorigenicity of SphK1-overexpressing ALDH-positive cell population . In
addition, chronic administration of S1PR3 antagonist inhibited the tumorigenicity of
SphK1-overexpressing ALDH-positive cell population .
Furthermore, we examined expression of S1PR3 using CSCs from breast cancer patients.
Similar to MCF-7 cells, S1PR3 was highly expressed in ALDH-positive cell population
derived from breast cancer patients. In addition, ALDH-positive cell population co-expressed
SphK1 and S1PR3. These data suggest that SphK1-S1P-S1PR3 signaling is present in
CSCs from breast cancer patients.
Taken together, our data suggest that S1P induces breast CSCs via S1PR3. Thus,
S1PR3 is a potential therapeutic target in breast CSCs.

P3-06
Analysis of alternative splicing in the cerebellum ofspinocerebellar ataxia type 6 knockin mice
Junya Hagiwara 1
Jhagi-info@umin.ac.jp

Kei Watase 3

keinuro@tmd.ac.jp

1

Masaki Morioka 2morioka-tky@umin.ac.jp

Hidehiro Mizusawa 4

h-mizusawa.nuro@tmd.ac.jp

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

Hiroshi Tanaka 2

tanaka@bioinfo.tmd.ac.jp

Department of Computational Biology, 2 Department of Bioinformat ics, 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 ataxias (SCAs) are a group of neurodegenerative diseases characterized by a lossof motor coordination and balance capability. Among them, SCA type 6 (SCA6) is a late-onset
autosomal dominant neurodegenerative disease caused by a polyglutamine (poly-Q) expansion in
Ca v2.1 voltage-gated calcium channel subunit. The development of effective therapy for SCA6 has
been long-awaited as it is one of the most prevalent inherited ataxias in Japan. We previously
reported that the poly-Q expansion causes Purkinje cells (PCs) degeneration without affecting thebasic properties of the channel, but the detailed pathogenesis is still unclear 1,2 . We hypothesizedthat the altered gene splicing may be involved in the disease pathogenesis of Sca6 MPI-118Q mice,in which 118 CAG repeats were inserted into the corresponding Cacna1a locus.
Here, we aimed to detect genes differentially transcribed or spliced between cerebellar tissues ofa wild-type mouse and an Sca6 MPI-118Q mouse by deep sequencing of transcriptome. The
TopHat package ( http://ccb.jhu .edu/software/tophat/) was used for the alignment of the shortreads to the mm10 reference genome, followed by Cufflinks ( http://cufflinks.cbcb.umd.edu/ ) and
MATS (http://rnaseq-mats.sourceforge.net/) for transcript assembly, read counting, and alternative
splicing analysis.
We found several genes differentially spliced between the wild-type and the SCA6 model mice by
CummeRbund and Integrative Genomics Viewer . We found several genes had alternative splicing
between the wild-type and the SCA6 model mice. For Example, a retained intron was found in thegene X, which is an enzyme necessary for glycolipid metabolism. Moreover, the gene X has beenreported to be associated with lysosomal diseases and neurodegenerative disorders. Further
validation study is required to identify the role of the gene X in the Sca6 and is being planned.
These genes might be associated with pathology of SCA6 such as degeneration of PCs and will bepossible novel targets for drug discovery.
[1] Watase K, Barrett CF, Miyazaki T, Ishiguro T, Ishikawa K, Hu Y, Unno T, Sun Y, Kasai S,
Watanabe M, Gomez CM, Mizusawa H, Tsien RW, Zoghbi HY. Spinocerebellar ataxia type 6
knockin mice develop a progressive neuronal dysfunction with age -dependent accumulation ofmutant Cav2.1 channels. Proc Natl Acad Sci USA, 105:11987-11992, 2008.
[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, 109:17693-8, 2012.

P3-07Prediction of gene expression networks in hepatocellular
carcinoma using genome-wide gene expression data
Norihiko Satake 1

Kaoru Mogushi 1

satacom@tmd.ac.jp

mogushi-k@umin.ac.jp

Masaki Morioka 1

Hiroshi Tanaka 1

morioka-tky@umin.ac.jp

1

tanaka@bioinfo.tmd.ac.jp

Department of Bioinformatics, Tokyo Medical and Dental University, Bunkyo -ku, Tokyo
113-8510, Japan

Keywords: Hepatocellular carcinoma, gene expression network, genie3
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality
worldwide in 2008. The number of patients of the hepatocellular carcinoma is increasing,
especially in Europe and the United States. Because molecular-targeted therapy for HCC iscurrently limited to sorafenib, novel drugs are strongly demanded to improve prognosis.
In this study, we used gene expression profiles from patients to identify pathway biomarkers,
which are the molecular networks representing the malignancy of HCC. We also aimed to obtainthe knowledge of candidate molecules for a novel therapeutic target. Identification of an abnormal
branch in the molecular networks can contribute significantly to the promotion of new therapies
development of HCC.
Gene expression profiles of 140 HCC specimens were obtained by DNA microarray (Affymetrix
HG-U133 Plus 2.0), and the data set is available at the Integrated Clinical Omics Database (iCOD).
We used 40 specimens for cancerous tissues including 20 specimens for each of good and poor
prognosis group, respectively. We also evaluated the gene expression patterns of 23 non-cancerous
specimens that contained eight specimens for good prognosis group and 13 for poor prognosis
group. In addition, 15 normal liver specimens of adjacent non-cancerous tissue from metastatic
liver cancer patients were also utilized for construction of a reference network. Co-expression
molecular networks were predicted using genie3 1 under R statistical software (version 3.1.1).
In HCC, growth factors such as vascular endothelial cell growth factor (VEGF), platelet -derived
growth factor (PDGF), epidermal growth factor (EGF), insulin -like growth factor (IGF), andhepatocyte growth factor (HGF) were found to be strongly interacted in cancerous specimens 2 .
Furthermore, interactions among a variety of signal transduction system such as ERK / MAPK
pathway, PI3K / AKT / mTOR pathway, and Wnt pathway were observed.
Focusing on these signal transduction systems, gene expression networks in HCC were visualizedusing a Cytoscape (version 3.1.1). By interpreting the difference of gene expression networks ineach group, molecular network abnormalities that possibly included the candidates for novel
therapeutic target were identified.
[1] Whittaker, S., Marais, R., and Zhu, A.X. The role of signaling pathways in the development andtreatment of hepatocellular carcinoma. Oncogene, 29:4989-5005, 2010.
[2] Huynh-Thu, V. A., Irrthum, A., Wehenkel, L., and Geurts, P. Inferring regulatory networks fromexpression data using tree-based methods. PLoS One. 2010 5:e12776, 2010.

P4-01
Basic approach to optimize the small drug seeds
with minimized CV risk on the early stage of drug
discovery by using small molecule-based in silico models
for prediction of hERG/Nav1.5 inhibitions
Tetsushi Dodo 1
t-dodo@hhc.eisai.co.jp

Megumi Ikemori (Kawada) 1
Takashi Yoshinaga

121Atsushi Inoue

1

Melvin Yu 2
Kohei Sawada 1

Eisai Co., Ltd., Tokodai 5-1-3 Tsukuba-shi Ibaraki 300-2635, Japan
Eisai Inc., 4 Corporate Drive Andover, MA 01810, USA

Keywords: small molecule-based in silico models, prediction of hERG/Nav1.5 inhibitions
It is generally known that many drugs, which show proarrhythmic effects including prolongationof QT/QTc interval and a ventricular tachycardia of torsade de pointes (TdP) type in clinical use,exert inhibitory effects on cardiac ion channels. Although the risk assessment of those channel
inhibitions has become more important in drug development, the elimination of the undesirable
effects is challenging despite the use of in vitro/vivo screening technologies. Under thesecircumstances, in silico models for ion channel inhibitions are receiving increasing attention as aneffective tool to select compounds with reduced risk of ion channel inhibition in the early stage ofdrug discovery.
At Eisai, we have an increasing need for a versatile prediction system that can screen a huge
number of structurally variable virtual compounds and that can search the structure-activity
relationship (SAR) for channel inhibition virtually. We are therefore applying two small
molecule-based in silico models of hERG/Nav1.5 inhibition to the early stage of drug discovery,
including providing predicted annotations of library compounds, comparing prediction results andactual wet data for the key family of compounds identified by high-throughput screenings both ofwhich will help find well-balanced seed compounds with minimized cardiac safety risks attributed
to the channel inhibitions. It is expected that this kind of in silico model can give concrete
directions for drug design to avoid CV liability. In this presentation, the details of the approach
to find/optimize the drug seeds by using in silico models developed in-house will be presented.

P4-02
Establishment of small molecule-based in silico models forprediction of hERG and Nav1.5 inhibitions for drug
discovery
Megumi Ikemori (Kawada) 1
m-kawada@hhc.eisai.co.jp

Tetsushi Dodo 1

12

Melvin Yu2

Takashi Yoshinaga 1

Atsushi Inoue 1

Kohei Sawada 1

Eisai Co., Ltd., Tokodai 5-1-3 Tsukuba-shi Ibaraki 300-2635, Japan
Eisai Inc., 4 Corporate Drive Andover, MA 01810, USA

Keywords: small molecule-based in silico models, prediction of hERG/Nav1.5 inhibitions
The struggle with cardiac ion channel inhib ition in drug discovery and development process
began more than 10 years ago when several non-cardiovascular drugs were reported to prolong the
QT/QTc interval and induce a severe ventricular arrhythmia (torsade de pointes - TdP) due tohuman ether-à-go-go-related gene (hERG) channel inhibition. Drug induced arrhythmia andcardiac dysfunction caused by cardiac sodium (Nav1.5) channel inhibition are also problems.
Therefore risk assessment of these channel inhibitions has become necessary in drug development.
To minimize the inhibitory effects on these ion channels and identify good drug candidates, weestablished in silico models of hERG and Nav1.5 inhibition to support drug discovery. Thesemodels have been constructed based on the platform of PipelinePilot (Accerlys Co.ltd.) and thetraining wet-data set of hERG and Nav1.5 has been obtained from in-house data by using
automated patch clamp systems (IonWorks and Q -patch) and/or a commercially available data set(BioPrint ® ). These established in silico models show high accuracy of around 80% for in-house
library compounds. In the results, in silico models of hERG and Nav1.5 inhibitions, which haveexpected specifications for drug discovery, were established in -house, so we will present about
details of the specifications in this presentation.

P4-03Visualisation of drug delivery by using high resolution
microscopic mass spectrometry
Masahiro Yasunaga 1 ,Masaru Furuta 2 , Koretsugu Ogata 2,
Yoshikatsu Koga 1,
mayasuna@east.ncc.go.jp furu@shimadzu.co.jp kogata@shimadzu.co.jp ykoga@east.ncc.go.jp
Yasuhiro Matsumura1
yhmatsum@east.ncc.go.jp
12

Developmental therapeutics, Research Center for Innovative Oncology, National Cancer
Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan
Analytical & Measuring Instruments Division, Shimadzu Corporation, 1,
Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan

Keywords: Drug delivery, Molecular imaging, MS imaging, Cancer
Pharmacokinetic (PK) and pharmacodynamic (PD) studies are important to evaluate the efficacy andtoxicity of the drugs. In these analyses, tissue homogenates are generally used for the quantification by
high-performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry(LC-MS). However, they lack the information regarding the drug distribution in a specific anatomicalarea. The information of the drug distribution allows us to optimize the drug design enabling more
efficient targeted delivery. We studied the tissue distribution of paclitaxel (PTX) and its micellar
formulation (NK105) using a microscopic mass spectroscopy (MMS). A MMS in which a microscope iscoupled with an atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) and
quadruple ion trap time-of-flight (TOF) analyser was used. The matrix-coated drug sample is ionised
and then separated on the basis of its mass-to-charge ratio (m/z). Images were acquired from imaging
mass spectrometry (IMS) or tandem mass spectrometry (MS/MS) data. (1) We established the drug imaging
system with enhanced resolution and sensitivity. In the analysis, MS and MS/MS were used for quantification andvalidation, respectively. (2) NK105 showed much stronger antitumor effects on a human pancreatic cancer
BxPC3 xenograft than PTX. In the drug imaging, we demonstrated that NK105 delivered more PTX to
the whole tumor tissue (including the center lesion). In the mouse model, PTX caused the peripheral
neurotoxicity but NK105 did not. Multiple high drug-signal areas surrounding and inside the caudal
nerve were observed in the case of PTX, whereas the signals after NK105 injection were significantly
low. We succeeded in corroborating the EPR effect using MMS. The data obtained by the drug imaging
may be useful for facilitating DDS-drug design.

[1] Yasunaga M, Furuta M, Ogata K, Koga Y, Yamamoto Y, Takigahira M, Matsumura Y. The
significance of microscopic mass spectrometry with high resolution in the visualisation of drug
distribution. Sci Rep . 3:3050. 2013.

P4-04
Evaluation of Human iPS cell-derived Hepatocytes for the
Application to ADME/Tox Tests in Drug Development
Seiichi Ishida, Takashi Kubo, Yukie Kuroda, Su-Ryang Kim, Yuko Sekino
ishida＠ nihs.go.jp

Division of Pharmacology, National Institute of Health S ciences,
1-18-1 Kamiyoga, Setagaya-Ku, Tokyo 1588501, Japan

Keywords: iPS cell derived hepatocyte, drug safety test, ADME/Tox, functional evaluation
Tests for the prediction of drug pharmacokinetics (ADME: absorption, distribution, metabolism,excretion), drug adverse effects, and drug induced liver injury are conducted at earlier stage ofdrug development process. Such tests are common and important for the development of a safe new
drug. Today, these depend on the human primary hepatocytes, however there are some difficulties
in them, such as the inter-individual differences of donors. To solve such difficulties, much effort
has been made to obtain hepatocytes from stem cells. The establishment of human iPS cells
accelerated such studies and hepatocytes differentiated from human iPS cells are available
commercially now.
Our laboratory has been evaluating human iPS cell derived hepatocytes about activities of drug
metabolizing enzymes, induction abilities by chemical inducers, and respo nses to hepato-toxic
chemicals. In this presentation, we will report the results of our evaluation of commerciallyavailable human iPS cell derived hepatocytes obtained from different venders together with thoseof HepaRG cells. In the evaluations, metabolic activities of CYP1A2 and CYP3A4 were measured
by P450-Glo Assay kit (Promega), and expressions of CYP1A1, CYP1A2, CYP2B6, CYP2C9,CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A7, GSTA2, UGT1A1, ABCB1, ABCC2, PXR,
CAR, RXR, AhR, ARNT, CEPB, and HNF4A were measured by TaqMan qPCR (LifeTechnologies). Induction of CYP1A2, CYP2B6, and CYP3A4 by typical inducers such as
omeprazole, phenobarbital and rifampicin were also evaluated. The toxicity test was done byexposing cells to the different concentrations of acetaminophen.
Based on our observations, we are going to discuss the possibilities of applying these cells to thedrug development process from the points of 1) stability of differentiation protocol, 2)
reproducibility of the hepatocyte products, and 3) accessibility to users.

P4-05Prediction of rat clearance using in vivo-in vitro
extrapolation and Genetic Algorithm-based multiple linear
regression
Kei Mayumi 1

Takanobu Matsuzaki 1

kei.mayumi@shionogi.co.jp

takanobu.matsuzaki@shionogi.co.jp

Ryosuke Watari 1

Ryosuke Shimizu 1

Shingo Sakamoto 1

ryosuke.watari@shionogi.co.jp ryosuke.shimizu@shionogi.co.jp shingo.sakamoto@shionogi.co.jp

1

Drug Metabolism & Pharmacokinetics, Research Laboratory for Development,
Shionogi & Co., Ltd. 3-1-1, Futaba-cho, Toyonaka-shi, Osaka, 561-0825, Japan

Keywords: Clearance, Intrinsic clearance, Prediction, In vitro-in vivo extrapolation, Multiple
linear regression
Drug clearance (CL) is a key determinant of animal and human pharmacokinetics (PK) for optimizing
its property during lead optimization stage of drug development. Recently, several CL predictivemodels have been developed; however, the in vivo-in vitro extrapolation (IVIVE) which was shown thegood accuracy required many in vitro experiences and statistical methods were difficult to identify
outliers. The aim of this study was developed better predictive CL model that could calculate byminimum in vitro experiments and show the applicability.
We applied multiple linear regression (MLR) to predict CL of rat which is widely used in lead
optimization stage. To minimize the experimental data used, we used rat hepatic intrinsic clearance(CLh) which was based on the basic well-stirred model as a predictor variable in the function.
Therefore, CLh was only calculated by in vitro t 1/2 in rat hepatic microsomes and unbound fraction in
rat serum (fu). Other predictor variables were selected from MOE 2D descriptors and kier-atom typedescriptors by Genetic Algorithm (GA). Additionally, we applied Biopharmaceutical Drug Disposition
Classification System (BDDCS) which was classified drugs according to their extent of metabolism
and solubility for the selection of training set [1]. The number of training set, test set and validation set
were 100, 26, 499 compounds, respectively. Validation set was used to define the applicable compoundclusters.
GA led to nine descriptor variables contributing to the MLR model, and these variables were assessedby the relative importance (RI). The most important descriptor was the Q_VSA_PNEG (RI = 1.0)
followed by total polar negative Van Der Waals surface area, and the next was the CLh (RI = 0.81).
This CL model showed good predictive accuracy (r 2 = 0.693, q 2 = 0.609, average fold error = 1.5 to
2.2-folds). Additionally, we could define applicable clusters for our model by specific features ofphysical property (extent of metabolism, solubility and vsa_pol) and functional groups (carboxyl andsulfonyl groups).
In conclusion, we developed a novel model for prediction of rat CL to combine GA-MLR with IVIVE
and BDDCS. Using our model could accelerate the optimization of CL in drug development to support
successful drug discovery.

[1] Benet, L.Z., et al., BDDCS applied to over 900 drugs, AAPS J, 13(4)519-47, 2011

P4-06
Detailed Analysis of the Binding Mode of Vanilloids toTransient Receptor Potential Vanilloid Type I (TRPV1) by
Mutational and Computational Study
Katsuya Ohbuchi 1
oobuchi_katsuya@mail.tsumura.co.jp

Kazuo Ogawa 1
ogawa_kazuo@mail.tsumura.co.jp

Yoshikazu Mori 1
mori_yoshikazu@mail.tsumura.co.jp

Masahiro Yamamoto 1
yamamoto_masahiro@mail.tsumura.co.jp

Takatsugu Hirokawa 2
t-hirokawa@aist.go.jp

1

2

Tsumura Research Laboratories, Tsumura and Co., 3586 Yoshiwara, Ami -machi Inashiki-gun,Ibaraki, 300-1192, Japan
Molecular Profiling Research Center for Drug Discovery, AIST Tokyo Waterfront Bio -IT
Research Building 2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan

Keywords: Structure property relationships, Pharmacophore,
Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel and amultimodal sensor protein. Although TRPV1 has been extensively characterized, detailed
structural information concerning the binding site is lacking. In this study, a human TRPV1
homology model was constructed to predict amino acid residues involved in the binding ofvanilloids. The involvement of these residues in ligand binding was then evaluated by mutationaland computational analyses. As a result, five amino acids (L515, I573, F587, F59 1 and L670) were
newly identified as being involved in ligand binding and/or modulation of proton sensitivity. The
locations of these residues range from TM3 to TM6, which indicated that the binding pocket of
TRPV1 consists of TM3-5 in the same monomer and TM5 and 6 in the adjacent monomer. These
results provide novel insights into the binding mode of TRPV1 and will be helpful in developing amode specific TRPV1 modulator.

P4-07
Transforming Quantity into Quality: a Highly Optimized
System to Rapidly Process High Throughput Ion-channel
Screening Data
Zsolt Lepp 1

Kaori Moriwaki 1

zsolt.lepp@genedata.com

kaori.moriwaki@genedata.com

Oliver Leven 2
oliver.leven@genedata.com

1
2

Genedata KK, Gotanda 1-7-11, Shinagawa-ku, 141-0022 Tokyo
Genedata AG, Margarethenstrasse 38, 4053 Basel, Switzerland

Keywords: Ion channel, Auto patch clamp, High throughput, Screening, Quality control, Time
trace, Electrophysiological, Parallel processing, Database
Electrophysiological Ion channel measurements generate a significant amount of signal datawith time traces often consisting of hundreds of thousands data points. Furthermore , multiple
events must be inspected, analyzed and combined for an optimal analysis result. This, together
with high-data volumes, makes data processing very time consuming and laborious . Recent
advances in automated electrophysiology enable the screening of thousands of compounds per
week. However, data handling/processing infrastructures have not kept pace with thesedevelopments. In the absence of efficient computational platforms, valuable information can beoverlooked while technology falls short of meeting its high-throughput discovery expectations.
Genedata has addressed these bottlenecks and challenges. T hrough extensive consultations withresearchers at some of the world’s largest pharmaceutical companies and with leading instrument
vendors, Genedata has developed a specific solution for automated, high-throughput
patch-clamping experiments. The Genedata Screener® software package integrates the trinity of
raw data – analysis – storage/reporting into one highly optimized workflow with a flexible
interface. This specialized and intuitive interface and the tight integration of components enables
scientists to tailor each step and detail with all changes quickly propagated throughout the entire
workflow.
This presentation will demonstrate how to simultaneously handle and analyze all plates andcompounds from an automated electrophysiology campaign, enabling thorough quality control andaccurate hit assessment. In total, over 3200 compounds were analyzed ranging over 10 384-wellplates with 18,000 time points per well, and 2 (pre/post) channels – for approximately 140 million data
points. The whole data processing procedure takes less than 30 minutes from raw data input to exportof detailed reports inclusive of quality control and hit identification . The software system also features
a flexible API, which enables the implementation of custom data processing algorithms, results export
to company data warehouses, and much more. Customized algorithm development examples will bepresented as well.

P4-08In Vitro Integrated Assessment for Prediction ofDrug-Induced Arrhythmia
Akiko Suzuki 1 Yukiko Arimura 1 Tomoko Yoshifuku 1
suzuki-akiko@snbl.co.jp

arimura-yukiko@snbl.co.jp

yoshifuku-tomoko@snbl.co.jp

Junko Matsuo 1 Masaaki Ban 1 Mihori Haruyama 1
matsuo-junko@snbl.co.jp

ban-masaaki@snbl.co.jp

haruyama-mihori@snbl.co.jp

Tatsuya Jikuzono 1 Yoshiki Deguchi 1 Hidenobu Sameshima 1
jikuzono-tatsuya@snbl.co.jp

1

deguchi-yoshiki@snbl.co.jp

sameshima-hidenobu@snbl.co.jp

Shin Nippon Biomedical Laboratories, Ltd. (SNBL) Kagoshima 891-1394, Japan

Keywords: Drug induced arrhythmia, Cardiac ion channels, Action potential, Trafficking
In vitro evaluation of the drug effects on a rapidly activating component of delayed rectifierpotassium current (I Kr ) is recommended in the ICH S7B guideline to assess the potential risk ofdrug candidates for QT interval prolongation in pre-clinical studies. However, the arrhythmogenic
mechanism is complicated and in vitro I Kr assay alone is not sufficient to identify drugs withproarrhythmic risk. Recently, a new paradigm focusing on a comprehensive assessment of effects
on multiple cardiac ion channels to predict the proarrhythmic risk of drugs has been proposed.
To predict proarrhythmic potential of drugs more properly, we investigated the effects of 10 drugs
on action potential (AP) and several cardiac ion channels [I Kr , L-type I Ca (I Ca, L ) and I Na ] in maleguinea pig cardiac muscles, and on human ether-a-go-go-related gene (hERG) currents, which areresponsible for I Kr , in hERG transfected CHO cells. In addition, we investigated the acute anddelayed effects of pentamidine, known as the hERG trafficking inhibitor, on hERG current in
hERG transfected CHO cells. [Methods] AP and each current measurement were performed using
microelectrode and patch clamp technique, respectively. [Results and Conclusion] Each drugchanged AP parameter depending on the inhibitory effects of I Kr , I Ca, L , and I Na . There is little
difference between the 50% inhibitory concentrations (IC 50 ) for I Kr and hERG current for each drug.
The IC 50 for I Kr [IC 50 (I Kr )] of TdP-negative drugs (verapamil, diltiazem, and lidocaine) werecomparable to IC 50 (I Ca, L ) or IC 50 (I Na ), and the IC 50 (I Ca, L )/ IC 50 (I Kr ) or IC 50 (I Na )/ IC 50 (I Kr ) values
were less than 7. The IC 50 (I Ca, L )/ IC 50 (I Kr ) and IC 50 (I Na )/ IC 50 (I Kr ) values of TdP-positive drugs(bepridil, quinidine, and astemizole) were greater than 60. These results showed that drugs have
IC 50 for I Ca, L or I Na similar to IC 50 for I Kr , did not induce TdP despite their potent inhibitory effecton I Kr . IC 50 (I Ca, L )/ IC 50 (I Kr ) and IC 50 (I Na )/ IC 50 (I Kr ) values were useful for the prediction of
drug-induced arrhythmia. Pentamidine has no acute inhibition of hERG current up to 10 µmol/L;
however, it induced delayed inhibition of hERG current with IC 50 of 4.2 µmol/L, the effectassumed to be due to the hERG trafficking inhibition. These results suggest that in vitro
assessment for drug candidates in multiple cardiac ion channel assays and hERG trafficking assay,in addition to in vitro I Kr assay, is useful for proper prediction of drug-induced arrhythmia.

P4-09
Molecular Dynamics Simulation of Shiga Toxin , II
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 [2].
In the previous meeting (CBI2013: C-1-08), we reported the direction of this study based on
the results of the short-time simulations (10-20 ns) about a single neutralizer, four
unconnected neutralizers, and a dendritic neutralizer with four connected peptides. In thismeeting, we will show the results of subsequent simulations of a dendritic neutralizer inwhich the simulation time was more than 100ns. The temporal analysis of the arrangementof the residues exhibited the following typical features.
(1) The number of residues which were bonded by electrostatic interactions increased as 7,
6, 6, 9, and 13 at time 10, 30, 50, 90, 130ns, respectively, providing the time scale of thebinding of the Stx and neutralizer.
(2) Among the 13 bonds at 130ns, the 9 bonds were already formed and fixed at 90ns.
(3) Although the 3-5 bonds of these 13 bonds were formed at 10, 30, and 50ns, thearrangement of the residues still varied and fluctuated.
The bonding of residues occurred frequently between arginine of the neutralizer andasparagine or aspartic acid of the Stx, and in addition, the bonding occurred between
methionine of the neutralizer and tryptophan of the Stx. This latter bonding results fromhydrophobic interaction of residues. In the meeting, quantitative analysis of the above
features will be presented in detail.
[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.

P4-10Comparison of in silico human ventricular model assay
with in vivo canine PK/PD analysis for predicting the
cardiac safety of antipsychotic drugs
Taeko Kubo 1, 2
taeko-kubo@ds-pharma.co.jp

Kenta Kadotsuji 1
kenta-kadotsuji@ds-pharma.co.jp

Atsushi Kobayashi 1
atsushi-kobayashi@ds-pharma.co.jp

Hitoshi Funabashi 1
hitoshi-funabashi@ds-pharma.co.jp
1

2

Takashi Ashihara 2
ash@belle.shiga-med.ac.jp

Yasunori Katsura 1
yasunori-katsura@ds-pharma.co.jp

Naruaki Nomura 1
naruaki-nomura@ds-pharma.co.jp

Minoru Horie 2
horie@belle.shiga-med.ac.jp

Preclinical Research Laboratories, Drug Research Division, Sumitomo Dainippon PharmaCo., Ltd., Osaka 554-0022 , Japan
Department of Cardiovascular Medicine, Heart Rhythm Center, Shiga University of
Medical Science, Otsu 520-2192 , Japan.

Keywords: ADMET, Computational toxicology, Pharmacokinetics, Pharmacodynamics
In new drug development, current approaches of cardiac safety assessment require in vitro I Kr
assay and in vivo QT assay in the preclinical stage and thorough QT/QTc prolongation test forsupratherapeutic drug exposures in the clinical stage. Such approaches have been shown, however,
not enough to predict the proarrhythmic potential of drugs. To improve the success rate of new
drug development by efficient and low-cost operation, we compared a new technique, in silico
prediction using human ventricular model, to an in vivo prediction based on the pharmacokinetics
and pharmacodynamics (PK/PD) analysis in anesthetized dogs at preclinical stage.
Transmural ventricular wall model, of which membrane kinetics was represented by modified
O’Hara-Rudy dynamic model of human ventricle, was constructed to simulate electrocardiogram.
To predict clinical effects of antipsychotic drugs on QTc, we incorporated experimental data on
their ion channel inhibition at unbound blood concentrations into the model. For PK/PD analysis,correlations between antipsychotic drug-induced QTc changes and plasma concentration of thedrugs in anesthetized dogs were examined. Their effects on QTc in human at the therapeutic
concentration were extrapolated based on the correlations. The QTc prolongation at the therapeutic
concentration was classified into 4 categories; none, mild, moderate, or severe degrees.
In cases of Zipracidone and Quetiapine, the human in silico model predicted clinically-observed
QTc prolongations more appropriately than the PK/PD analysis. In case of Thiolidazine, of whichmetabolites are suggested to contribute to the QTc prolongation, both human in silico and canine
PK/PD analysis models underestimated the clinically-observed QTc prolongation at the therapeutic
concentration.
Although the current version of the in silico human ventricular model needs to be improved , we canat least conclude that in silico assay would offer a powerful tool compared to canine in vivo
conventional QT assay for predicting cardiac safety of new drugs at their early discovery stage.
[1] O’Hara, T., Virag, L., Varro, A., and Rudy, Y., Simulation of the Undiseased Human Cardiac
Ventricular Action Potential: Model Formulation and Experimental Validation, PLoS
Computational Biology, 7(5): e1002061, 2011.

P4-11QSAR analyses for thermodynamic driving force ofantihistamines in their binding affinity for human H1
receptors
Yoshihiro Uesawa 1
uesawa@my-pharm.ac.jp

1

2

3

Hiroyuki Fukui 3

Kenta Sugawara 2
k.sugawara@spec-lab.com

hfukui@tokushima-u.ac.jp

Shigeru Hishinuma 2

Masaru Shoji 2hishi@my-pharm.ac.jp

msji@my-pharm.ac.jp

Department of Clinical Pharmaceutics, Meiji Pharmaceutical University, 2 -522-1 Kiyose,Tokyo 204-8588, Japan
Department of Pharmacodynamics, Meiji Pharmaceutical University, 2 -522-1 Kiyose, Tokyo
204-8588, Japan
Department of Studies for Incurable Diseases, Institute of Health Biosciences, The Universityof Tokushima Graduate School, 1-78-1 Shomachi, Tokushima 770-8505, Japan

Keywords: Affinity, Antihistamines, Enthalpy, Entropy, Histamine H 1 receptor, QSAR
[Introduction]
Differences between the thermodynamic driving forces of first and second
generation
antihistamines for human H1 receptors and their structural determinants were evaluated withquantitative structure-activity relationship (QSAR) analyses for the thermodynamic driving force.
[Methods]
The binding enthalpy and entropy of 20 antihistamines were estimated with the van't Hoff equation
using their dissociation constants obtained from their displacement curves against the binding oftritium-labeled mepyramine, a typical antihistamine, to membrane preparations of Chinese hamster
ovary cells expressing human H1 receptors at various temperatures. Structural determinants ofantihistamines for their thermodynamic binding properties were assessed by QSAR analyses withgenetic algorithm-partial least squares (GA-PLS) regression.
[Results]
Entropy-dependent binding was more evident in second than in first generation antihistamines,
resulting in enthalpy-entropy compensation between the binding forces of first and second
generation antihistamines. QSAR analyses indicated that enthalpy -entropy compensation wasdetermined by the sum of degrees, maximal electrostatic potentials, water -accessible surface areaand hydrogen binding acceptor count of antihistamines to regulate their affinity for receptors.
[Discussion]
It was revealed that entropy-dependent hydrophobic interaction was more important in the bindingof second-generation antihistamines. Furthermore, the responsible structural determinants forenthalpy-entropy compensation were explored. These findings may contribute to understanding thefundamental mechanisms of how the affinity of ligands for their receptors is regulated.
[1] Hishinuma S, Sugawara K, Uesawa Y, Fukui H, Shoji M., Biochem. Pharmacol. 91(2):231 -41,
2014.
[2] Uesawa Y, Hishinuma S, Shoji M., J. Pharmacol. Sci., 124(2):160 -8, 2014

P5-01
In vivo quantification of pharmacologically blockedneuronal slow K + current and compensatory changes of
HCN and persistent Na + currents
Hiroyuki Nodera, M.D.
hnodera@tokushima-u.ac.jp

Department of Clinical Neuroscience, Tokushima University,
3-18-15 Kuramotocho, Tokushima, 770-8503, Japan

Keywords: ion channel, threshold tracking, slow potassium current, HCN current, persistentsodium current
Background: Axonal excitability is tightly balanced by multiple ion channels such as slow K +channels, persistent Na + channels (INap), and HCN channels (Ih). For drug development, an
ion-channel modulating drug is clinically promising. Quantitative in vivo assessment of ionchannel functions is useful to confirm the intended effect. Threshold tracking (QTRAC) has
elucidated dysfunction of ion channels in various neurological diseases.
Objective: To quantitate the in vivo effects of blocking slow K + channels on other ion channels
Methods: BaCl 2 (20 mg/kg) was given i.p. to seven normal, 6-week-old, male mice. Sensory
nerve action potential was recorded at the tail. Axonal excitability was recorded by thresholdtracking (QtracW, Digitimer, UK) before and 30 min post-infusion. Mathematical modelingassessed the interval changes of the ion channel functions.
Results: After the injection of BaCl 2 , there were the following interval changes: (1) increased
supernormality (P = 0.001) and decreased late subexcitability (P = 0.009) (recovery cycle), (2)increased threshold by long hyperpolarization (threshold electrotonus (P = 0.01)). Modelingshowed the following changes of the channel parameters: 82% decrease of nodal slow K + current,
55% decrease of INap, and 56% decrease of Ih.
Conclusions: (1) Inhibition of neuronal slow K + current could be assessed in vivo, which wascounterbalanced by reduction of INap and Ih. (2) Accommodation by other channels results in tightmaintenance of axonal excitability. (3) In vivo axonal excitability testing is a non-invasive
technique that can be easily applicable to human and animals. It can be a useful biomarker tomonitor effects of ion channels by channel modulating drugs.

P5-02Estimating fitness values and appearance time of evolvableself-replicating molecules from time series of frequencies
in an evolution reactor
Takuyo Aita 1

Norikazu Ichihashi 1,2

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

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

Motooka Daisuke 4

Nakamura Shota 4

daisukem@gen-info.osaka-u.ac.jp

nshota@gen-info.osaka-u.ac.jp

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

2

3

4

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
Research Institute of Microbial Diseases, Osaka University, Yamadaoka 1-5, Suita, Osaka,Japan

Keywords: data mining; next generation sequencing;quasispecies; evolutionary dynamics; synthetic biology

experimental

evolution;

fitness;

We have established a translation-coupled RNA replication system within a cell-likecompartment, and conducted an experimental evolution of RNAs in the system [1]. As a result,
we obtained a time series of the occurrence frequencies of various individual genotypes throughnext-generation sequencing technique. The time series showed a complex clonal interference [2]and a polymorphic population called the “quasispecies” [3]. Let Yi(t) be the observed mole
fraction of a genotype i at generation t. By analyzing the time series of Yi(t), we estimated thefitness value and appearance time for each of the major genotypes. The analysis was conducted
as follows. First, we assumed that the dynamics of a genotype i obeys the simple differential
equation, dXi(t)/dt=(Fi-D(t))*Xi(t), where Xi(t) and Fi represent the mole fraction at generationt and propagation rate constant (=fitness) of the genotype i, respectively. D(t) is the dilution rate.
Next, defining the sum of the relative entropy over all observed generations, S = Σ_t Σ_i
Yi(t) log Yi(t)/Xi(t), and minimizing the value of S, we obtained the estimated fitness value Fi
and appearance time for each genotype. As a result of the minimization, Xi(t) was well fitted toYi(t) showing the correlation coefficient between Yi(t) and Xi(t) was 0.984. The estimatedfitness values for 11 genotypes arbitrarily chosen were well consistent with the measured onesby the clonal measurement. These results support the validity of our estimation. We thenanalyzed the sequence-fitness relationship and concluded that the mutational effect is roughly
additive.
This approach can clarify the evolutionary dynamics of quasispecies of viruses or otherpathogens, and it may contribute to the drug design against the quasispecies.
[1] Ichihashi et al., Darwinian evolution in a translation-coupled RNA replication system within acell-like compartment. Nat. Commun., 4: 2494, 2013.
[2] Gerrish & Lenski, The fate of competing beneficial mutations in an asexual population.
Genetica, 102-103, 127-144, 1998.
[3] Eigen, Macromolecular evolution: dynamical ordering in sequence space. Ber. Bunsenges. Phys.
Chem., 89, 658-667, 1985.

P5-03
Bifurcation Analysis in Reprogramming Process ofSomatic Cells
Masaki Inoue 1

Jun-ichi Imura 2

Hikaru Ikuta 1

minoue@appi.keio.ac.jp

imura@mei.titech.ac.jp

hikuta@z5.keio.jp

Kenji Kashima 3
kashima@amp.i.kyoto-u.ac.jp

1

2

3

4

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

Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku,
Yokohama-city, Kanagawa, 223-8522, Japan
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 Informatics, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto,606-8501, Japan
Institute of Industrial Science, the University of Tokyo, 4 -6-1, Komaba, Meguro-ku, Tokyo
153-8505, Japan

Keywords: Systems biology, Reprogramming, Bifurcation theory, Robust control theory
We study a mathematical model [1] that represents a mechanism for generating induced pluripotent
stem cells (iPS cells). From dynamical systems theory, we find that saddle-node bifurcation
occurs in the iPS model. We further address the robustness analysis problem to show that thebifurcation plays an essential role for inducing pluripotency even in actual iPS cells.
[1] Shu J. et al., Cell, 153:963-975, 2013.
[2] Inoue M., Imura J.-I., Kashima K., Aihara K., International Journal of Bifurcation and Chaos ,23:1350157, 2013.
[3] Inoue M., Imura J.-I., Arai T., Kashima K., Aihara K., Proceedings of the 53rd IEEE
Conference on Decision and Control, 2014 (to appear).

P5-04
Oriented reconstitution of an ion channel in a cell-sized
liposome
Miho Yanagisawa 1

Shigetoshi Oiki 2

myanagi@cc.tuat.ac.jp

oiki@u-fukui.ac.jp

Kenichi Yoshikawa 3
keyoshik@mail.doshisha.ac.jp

1

2

3

Department of Applied Physics, Tokyo University of Agriculture and Technology. Naka-cho
2-24-16, Koganei, Tokyo 184-8588, Japan.
Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, Universityof Fukui. Matsuokashimoaizuki 23-3, Eiheiji, Yoshida, Fukui 910-1193, Japan.
Graduate School of Life and Medical Sciences, Doshisha University. Tatara Miyakodori 1-3,
Kyotanabe 610-0394, Kyoto, Japan.

Keywords: Model cells, Membrane proteins, Vesicles, Molecular robots.
We introduce a method for the reconstitution of the KcsA potassium channel with an appropriateorientation in cell-sized liposomes, using droplet-transfer technique. First, we prepared cell-sizedwater-in-oil (W/O) droplets covered with a lipid monolayer. When solubilized KcsA wasencapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) andphosphoethanolamine (PE). The droplet was then transferred through an oil/water interface having
a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer
liposome. By creating chemically different lipid-layers at the droplet and oil/water interface, we
obtained liposomes with asymmetric lipid compositions in the outer and inner leaflets. KcsA was
spontaneously inserted into liposomes from the inside or outside. Integrated insertion into theliposome membrane and the KcsA orientation was examined by functional assay, exploiting the pH
sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces
toward acidic media. KcsA loaded from the inside of the PG-containing liposomes becomes
permeable only when the intra-vesicular pH is acidic, and the KcsA loaded from the outside
becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external
insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic
CPD in the added aqueous side [1].
Recently, self-moving liposomes are trying to construct as a molecular robot. It is known that
liposomes tabulate their membranes in presence of membrane proteins or under hypertonic
conditions [2-3]. Oriented reconstitution of ion channels to the tabulated liposomes using thismethod may allow them to achieve ameboid movements and illustrate the self-moving liposomes.
[1] M. Yanagisawa, et al., Oriented reconstitution of a membrane protein in a giant unilamellar
vesicle: Experimental verification with the potassium channel KcsA. Journal of the American
Chemical Society, 133:11774, 2011.
[2] Y. T.-Takiguchi, et al., Physicochemical analysis from real-time imaging of liposome tubulation
reveals the characteristics of individual F-BAR domain proteins. Langmuir, 29:328, 2012.
[3] K. Fujiwara & M. Yanagisawa. Generation of giant unilamellar liposomes containing
biomacromolecules at physiological intracellular concentrations using hypertonic conditions. ACS
Synthetic Biology, in press.

P5-05
Positive Quadratic Systems Modeling and Stability
Analysis of Chemical Reactions in Cells
Yuji Okamoto 1

okamoto@cyb.mei.titech.ac.jp

Mariko Okada-Hatakeyama 2mariko@rcai.riken.jp

Jun-ichi Imura 1

imura@mei.titech.ac.jp

12

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, Japan
Laboratory for Integrated Cellular Systems RIKEN Center for Integrative Medical Sciences(IMS-RCAI), 1-7-22-W518, Tsurumi-ku, Yokohama, Kagawa, Japan

Keywords: Chemical reactions, Nonlinear Systems, Positive Systems
Chemical properties in a cell are analyzed by statistic, information theory, mechanics system, and etc.
In particular, the intensive study on the relationships between signals outside a cell (extracellularsignals such as antigen, growth factor, cytokine) and properties inside the cell (dynamics ofintracellular signal transduction pathway, responsible for cell growth, differentiation or cell death) has
been performed in the field of systems biology, where several dynamical models of chemical reactionsin the cell including the signals outside the cell [1]: S-systems [2-4] or ordinary differential equations(ODEs) consisting of Hill functions [5].
It is one of important problems to “control” chemical properties in a ce ll through stimulus outside thecell as a control input. However, few models of chemical reactions in a cell that is suitable to design an
optimal control input, more specifically, that expresses specified properties on chemical reactions in a
simple and explicit way. We propose a new model of chemical reactions in a cell to express twobiological properties: concentrations of chemical materials are non-negative and the dynamics of thechemical reactions depend on some multiplications of chemical material concentrations. Next weanalyze stability of the new model by using a positive system analysis [6] and the LaSall invariant
theorem [7].

[1] Tony Yu-Chen Tsai, Yoon Sup Choi, Wenzhe Ma, Joseph R. Pomerening, Chao Tang, andJames E. Ferrell Jr, “Robust, tunable biological oscillations from interlinked positive andnegative feed- back loops,” Science, 321:126–129, 2008.
[2] M. A. Savageau, “Biochemical systems analysis I. some mathematical properties of the rate law
for the compo- nent enzymatic reactions,” J. Theor. Biol., 25:365–369, 1969.
[3] M. A. Savageau, “Biochemical systems analysis II. the steady-state solutions for an n-poolsystem using a power-law approximation,” J. Theor. Biol., 25:370–379, 1969.
[4] M. A. Savageau, “Biochemical systems analysis III. dynamic solutions using a power-law
approximation,” J. Theor. Biol., 26:215–226, 1970.
[5] Tony Yu-Chen Tsai, Yoon Sup Choi, Wenzhe Ma, Josepha R. Pomerening, Chao Tang, andJames E. Ferrell Jr, “Robust, tunable biological oscillations from interlinked positive andnegative feedback loops,” Science, 321:126-129, 2008.
[6] L. Farina and S. Rinaldi, Positive Linear Systems Theory and Applications, John Wiley & Sons
Inc., 2000.
[7] H. Khalil, Nonlinear Systems, 3rd ed. Prentice Hall, 2002.

P5-06
Establishment of neuron-specific toxicity evaluation systemusing human induced pluripotent stem cell-derived neurons
Kanako Takahashi 1

Yukari Shigemoto-Mogami 1

ktakahashi@nihs.go.jp

shigemot@nihs.go.jp

1

2

3

Kanae Ohtsu 1

Yohei Okada 2, 3
yohei@aichi-med-u.ac.jp

Hideyuki Okano 2

Yuko Sekino 1

Kaoru Sato 1

hidokano@a2.keio.jp

yukos@nihs.go.jp

kasato@nihs.go.jp

Division of Pharmacology, National Institute of Health Sciences, Setagaya-ku,
Tokyo 158-8501, Japan
Department of Physiology, Keio University School of Medicine, Shinjuku-ku,
Tokyo 160-8582, Japan
Department of Neurology, School of Medicine, Aichi Medical University, Nagakute
Aichi 480-1195, Japan

Keywords: Human induced pluripotent stem cell-derived neurons , Excitotoxicity,
L-glutamate , Toxicity evaluation system
Human induced pluripotent stem cells (hiPSCs) hold potent possibility in the drug development, because
they possibly overcome difference between species in drug efficacy and toxicity. Neurons have specificdamage process called ‘exictotoxicity', which is caused by excessive stimulation of excitatoryneurotransmitter receptors. Because Ca 2+ influx via NMDA type L-glutamate (L-Glu) receptors play a mainrole in this process, we have been searching for the hiPSC-derived neurons which stably express functional
NMDA receptors. We first confirmed the correlation of NMDA receptors with the excitotoxicity in
hiPSC-derived neurons by fura-2 Ca 2+ imaging of neurons differentiated from 253G1, andcommercially-available hiPSC-derived neurons (iCell neurons [iNeurons]). 253G1-derived neurons
showed stable but weak Ca 2+ responses to L-Glu (100 M) from 10 days in vitro (DIV). They did notexhibit the responsiveness to NMDA (100 M) until 40 DIV. On the other hand, iNeurons showed sharp
Ca 2+ responses to L-Glu from 1 DIV. The L-Glu responses were sometimes inhibited by AP5, an
NMDA-specific antagonist, however, the efficacy was not stable. We then tried to evaluate theexcitotoxicity using these two lines. The toxicity was quantified by three parameters, i.e., propidium iodide(PI) / calcein staining, lactate dehydrogenase (LDH) release, and reduction of MTT. Although L-Glu (100M, 1 hr) did not cause cell damage in 253G1-derived neurons (three independent experiments), it
caused significant cell damage in iNeuron-culture batches in which AP5 responsiveness had been
detected. Staurosporine, an apoptosis inducer, caused significant toxicity to both of 253G1-derived
neurons and iNeurons. These results indicate that the hiPSC-derived neurons which stably express
NMDA receptors are necessary for the excitotoxicity evaluation system. We have examined 6 lines of
hiPSC-derived neurons so far, and found two new lines most of which cells have functional NMDA
receptors.
This work was partly supported by a Grant-in-Aid for Young Scientists from MEXT, Japan (KAKENHI 21700422),
the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, Japan, a Health and Labor Science
Research Grant for Research on Risks of Chemicals, a Labor Science Research Grant for Research on New Drug
Development from the MHLW, Japan, awarded to K.S., and a Health and Labor Science Research Grant for Researchon Publicly Essential Drugs and Medical Devices, Japan, awarded to Y.S.

P5-07
Biomarker-responsive supramolecular hydrogels
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: Hydrogel, Stimuli-response
Soft matters such as supramolecular hydrogels composed of self-assembled nanofibers exhibiting
stimuli-response under aqueous conditions are attractive owing to their numerous potential
bio-applications. However, installing macroscopic response toward structurally complex biochemical
stimuli into them still remains a challenge. Here we show that redox-responsive peptide-basedhydrogels [1] have the ability to encapsulate enzymes whilst retaining their activities and co-operative
coupling of enzymatic reactions with the gel response allows us to construct unique stimuli responsive
soft materials capable of sensing a variety of disease-related biomarkers. The programmable gel-sol
response even to biological samples is visible to the naked eye. Furthermore, we built the Boolean
logic gates, OR and AND gates into the hydrogel-enzyme hybrid materials, which were able tosimultaneously sense plural specific biochemicals and execute a controlled drug release through thelogical calculation. [2] The intelligent soft materials that we developed may prove valuable in future
medical diagnostics or treatments.

Figure 1 Schematic representation of Ox⊂BPmoc-F 3 hybrid gels. Information in the form ofmolecular input is received by enzyme (oxidases) and converted into H 2 O 2 , which eventually gives rise
to gel-sol change as output through the degradation of matrix consisting of H 2 O 2 -responsive nanofiber.

[1] M. Ikeda, T. Tanida, T. Yoshii, I. Hamachi, Adv. Mater., 2011, 23, 2819–2822.
[2] M. Ikeda, T. Tanida, T. Yoshii, K. Kurotani, S. Onogi, K. Urayama, I. Hamachi, Nat. Chem.,
2014, 6, 511–518.

P5-08Live-Cell Super-Resolution Imaging with
Spontaneously Blinking Fluorophores
Shin-nosuke Uno 1

Mako Kamiya 1

Toshitada Yoshihara 2

suno@m.u-tokyo.ac.jp

mkamiya@m.u-tokyo.ac.jp

yoshihara@gunma-u.ac.jp

Ko Sugawara 3

Kohki Okabe 3,46198934712@mail.ecc.u-tokyo.ac.jp okabe@mol.f.u-tokyo.ac.jp

Hiroyuki Fujita 5
hfujita@iis.u-tokyo.ac.jp

Seiji Tobita 2
tobita@gunma-u.ac.jp

Mehmet C. Tarhan 5
mctarhan@iis.u-tokyo.ac.jp

Takashi Funatsu 3
funatsu@mol.f.u-tokyo.ac.jp

Yasushi Okada 6
y.okada@riken.jp

Yasuteru Urano 1, 3, 7
uranokun@m.u-tokyo.ac.jp

1

Graduate School of Medicine, The University of Tokyo, Tokyo 113 -0033, Japan.

2

Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu
376-8515, Japan
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113 -0033, Japan.
JST, PRESTO, Saitama 332-0012, Japan
Center for International Research on Micronano Mechatronics, Institute of Industrial Science,
The University of Tokyo, Tokyo 153-8505, Japan.
Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN, Suita, 565 -0874,
Japan
Basic Research Program, Japan Science and Technology Agency, Tokyo 102 -0075, Japan

3
4
5

6

7

Keywords: Super-resolution imaging, Fluorescent probe, Rhodamine
Single-molecule localization microscopy (SLM) is used to construct super -resolution images by
repeated detection and high-precision localization of individual fluorophores. However, to achieve
an initial dark state and subsequent blinking of conventional fluorophores for SLM, additives suchas thiols, an oxygen scavenging system, and high-intensity laser irradiation prior to measurement
are needed, and these requirements are unfavorable for live -cell imaging. From this point of view,
fluorophores that spontaneously blink in the absence of any additive and without the need forspecial conditions are highly desirable. Herein, we report a ne w class of spontaneously blinking
fluorophore, utilizing intramolecular spirocyclization, which is suitable for live -cell SLM without
additives or special conditions [1] . We focused on the phenomenon that rhodamine derivatives
bearing an intramolecular nucleophile exist in thermal equilibrium between a fluorescent open
form and a non-fluorescent spirocyclic form in the ground state. In order to utilize this thermal
equilibrium to achieve spontaneous fluorescence blinking suitable for SLM, we optimized two
parameters, the ratio of bright state and the duration until the open form reverts to the closed form.
We prepared a series of rhodamine derivatives bearing various intramolecular n ucleophiles and/or
fluorophores, evaluated the properties, and selected HMSiR as an appropriate fluorophore. Then,we performed live-cell SLM of microtubules in HeLa cells with HMSiR. -Tubulin-SNAP wasexpressed and labeled with benzylguanine derivative of HMSiR, and SLM was carried out inculture medium after washing. Spontaneous blinking of HMSiR was observed in the absence of
any additive in the intracellular environment and microtubule structures were successfully
constructed at far higher resolution than the projection image.
[1] Uno, S. et al. A spontaneously blinking fluorophore based on intramolecular spirocyclization
for live-cell super-resolution imaging. Nature Chemistry 6:681-689, 2014.

P5-09Introduction of DNA nanostructures and multiple µm-sized
objects into live cells by a novel cell-GUV electrofusion
methodAkira C. Saito1, Toshihiko Ogura2, Satoshi Murata1, and Shin-ichiro M. Nomura1
saito@molbot.mech.tohoku.ac.jp

ogura@idac.tohoku.ac.jp

murata@molbot.mech.tohoku.ac.jp

nomura@molbot.mech.tohoku.ac.jp

1

Department of Bioengineering and Robotics, Tohoku University, 6-6-01 Aramaki Aza-Aoba,
Aobaku, Sendai, Miyagi, Japan.
2
Department of Developmental Neurobiology, IDAC, Tohoku University, 4-1 Seiryo, Aobaku,
Sendai, Miyagi, Japan.

Keywords: GUV, electrofusion, artificial cell
Here we report a novel method for introducing large objects, up to a micrometerlevel, into live HeLa cells by electrofusion with an artificial giant unilamellar vesicle(GUV) [1]. We prepared the GUVs using the water-in-oil (w/o) emulsion centrifugation
method [2], by which artificial objects were encapsulated in them. HeLa cells and GUVs
were placed into an electrofusion chamber, then exposed to an AC field to align cells
and GUVs linearly. After this positioning, DC pulses were applied to induce transient
electrofusion and rapid transfer of objects from GUVs into cells. We successfully
demonstrated that plasmids (GFP, mCherry), designed DNA nanostructure (DNAorigami) and magnetic beads can be introduced into live cells by this approach. We also
confirmed that multiple artificial objects (an expression plasmid for mCherry protein and1 µm-sized fluorescent beads) can be transferred simultaneously into live cell. The
treated cells divided normally and reached confluency in culture without significant
damages. In addition, Hela cells, in which magnetic beads were introduced by thismethod, can be moved externally by a neodymium magnet.
Based on these results, we believe that our method can be used for
simultaneous transfer of multiple genes and artificial objects, which paves a new way for
elucidation of cell mechanisms, drag delivery system, and even creation of artificial
cells.

[1] Saito AC et al., Introducing micrometer-sized artificial objects into live cells, PLOS
ONE, In press.
[2] Natsume Y et al., Giant vesicles containing microspheres with high volume fraction
prepared by water-in-oil emulsion centrifugation, Chem Lett, 42, 2013.

P5-10A bright future: optogenetics and the FLIPR for voltage
gated ion channels
Sabrina Corazza, Alberto di Silvio, Sara Tremolada, Jean-Francois Rolland, Katharina
Montag, Viviana Agus, Loredana Redaelli, Lia Scarabottolo, Michela Stucchi, Stefan Lohmer
1

Axxam SpA, via Meucci 3, 20091 Bresso (Milano, Italy).

Keywords: optical, Ion channel, screening
Channelrhodopsin-2 (ChR2) is a light-activated microbial nonspecific cation channel, which canbe used to depolarize neurons through the incidence of blue light.[1] In this regard it is possible
to optically control the plasma membrane potential. This process opens new and interesting
perspectives for the characterization of voltage -gated ion channels and the search for modulators.
A proof of principle study was performed to verify the applicability of this tool to the developmentof cell based assays suitable for High Throughput Screening (HTS).
We generated a stable cell line, which expresses both ChR2 and the human voltage -gated calcium
channel Cav1.3 (hCav1.3). In addition, the inward rectifier hKir2.3 channel was stablytransfected to control the plasma membrane potential by adjusting the extracellular K +concentration.
We used a ChR2 variant which carries a single amino acid mutation. This results i n a prolonged
lifetime of the ChR2 conducting state and reduces the light energy required for its activation.
Two FLIPR TETRA protocols were created to perform light stimulation of ChR2, with subsequent
Cav1.3 activation, and also to induce a light depende nt Cav1.3 partial inactivation. Known Cav1.3
state dependent blockers were tested in the resting versus the partial inactivated conditions and theobtained results were in very good agreement with the ones obtained with both the classical“external K + protocol” and patch clamp experiments (Q-Patch).
Our study shows for the first time the application of an optogenetic tool on the FLIPR instrument,
for the purpose of running screening on voltage -gated ion channels in physiological extracellularpotassium concentration.
[1] Deisseroth K, Feng G, Majewska AK, et al.: Next-generation optical technologies forilluminating genetically targeted brain circuits. J. Neurosci. 2006; 26(41):10380-10386.

P5-11
Theoretical analysis of the formation of membrane tubes ingiant liposomes induced by electrostatic effectTamiki Umeda 1

umeda@maritime.kobe-u.ac.jp

	
  	
  	
  	
  Yohko Tanaka-Takiguchi 2

	
  	
 

Kingo Takiguchi 3
1

Graduate School of Maritime Sciences, Kobe University, Fukae-minami-machi
	  Higashinada-ku , Kobe 658-0022 Japan
2
Structural Biology Research Center, Nagoya University, Furo-cho, Chikusa-ku
Nagoya 464-8602 Japan
3
Graduate School of Sciences, Nagoya University, Furo-cho, Chikusa-ku
Nagoya 464-8602 Japan

Keywords: Lipid bilayer membranes, shape transformation, electrolyte, numerical calculations
Liposomes are closed vesicles formed by lipid bilayer membranes. They have been well studied as
simplified models of biological membranes, and are considered as a possible component formolecular robots. It is well known that some cytoskeletal proteins and cytosolic proteins induce
shape transformation of liposomes, such as the formation of long and thin membrane tubes from
globular vesicles. However, membranes can undergo spontaneous tubulation without proteins. We
observed giant liposomes composed of negatively charged lipids (PC/PI) and containing a sugar
solution, and found that when they were exposed to a hypertonic KCl solution, very thin membranetubes protruded into the interior of the spherical liposomes. To investigate the mechanism of thisphenomenon, we numerically calculated the free energy of curved membranes, which isdetermined from the surface charge density, the distribution of electrolytes in the aqueous solution,and the membrane elasticity. The result showed that unless the lipid molecules have bulky heads, acylindrical charged membrane may have lower energy than a flat one. This fact suggests that thethin tubes observed in the experiment may be induced by the electrostatic effect of the membranecharge.

P5-12
Loosely-stabilizing Algorithms for Leader Electionin Population Protocols

1

2

Fukuhito Ooshita 1

Yuichi Sudo 2

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

y-sudou@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, JapanNTT Secure Platform Laboratories, Midori-cho, Musashino, Tokyo 180-8585, Japan

Keywords: Distributed algorithm, Loose-stabilization, Population protocols, Leader election
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 distributed systems, where computing devices operate based on local information but they work
cooperatively as a system. For this reason, it is natural to apply distributed algorithms (i.e.,algorithms for distributed systems) to molecular robotics.
As a model to design distributed algorithms for molecular robotics, we focus on the population
protocol (PP) model [2]. In the PP model, devices are passively moved, and two devices
communicate with each other only when they come sufficiently close to each other (we call thisevent an interaction). The PP model is suitable to molecular robotics because typical moleculardevices are not fixed and they can sense only nearby devices.
In this poster, we introduce loosely-stabilizing leader election algorithms in the PP model. A
loosely-stabilizing algorithm [3] ensures that, starting from any initial configuration, a system
satisfies the problem specification within a relatively short time; after that, the system holds the
specification for a sufficiently long time. Such an algorithm is highly reliable because, even if
states of devices are changed due to some fault, the system recovers within a relatively short time.
Since molecular devices can fail, high reliability of algorithms is important. We introduce twoloosely-stabilizing leader election algorithms: Both algorithms elect a device as a leader within apolynomial time and keep the unique leader for an exponential time. The first algorithm [3]assumes that interaction between any pair of devices happens uniformly at random, and the second
algorithm weakens this assumption.
[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] Angluin, D., Aspnes, J., Diamadi, Z., Fischer, M. J., and Peralta, R., Computation in networksof passively mobile finite-state sensors, Distributed Computing, 18(4), 235-253, 2006.
[3] Sudo, Y., Nakamura, J., Yamauchi, Y., Ooshita, F., Kakugawa, H., and Masuzawa, T.,
Loosely-stabilizing leader election in population protocol model , Theoretical Computer
Sciences, 444, 100-112, 2012.

P5-13
pH-Responsive High-Density Lipoprotein for Drug
Delivery System

1

2

Tatsuya Murakami 1

Hyungjin Kim 1

murakami@icems.kyoto-u.ac.jp

hkim@icems.kyoto-u.ac.jp

Haruki Okamoto 1

Arnaud E. Felber 2

hokamoto@icems.kyoto-u.ac.jp

arnaud.felber@gmail.com

Anna Palomska 2

Jena-Christophe Leroux 2anna.palomska@pharma.ethz.chjean-christophe.leroux@pharma.ethz.ch

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University,
Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich,
Switzerland

Keywords: Nanobiotechnology, Nanoparticle, Internalization, Endosomal escapeEndosomal escape of drug-loaded nanoparticles is a key step to achieve the efficacy andspecificity of the drugs. High-density lipoprotein (HDL) is a natural drug carrier, and we
succeeded in development of a cell-penetrating high-density lipoprotein (cpHDL) via genetic
fusion with a cationic peptide derived from human immunodeficiency virus [1]. cpHDL was able todeliver an anticancer drug [1], an photothermal dye [2], and metal nanoparticles [3] into humancancer cells, and to potentiate their therapeutic actions. However, the majority of the cargoesdelivered by cpHDL was entrapped in the endosomes. Polyanions (PA) bearing both pendant
carboxylate groups and alkyl chains are able to destabilize the endosomal membrane in response to
mildly acidic endosomal pH. Here we sought to develop cpHDL/PA complexes for cytosolic drugdelivery by cpHDL.
[1] Murakami, T., Wijagkanalan, W., Hashida, M., Tsuchida, K., Intracellular drug delivery by
genetically engineered high density lipoprotein nanoparticles, Nanomedicine (London, U. K.),
5:867-879, 2010.
[2] Mathew, S., Murakami, T., Nakatsuji, H., Okamoto, H., Morone, N., Heuser, J. E., Hashida, M.,
Imahori, H., Exclusive photothermal heat generation by a gadlinium bis(naphthalocyanine)complex and inclusion into modified high-density lipoprotein nanocarriers for therapeutic
applications, ACS Nano, 7:8908-8916, 2013.
[3] Murakami, T., Nakatsuji, H., Morone, N., Heuser, J. E., Ishidate, F., Hashida, M., Imahori, H,
Mesoscopic metal nanoparticles doubly functionalized with natural and engineered lipidic
dispersants for therapeutics, ACS Nano, 8:7370-7376, 2014.

P5-14
Mathematical models of RNP-mediated synthetic translational
systems for precise control of the differentiation of iPS cells
Shota Suzuki1

suzuki@fun.bio.keio.ac.jp

Shunsuke Kawasaki2

kawasaki.shunsuke.44x@st.kyoto-u.ac.jp

Hirohide Saito2
hirohide.saito@cira.kyoto-u.ac.jp12Noriko Hiroi1

hiroi@bio.keio.ac.jp

Akira Funahashi1
funa@bio.keio.ac.jp

Funahashi-Hiroi Lab, Department of Biosciences and Informatics, Keio University,3-14-1, Hiyoshi,
Kohoku-ku Yokohama-shi, Kanagawa, 223-0061, Japan
Saito Lab, Center for iPS Cell Research and Application/The Hakubi Center, Kyoto University, 53,
Shogoin Kawaharacho, Sakyo-ku Kyoto-shi, Kyoto, 606-8397, Japan

Keywords: RNP-mediated synthetic translational systems, mathematical modeling, synthetic biology
Recently, it is possible to induce cellular differentiation and to develop various tissues and organs with iPS
cells which are generated from human somatic cells, such as epithelial cells. The application to regenerative
medicine of this biotechnology is expected. However, because of dispersion of the states of iPS cells or errorof the handling, efficient induction of differentiation is difficult to achieve.
Here we exhibit the strategy to induce aimed differentiation of iPS cells by applying RNP-mediated synthetictranslational system. Our synthetic translational systems detect the cellular state autonomously, and will
provide appropriate stimuli for differentiation at the each stage of the cell, respectively.
To design a desired synthetic translational system, we developed mathematical models which can simulate
the behavior of its dynamics in a cell. There exists an RNA/protein interaction-based synthetic translationalswitch to create a feedback system that tightly controls the expression of proteins [1]. It is the simplest andthe most efficient system among possible synthetic switch, however there is no RNA/protein interactionbased synthetic translational switch which may behave as a positive feedback system. Based on this fact, first
we designed a desired synthetic systems by applying artificial genetic circuits which consist of cis- and transregulatory modules [2] in addition to RNP-mediated synthetic translational systems. We simulated the
behavior of sequential and oscillation circuit.
Simulation results suggested that time delay or nonlinear systems are required to produce sequential andoscillation circuit. Our goal is to establish the new strategy which can provide safe and precise autonomous
system to control the fate of cells by RNP-mediated synthetic translational systems.
[1] “Feedback Control of Protein Expression in Mammalian Cells by Tunable Synthetic Translational
Inhibition”, James A Stapleton, Kei Endo, Yoshihiko Fujita, Karin Hayashi, Masahiro Takinoue, Hirohide
Saito and Tan Inoue, ACS Synthetic Biollogy, 1:3, 83-88, (2011).
[2] “A versatile cis-acting inverter module for synthetic translational switches”, Kei Endo, Karin Hayashi,
Tan Inoue, and Hirohide Saito, Nature Communications, 4:2393, (2013).

P5-15Novel voltage protocol for the characterization of hNav1.5
channel inhibition of drug candidates
Takashi Yoshinaga
t2-yoshinaga@hhc.eisai.co.jp

Kohei Sawada
k-sawada@hhc.eisai.co.jp

Global CV Assessment, Biopharmaceutical Assessments CFU, Eisai Co., Ltd., 5-1-3 Tokodai
Tsukuba-shi, Ibaraki 300-2635, Japan

Keywords: Drug screening, Ion channel, Automated-patch clamp system
The human cardiac sodium channel (hNav1.5) plays important roles such as generation of action
potential and propagation of excitation-conduction in the heart. Blockade of sodium channel canbe the causes of arrhythmia or cardiac dysfunction in clinical use. Therefore, hNav1.5 inhibition
may be considered as a risk factor in drug development. Detection and mitigation of Nav1.5
inhibition for drug candidates at early stage of drug development is very important. The patch
clamp assay was conducted using hNav1.5-CHL cells and the automated patch-clamp system,
IonWorks-Quattro. The voltage protocol used in this assay was a double pulse protocol which
including 1 sec duration of recovery period at -100mV to evaluate the state-dependency andbinding kinetics. The inhibitory activities of 6 typical sodium channel inhibitors such as
disopyramide (Dis), flecainide (Fle), lidocaine (Lid), mexiletine (Mex), propafenone (Pro) andquinidine (Qui) were evaluated and the peak amplitude of sodium current at 1st and 2nd
depolarization pulses was analyzed. The IC50 values at 1st pulse and 2nd pulse of Dis, Fle, Lid,
Mex, Pro and Qui were 183.4 and 217.0 uM, 2.0 and 3.0 uM, 107.1 and 466.0 uM, 5.4 and 69.1 uM,
0.3 and 0.7 uM, 7.4 and 9.8 uM, respectively. The concentration-inhibition curves for the 1st pulseof Lid and Mex were dramatically shifted to the left, but those of Dis, Fle and Qui were not shifted
so much. These variations of curve shift are thought to be due to the differences of dissociation
kinetics of each drug at -100 mV. It is concluded that the double pulse protocol is useful for
elucidation of Na channel inhibition kinetics and will contribute to the integrated risk assessment
for the cardiac function.

P5-16
In Vitro Selection of Functional Peptides with
Pore-Forming Ability to Liposomes
Toshiki Miyajima 1

Yuki Yoshokawa 1,§

S14mp232@mail.saitama-u.ac.jp

yoshikawa@tetrapak.com

Naoto Nemoto 1
nemoto@fms.saitama-u.ac.jp

1

Graduate school of Science and Engineering, Saitama University, 255 Shimo-Okubo,
Sakura-ku, Saitama 338-8570, Japan§ Present Address: Nihon Tetra Pak K.K., 755-1 Jimba-Uenohara, Gotemba-shi, Shizuoka
412-0047, Japan

Keywords: Antimicrobial peptide, cDNA display, liposome
Antimicrobial peptides that bind to the cell membrane of the bacteria, indicating bactericidal
activity are found naturally in a wide variety of organisms, including amphibians, insects, humans,and plants 1 .This mechanisms of action is due to the fact that these peptides form pores in the cell
membrane, and to flow out the cytoplasm. Recently these peptides are also attracting attention in
the application to the Drug Delivery System and new therapeutic drug development 2 . Structuralfeatures of antimicrobial peptides are amphipathic with hydrophobic and hydrophilic clusters, areto take the secondary structures.
By using cDNA display method which is an in vitro display technology, we have alreadysucceeded in vitro selection of functional peptides having liposome membrane anchoring ability
from a random peptide library of 28 amino acids in length . Then we discuss about the next
experimental system to obtain the functional peptide with pore-forming ability to the liposome
membrane by considering the application to Drug Delivery, by cDNA display method using a
Fluorescence Activated Cell Sorting (FACS).
[1] Diamond, G., Beckloff, N., Weinberg, A., kisich, K. O., The roles of antimicrobial peptides ininnate host defense. Curr. Pharm. Design, 15:2377-2392, 2009.
[2] Gordon, Y.J., Romanowski, E.G. and McDermott, A.M., A review of antimicrobial peptides andtheir therapeutic potential as anti-infective drugs. Curr Eye Res, 30:505-515, 2005.
[3] Mochizuki, Y., Biyani, M., Tsuji-Ueno, S., Suzuki, M., Nishigaki, K., Husimi, Y. and Nemoto,N., One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin -linker
DNA. ACS Comb Sci, 13:478-485, 2011.

P5-17
Image-based drug profiling reveals a dual inhibitor of
EGF receptor tyrosine kinase and microtubules
Kenji Tanabe 1

tanabe.kenji@twmu.ac.jp

1

Medical Research Institute, Tokyo Women’s Medical University, 8-1
Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

Keywords: High contents analysis, Drug profiling, Membrane traffic, Signal transduction
Small-molecule inhibitors are widely used as tools for biology and therapeutic drugs, anduncovering the target specificity provides a valuable insight into both basic and clinical studies.
Here, we present image-based inhibitor profiling of EGF receptor pathway by quantitating theireffect on both receptor trafficking and signal transduction. Unbiased multivariate analysis
classified fourteen inhibitors into four clusters. Interestingly, EGFR inhibitor, a highly
uni-specific tyrosine kinase inhibitor, was classified with a microtubule depolymerizer. Actually,
EGFR inhibitor had microtubule depolymerizing activity both in vivo and in vitro, and also had anantimitotic activity. Our work indicates that image-based multivariate analysis is a powerful toolfor discovering an unexpected drug properties, and EGFR inhibitor become as a novel seeds for
multi-targeting cancer drug.

P5-18Novel Cell- or Tissue-Markers Based on Giant vesicles
Taro Toyota 1,2
cttoyota@mail.ecc.u-tokyo.ac.jp

12

Department of Basic Science, Graduate School of Arts and Sciences, The Universityof Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
Department of Bioinformatics, Graduate School of Medicine, Chiba University, 1 -8-1
Inohana, Chuo-ku, Chiba 260-8670, Japan

Keywords: Giant vesicle, Magnetic resonance imaging, Infrared fluorescence imaging
The development of new technologies for imaging of li ving bodies or organs using celland tissue-markers to ensure good spatial resolution, sensitivity, and detectability is one of themost important subjects in pharmaceutical and medical treatment research. Here we introduce cellor tissue-markers based on giant vesicles (GVs) developed by our group.
High-field magnetic resonance imaging (MRI) with contrast medium is already usedclinically, and the design of new MRI probes for pharmaceutical and medical applications has
attracted significant attention. Super-paramagnetic iron oxide (SPIO) is a promising candidates forthe development of smart and functional probes. Here GVs that encapsulate SPIO particles at highnumber density have become a research focus. [1] Phospholipid membranes are natural membranes
that are degradable in vivo. The encapsulation of SPIO particles at a high number density in GVs
yields SPIO-containing GVs (SPIO-GVs) that can be used as highly sensitive MRI probes. The
SPIO-GVs were composed of a single lamellar membrane with a diameter of 4.7 ± 2.2 μm. Afterinjection of the SPIO-GV into one cell of a 4-cell stage medaka embryo with a glass capillary andfixing with paraformaldehyde, dark spots (typical diameter, 200 μm) were observed at theblastomeres of the injected cells in micro MRI. This indicated that the GV-based SPIO MRI
contrast agent could be effective for enhancement of the MRI signal at the cellular level.
Rapid in situ imaging of human tissues is required for surgical applications. Specifically,various techniques using preoperative or intraoperative gastrointestinal endoscopies to assist with
the intraoperative localization of tumors have been proposed. Near-infrared (NIR) fluorescentmolecules, such as indocyanine green (ICG) [2] , have drawn much attention because the NIR light
can permeate organ tissues and can be excited and detected with a fluorescence CCD camera.
GV-based tissue markers, including ICG, are prepared using three steps [3] : (i) mixing of ICG withlecithin GVs;, (ii) suspension of the ICG-containing giant vesicles (ICG-GV) in an oil phasedissolving polyglycerol-polyricinoleate (PGPR), and (iii) centrifugation of the suspension layeredon a buffered solution to obtain a giant polymer vesicle (polymerasome) containing ICG -GV.
When the tissue marker was injected into the inner gastric surface of an anesthetized pig using an
endoscopic syringe and the injection site was observed using a laparoscopic fluorescence camera,
the diameter of the spot blur was approximately 2 cm over a 5 -h period, which can be used todetermine the location of early-stage tumors of several centimeters in diameter.
[1] Toyota, T., Ohguri, N., Maruyama, K., Fujinami, M., Saga, T., Aoki, I., Giant Vesicles
Containing Superparamagnetic Iron Oxide as Biodegr adable Cell-Tracking MRI Probes, Anal.
Chem. 84:3952-3957, 2012.
[2] Landsman, M.L., Kwant, G., Mook, G.A., Zijlstra, W.G., Light-absorbing properties, stability,
and spectral stabilization of indocyanine green, J. Appl. Physiol. 40:575-583, 1976.
[3] Hatayama, H., Toyota, T., Hayashi, H., Nomoto, T., Fujinami, M., Application of a Novel Near
Infrared-Fluorescence Giant Vesicle-and Polymerasome-based Tissue Marker for Endoscopic
and Laparoscopic Navigation, Anal. Sci. 230:225-230, 2014.

P5-19Hybrid Systems as Controllers of Molecular Robots--- A Case Study
Shaoyu Wang 1 Nathanael Aubert-Kato 2 Masami Hagiya 1
1
2

Department of Computer Science, University of Tokyo
Leading Graduate School Promotion Center, Ochanomizu University

Keywords: Hybrid systems, molecular robotics, DNA computing, DNA PEN toolbox, Seesaw gate
Hybrid systems such as hybrid automata are often used tomodel controllers that process continuous values from sensors
measuring the environment. Controllers for molecular robots
are no exceptions. In this case study, we design a hybrid systemthat controls a “Paramecium Robot”, which senses theconcentration of “food”, and moves on until the position withthe highest concentration is found.
The robot senses the concentration of “food” at its current
position. When the food concentration “x” gets higher, therobot stays in the state A (see Figure) and goes straightforwards. In this state, the concentration of “r” is used to store
the value of “x”. When the food concentration gets lower or the
robot stays in A for a long time, it transfers to B and tumbles todirection. In this state, “r” remains constant. Furthermore, after
the state B becomes unstable and the robot returns to A.

changeitssome delay,

We use the PEN Toolbox[1] to realize
the states and state transitions. A typicalbi-stable system[2] consisting of thestates A and B is used, where thetransitions between the two states arecontrolled by “r” and “x”. We use theSeesaw Gate[3] to approximate theassignment of “r:=x”. The delay wasrealized by making the bi-stable system
asymmetric so that B is weaker than A.
Time trace for specific distribution of “x”
is shown to the right.
However, while this system can be
implemented by chemical reactions, itcannot be formulated as a hybrid
automaton, prompting us to design another model with three states. Both the states A and C are for“going straight forwards” and B is for “tumbling to change direction”. The value of “r” is updated(by r:=x) in B and C. When “x” falls down, the robot transfers to B, and when “x” rises, it transfers
to C. After some delay, it returns to A.
[1] K. Montagne, R. Plasson, Y. Sakai, T. Fujii, and Y. Rondelez. "Programming an in vitro DNA
oscillator using a molecular networking strategy." Molecular systems biology 7, no. 1 (2011)[2] A. Padirac, T. Fujii, and Y. Rondelez. "Bottom-up construction of in vitro switchable
memories." PNAS109, 47 (2012)[3] L. Qian, E. Winfree, "Scaling up digital circuit computation with DNA strand displacement
cascades." Science 332, no. 6034 (2011)

P5-20Creation and manipulation of an artificial cellular
membrane
Tsutomu Hamadat-hamada@jaist.ac.jp

School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST)
1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan

Keywords: lipid bilayer, photo-control, membrane interaction, vesicle transformation
In living cells, bio-molecules self-assemble in a predetermined fashion under specific conditions
into highly organized supramolecular structures with specific functions. The constructive andsynthetic approach to biological cells is an important and interesting challenge that spans acrossseveral disciplines, including physics, chemistry and biology. A closed two-dimensional surface
consisting of a lipid bilayer is an essential compartment of living organisms. We have developed
an artificial cellular membrane that exhibits biological functions [1]. We utilized synthetic
photosensitive molecules to achieve the photo-based manipulation of membrane transformations[2]. The successful photo-manipulation of mesoscopic membrane structures in a noncontact andreversible manner may see wider application, such as in micro-reactors and smart drug-delivery
systems (SDDS). We then investigated the association of nano-materials, such as nano-particles(NPs), on a model membrane surface [3]. We found that lateral heterogeneity in the membrane
mediates the partitioning of NPs in a size-dependent manner. These studies may lead to a better
understanding of the basic mechanisms that underlie the association of nano-materials within a cellsurface, and to a design of artificial cell systems that recognize contacting objects.
[1] Hamada, T. & Yoshikawa, K., Cell-sized liposomes and droplets: real-world modeling of living
cells, Materials, 5:2292-2305, 2012., Himeno, H. et al., "Charge-induced phase separation inlipid membranes" Soft Matter (in press)., Morita, M. et al., "Endo- and Exocytic Budding
Transformation of Slow-Diffusing Membrane Domains Induced by Alzheimer’s Amyloid Beta"
Phys. Chem. Chem. Phys., 16:8773-8777, 2014.
[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.

P5-21
Oscillatory Population Protocols
Colin Cooper 1
Colin.cooper@kcl.ac.uk

Giovanni Viglietta 3
viglietta@gmail.com

Anissa Lamani 2lamani@csce.kyushu-u.ac.jp

Masafumi Yamashita 2
mak@csce.kyushu-u.ac.jp

Yukiko Yamauchi 2
yamauchi@inf.kyushu-u.ac.jp
1

Department of informatics, Kings College, UK
Graduation school of information science and electrical engineering, Kyushu University, Japan3
School of computer science, Carleton University, Ottawa, Canada
2

Keywords: Population protocols, Oscillatory behavior, Distributed algorithms, Molecular robots
Understanding how autonomy emerges in biological systems and applying it in giving artificial
distributed systems autonomous properties motivate our study. Precisely, we focus on
self-oscillations that play crucial roles in autonomous biological reactions, and investigate them asa phenomenon in distributed computing. Self-oscillations are often understood as a chemical
oscillator provided, for example, by the Belousov–Zhabotinsky reaction. In biological systems, theoscillatory behavior is used as a natural clock to transmit signals and hence transfer information.
In artificial distributed systems, self-oscillations could be used to distributely and autonomously
implement a clock. This problem emerges in the project of designing molecular robots [2].
In our investigation, we use the population protocol (PP) model introduced by Angluin et al. [1].
PP is used as a theoretical model of a collection of finite-state mobile agents that interact witheach other in order to solve a given problem in a cooperative fashion. Computations are done
through pairwise interactions and the interaction pattern is unpredictable. PPs can represent not
only artificial distributed systems as sensor networks and mobile agent systems, but also natural
distributed systems such as chemical reactions and biological systems. We aim in our work at
designing algorithms that make a given population exhibits an oscillatory behavior by itself
whatever its initial state.
[1] D. Angluin, J. Aspnes, Z. Diamadi, M J. Fischer, and R. Peralta. Computation in networks ofpassively mobile finite-state sensors. In Proc of PODC, pp 290–299, 2004.
[2] S. Murata, A. Konagaya, S. Kobayashi, H. Saito, and M. Hagiya. Molecular robotics: A newparadigm for artifacts. New Generation Computing, 31(1):27–45, 2013.

P5-22Development of a hybrid potassium ion probe for detectionof local potassium ion transition on cellular membrane
Tomoya Hirata 14415682657@mail.ecc.u-tokyo.ac.jp

Tetsuo Nagano 3
tlong@mol.f.u-tokyo.ac.jp

Takuya Terai 1
t-terai@mol.f.u-tokyo.ac.jp

Yasuteru Urano 1,2
uranokun@m.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
Graduate School of Medicine, the University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan
3
Open Innovation Center for Drug Discovery, the University of Tokyo , 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan

Keywords: fluorescent probe, potassium channel, protein labeling, imaging
Potassium ion (K + ) is the most abundant metal cation inside living ce lls, which determines the levelof resting membrane potential. Hence, real-time analysis of potassium ion (K + ) dynamics near cellular
membrane brings us important biological insights. Lately, several fluorescent K + probes have beenreported, but they are not capable of telling the difference between average extracellular [K + ] change
and local [K + ] transition around the cells of interest, due to the suboptimum localization of the probes.
To solve this problem, we conceived a strategy to selectively tether a fluorescent K + probe to the outersurface of plasma membrane by HaloTag technology (Figure 1a).
We synthesized TLShalo, a K + -selective, photoinduced electron transfer (PeT) -regulated fluorescent
Halo ligand. As fluorophore we adopted boron dipyrromethene (BODIPY) derivative, which has
excellent optical properties for cell imaging. As a chelator moiety we selected triazacryptand (TAC)
structure, which was reported to have favorable affinity for K + and selectivity against Na + . To give
sufficient membrane-impermeability to the probe, we introduced multiple anionic functional groups tothe structure. As a result, fluorescence intensity of TLShalo elevated up to 10 -fold as K + concentration
increased from 0 to 150 mM, even in the presence of Na + (Figure 1b). TLShalo selectively labelledcellular surface of HeLa cells expressing HaloTag-fused protein and detected K + concentration change
in extracellular medium. With the system, we could detect K + efflux induced by K + ionophore on thecellular membrane of HeLa cells under a cover glass that restricted K + diffusion. Further, wedemonstrated that TLShalo could monitor local [K + ] transition near the cellular surface at subcellular
resolution by using microinjection apparatus (Figure 1c, d).
We are now trying to detect real-time K + channel activity by labeling potassium channel directlywith TLShalo.
Figure 1 (a) Schematic image offluorescentK+probe-based
K+
detection system. (b) Emission spectraof 1 M TLShalo in HEPES buffer
containing indicated concentration of
K + . (c)Epi fluorescent microscopic
image of HeLa cells labelled with 2
M
TLShalo.
(d)Fluorescenceintensity ratio (F/F0) transition ineach ROIs as a function of time.
During experiment, 150 mM KCl aq.
was effused every 2.5 seconds from a
micro-injector needle.

P5-23
Reaction Graphs Controlled by External Signals
Youji Hasegawa 1
h1331084@edu.uec.ac.jp

1

Satoshi Kobayashi 1
kobayashi.satoshi@uec.ac.jp

Depertment of Communication Engineering and Informatics
Graduate School of Informatics and Engineering,
The University of Electro-Communications,
1-5-1, Chofugaoka, Chofu, Tokyo 182-8585, JAPAN

Keywords: Molecular robotics, DNA nanotechnology,
In the field of molecular computing, researchers aim to implement information processing bychemical reaction of biopolymers. Especially computation using DNA is prosperous. There has been
proposed many logic circuits using DNA([1], [2], [3], etc.). In particular, seesaw gate advanced by
Qian and Winfree is a very promising molecular device with which we can construct large scale logic
circuits. The progress of DNA computing technology may contribute to that of DNA nanotechnology.
Yin, et al. developed an elegant DNA computing system which controls, or programs, bio -molecular
self-assembly pathways of DNA nanostructures. They formulated the self -assembly reaction by the useof graphs, where each node represents DNA motif and each edge represents assembly or disassembly
reaction. The system is called a "reaction graph".
Photochemical devices are also regarded as important nano-tools for DNA computing. There has
been some studies on the application of photo-reactive devices to the implementation of molecular
computing models. For example, Fujimoto's photo-reactive devices were used to construct a DNA logic
circuit, full adder([5]). Tanida, et al., utilized azobenzene to the implementation of photonic
automaton([6]). Thus, it is interesting to apply photochemical devices to the control of self -assembly
pathways of DNA nanostructures. In this poster presentation, we will study on the computational modelfor the control of self-assembly pathways of DNA nanostructures by external signal. As externalsignals, we consider photo-irradiation of specific wavelength or temperature change, etc. In this study,
we start from the computational model, "reaction graph", proposed by Yin, et al., and extend it so thatit can deal with signals from outside the system.

[1] Seelig, G., Soloveichik, D., Zhang, D.Y., and Winfree, E., Enzyme-free nucleic acid logic
circuits, Science, 314, 1585-1588, 2006
[2] Stojanovic, M. N., Mitchell, T. E., and Stefanovic, D., Deoxyribozyme-based logic gates, J Am
Chem Soc, 124, 3555-3561, 2002
[3] Lulu Quian, Erik Winfree, Scaling Up Digital Circuit Computation with DNA Strand
Displacement Cascades, Science, 332, 1196, 2011[4] Peng Yin, Harry M.T. Choi, et al, Programming biomolecular self-assembly pathways, Letters,
Nature, Vol.451, 2008[5] Shinzi Ogasawara, Takehiro Ami, and Kenzo Fujimoto, Autunomous DNA Computing Machine
Based on Photochemical Gate Transition, J Am Chiem Soc, 2008
[6] Hiroto Sakai, Yusuke Ogawa, and Jun Tanida, Positional State Representation and Its
Transition Control for Photonic DNA Automaton, DNA15, LNCS 5877. pp. 126-136, 2009

P5-24
Automated Microtubule Path Tracking on Gliding Assay
Using Dynamic Programming
Bulibuli Mahemuti 1
bulbul.aa@m.titech.ac.jp

Yuexing Han 1, 2
hanyuexing@gmail.com

Daisuke Inoue 3
daisukeinoue@mail.sci.hokudai.ac.jp

Akira Kakugo 3, 4
kakugo@sci.hokudai.ac.jp

Akihiko Konagaya 1
Lastname3@cbi-society.org

1

2

3
4

Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama, 226-8502, Japan
School of Computer Engineering and Science, Shanghai University, 99 Shangda Road, BaoShan
District, Shanghai, 200444, China
Faculty of Science, Hokkaido University, Sapporo 060 -0810, Japan
Graduate School of Chemical Sciences and Engineering, Hokkaido Universit y, Sapporo
060-0810, Japan

Keywords: Bio-image informatics, object tracking, microtubule gliding assay, microtubule
movement mechanism
Microtubule gliding assay is a biological experiment observing the dynamics of microtubules
driven by motor proteins fixed on a glass surface when ATPs are dosed, video data of
microtubules’ movement can be obtain from gliding assay. In order to analyze the microtubule
gliding mechanism from video data, computational methodologies that can automatically track thepaths in microtubule gliding assay video data are urgently needed.
In microtubule gliding assays, object tracking becomes non -trivial due to the occurrences ofcompound objects such as crossing and snuggling of microtubules as well as sudden appearance
and disappearance of microtubules. In order to solve these issues, we discuss the object tracking
methodology using a dynamic programming.
These microtubule tracking paths can enhance our understanding of the dynamics of microtubule
movement. Our future work will focus on the improvement of the recognition accuracy andmechanism of microtubule turning.
[1] A. Kakugo, J. P. Gong et al., Formation of ring-shaped assembly of microtubules with a narrowsize distribution at an air–buffer interface. Soft Matter, 8:10863-10867, 2012.
[2] B. Mahemuti, Y. Han, D. Inoue, A. Kakugo, A. Konagaya , Automated Microtubule Path
Tracking on Gliding Assay Using Hidden Markov Model, oral presentation in International
Conference on Bioinformatics (InCoB 2014), July 31-Aug. 2, Sydney, Australia, 2014.

P5-25Comparison of field potential measurement and clinicalinformation with in vitro system to evaluate
arrhythmogenic potentials of compounds using human
iPS-cell derived cardiomyocytes
Tetsuo Kitamura 1

Mototsugu Sakakibara 1

Kitamura.Tetsuo@mh.medience.co.jp

Sakakibara.Mototsugu@mx.medience.co.jp

Fumihide Bunai 1

Daisuke Minami 1

Bunai.Fumihide@mv.medience.co.jp

Minami.Daisuke@mg.medience.co.jp

Mayumi Obo 1

Tomoharu Osada 1

Obo.Mayumi@ms.medience.co.jp

Osada.Tomoharu@mg.medience.co.jp

Hiroaki Inoue 1

Yasuyuki Oonishi 1

inoue.hiroaki@mb.medience.co.jp

Oonishi.Yasuyuki@mp.medience.co.jp

Hideaki Hiratsuka 1
Hiratsuka.hideaki@mg.medience.co.jp
1

Nonclinical Research Center, Drug Development Service Segment, LSI Medience Corp. 14-1
Sunayama, Kamisu, Ibaraki, 314-0255, Japan

Keywords: drug development, safety pharmacology, human iPS-cell derived cardiomyocytes, fieldpotential measurement, arrhythmogenic
To provide a better in vitro assay system to predict arrhythmogenic during the early stages of drug
development including preclinical study, we propose here a drug screening system using the fieldpotential (FP) measurement with human induced pluripotent (iPS) stem cell-derived
cardiomyocytes. To accurately predict arrhythmogenic property of test compounds, several indicesof FP have been proposed. In this study, we examined whether FPD prolongation with STV
increase and expression of early afterdepolarization (EAD) were effective for the prediction ofarrhythmogenicity in vitro.
We collected FP recording data from 36 compounds. Exposure of 31 compounds to the
iPS-derived cardiomyocytes was conducted according to plasma concentrations in clinical studies.
EAD or triggered activity was detected at concentrations within 1000 times more than humanplasma concentration in 15 of 18 drugs, which are characterized as arrhythmogenic. Combinedwith the observation of FPD prolongation and STV increase, all 18 drugs were evaluated asarrhythmogenic. On the other hand, EAD or triggered activity was detected in only 1 of 13 drugs,in the interview form of which, no description on the risk of torsades de pointes was seen. Insummary, concordance rate was found at 30/31 (97%), the false positive was 1/31 (3%) and thefalse negative was 0/31 (0%).
The screening system presented here showed an ability to predict the arrhythmogenic property ofknown compounds under the indices taken here. In this study, antiarrhythmic drugs were mainly
tested in order to obtain a fundamental data in initial trials. Other compounds including anti-cancer
or anti-psychiatric drugs will be the next challenge to improve sensitivity in the prediction ofarrhythmogenicity with the system.

P5-26Chronic probucol treatment decreases the slow componentof the delayed-rectifier potassium current
in CHO cells transfected with KCNQ1 and KCNE1
Tomohiko Taniguchi 1t2-taniguchi@hhc.eisai.co.jp

Taro Hihara 1
t2-hihara@hhc.eisai.co.jp

Kohei Sawada 1
k-sawada@hhc.eisai.co.jp

1

Eisai Product Creation Systems, Eisai Co., Ltd., Tokodai 5-1-3, Tsukubashi,
Ibaraki 300-2635, Japan

Keywords: Ion channel, cardiac safety and pharmacology
Voltage-gated potassium (KV) channels can form heteromultimeric complexes with a variety ofaccessory proteins, including KCNE subunit. KCNQ1 (KV7.1) together with KCNE1 generates theslow component of delayed rectifier potassium current (IKs) that important for cardiac
repolarization. We had reported that acute treatment of an anti-hyperlipidemic drug probucol did
not inhibit IKs current in CHO cells coexpressing KCNQ1 and KCNE1 but chronic treatment of the
probucol inhibited IKs current of the cells dose-dependent manner. Western blotting analysis
suggested the disruption of the heteromeric complex formation could be the inhibition mechanism.
Then, we had tried to reveal in detailed mechanism of chronic probucol’s effect on the IKs current.
Heterologous expression studies have demonstrated different functional modulation effects ofprobucol between IKs complex and KCNQ1 molecule. Probucol completely inhibited the IKs
current in CHO cells expressing both KCNQ1 and KCNE1 genes (KCNQ1/KCNE1-CHO) at aconcentration of 1 μM. However, KCNQ1 current in CHO cells expressing KCNQ1 gene alone(KCNQ1-CHO) was not reduced by probucol at concentrations up to 10 μM. These results suggestthat chronic probucol treatment may contribute to KCNE1 molecule mediated IKs function. Then,because of probucol has cholesterol-lowering effect in the cells; we examined the effects ofanother type of cholesterol-lowering drugs, simvastatin and triparanol, on the IKs and KCNQ1
current using KCNQ1/KCNE1-CHO and KCNQ1-CHO cells, respectively to determine detailed
inhibition mechanisms of the probucol on the IKs current.
[1] Taniguchi, T., Uesugi, M., Arai T., Yoshinaga, T., Miyamoto, N., and Sawada, K., Chronic
Probucol Treatment Decreases the Slow Component of the Delayed-Rectifier Potassium Current
in CHO Cells Transfected With KCNQ1 and KCNE1: A Novel Mechanism of QT Prolongation, J
Cardiovasc Pharmacol, 377-386, 2012.
[2] Hihara, T., Taniguchi, T., Ueda, M., Yoshinaga, T., Miyamoto, N., and Sawada, K., Probucol
and the cholesterol synthesis inhibitors simvastatin and triparanol regulate Iks channel function
differently, Human and Experimental Toxicology, 1028-1037, 2013.

P5-27

Monitoring Cellular Response to Ion Channel Blocker with
Motion Vector Prediction Method

Kazuhiro Nakagawa
Kazuhiro.Nakagawa@jp.sony.com

Tomohiro Hayakawa
Tomohiro.Hayakawa@jp.sony.com

Shiori Oshima
Shiori.Oshima@jp.sony.com

Eriko Matsui
Eriko.Matsui@jp.sony.com

LCI Business Dept. Life Science Business Div. Medical Business Unit, Sony Corporation, 1-7-1
Konan, Minato-ku, Tokyo 108-0075, Japan

Keywords: Cellular motion, Ion channel, Induced pluripotent stem cell
We present a noninvasive method for the characterization of cellular motion behavior and its
applicability for cardiac and neural cell. Motion Vector Prediction (MVP) method based on a blockmatching algorithm calculates motion vectors, which have a velocity dimension, with various
spatiotemporal resolutions of the series of time-lapse phase contrast microscopic images.
Especially, the MVP method with high spatiotemporal resolution images allows us to analyze themotion vector length with sub-micrometer resolution. We have established an evaluation forcontractile behaviors of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs).
Simultaneous measurements of contractile motion and field potential revealed that theelectrophysiological behaviors of hiPS-CMs are quantitatively reflected in the contractile motion.
We also demonstrated that contractile motion prolife of hiPS-CMs was affected by various ionchannel blockers in a dose-dependent manner. Furthermore, by using this method, we could detectsub-micrometer motion of human iPS cell-derived neural cells (hiPS-Neurons) and rat primarycultured cortical neural cells. Surprisingly, voltage-gated and ligand-gated Na + channel blockers
inhibited the neural cell motion. Extracellular Na + deprivation also inhibited neural cell motion,suggesting that cellular ion flux affected neural cell motion. We will discuss the biological basis ofneural cell motion and the applications of our high spatiotemporal resolution motion analysis tomonitor cellular ion flux in various cells.
[1] Hayakawa, T., Kunihiro, T., Dowaki, S., Uno, H., Matsui, E., Uchida, M., Kobayashi, S.,
Yasuda, A., Shimizu, T., Okano, T., Noninvasive evaluation of contractile behavior of
cardiomyocyte monolayers based on motion vector analysis, Tissue Eng. Part C Method, 18(1):21-32, 2011.
[2] Hayakawa, T., Kunihiro, T., Ando, T., Kobayashi, S., Matsui, E., Yada, H., Kanda, Y.,
Kurokawa, J., Furukawa, T., Image-based evaluation of contraction-relaxation kinetics ofhuman-induced pluripotent stem cell-derived cardiomyocytes: correlation and complementarity
with extracellular electrophysiology, J. Mol. Cell. Cardiology, in press

P5-28Contribution of quantitative change in funny current (If) toelectrophysiological properties of developing embryonicventricular cells: a simulation study
Maiko Wakita1,2
t13996mw@sfc.keio.ac.jp

Hitomi I. Sano1,2
ducky@sfc.keio.ac.jp

Yasuhiro Naito1,2,3

Masaru Tomita1,2,3

ynaito@sfc.keio.ac.jp

mt@sfc.keio.ac.jp1

Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa 252-0882, Japan
Department of Environment and Information Studies, Keio University, Fujisawa, Kanagawa
252-0882, Japan
3 Systems Biology Program, Graduate School of Media and Governance, Keio University,
Fujisawa, Kanagawa 252-0882, Japan
2

Keywords: myocardium, pacemaker channel, embryonic development, mathematical modelIn early embryonic development, the heart develops and gains new functions while continuously pumping blood,and heart abnormalities progress to congenital heart malformations; therefore, the developmental program of theheart, including the expression of the genes responsible for various ionic channels, is likely to be tightly regulated.
The quantitative changes in ionic channels, pumps, exchangers, and sarcoplasmic reticulum Ca2+ kinetics are
responsible for the changes in electrophysiological properties of the developing cardiomyocytes. Previously, wedemonstrated that the developmental changes in action potentials (AP) of ventricular myocytes were well
represented, as Na+ current (INa) increased before the disappearance of and funny current (If), followed by a 10fold increase in inward rectifier K+ current [1] via simulation using a comprehensive cardiomyocyte model, theKyoto model [2]; briefly, the relative conductances of the 9 components were switched between early embryonic(EE) and late embryonic (LE) values and simulated the 512 (29) combinations of the model. Here, we constructeda model to represent “middle” embryonic (ME) stage of guinea pig ventricular cell on the basis of experimental
data, shifted relative current densities of the 9 components among EE, ME, and LE stages, in order to further
overview the functional landscape of developmental changes in embryonic ventricular cells. As a result, we showthat the relative conductance of If should be decreased by 80% by ME stage in order to avoid unphysiologicalbehavior of ventricular cell in latter half of embryonic development.

[1] Okubo C, Sano HI, Naito Y, Tomita M. Contribution of quantitative changes in individual ionic current
systems to the embryonic development of ventricular myocytes: a simulation study. J Physiol Sci,
63:355-367, 2013.
[2] Kuzumoto M, Takeuchi A, Nakai H, Oka C, Noma A, Matsuoka S Simulation analysis of intracellular Na+ and Cl- homeostasis during bata1-adrenergic stimulation of cardiac myocyte. Progress in Biophysics andMolecular Biology, 96:171– 186, 2008.

P5-29
Revised Modeling of Self-oscillating Gel
including Experimental Results

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 the gel which pulsates spontaneously itself using the
dynamics of BZ reaction inside the gel. Recently, it is applied to several kinds of devices
as an actuator or micro pump. To clarify its mechanism, we have made the model basedon dissipative particle dynamics method, and perform the simulations of self oscillatinggel.
In our previous model, the oscillation occurs along the change of the interaction
parameter between polymer and solvent, and its value is changed along the sin-type
oscillation according to our computational convenience. In the real self oscillating gel,
BZ reaction occurs in the reaction-diffusion system, and the real oscillation of BZ
reaction is not like the sin-type oscillation. Therefore we need the revision of oursimulation model.
To represent self oscillating gel more realistically, we must include the realoscillation of BZ reaction from the experimental results. In the BZ reaction, change ofreaction step can be observed by its color. Color is changed periodically from yellow togreen. In the self oscillating gel, the changes of color also occur. In the shrinking state,color is from pale orange to yellow. In the swelling state, the colors change in an
opposite manner. These changes of color are analyzed using our OCTA system. The
color of gel is observed as a RGB value using the snapshots in each steps from themovie of self oscillating gel. From our analysis, in the swelling steps, the color changes
rapidly, and in the shrinking steps, the color changes much slower.
These changes are included in our revised model. The change of interaction
parameter is set along the change of RGB value. Using this parameter changes, we
perform the DPD simulation. From our simulations, the result of change of thickness in
swelling-shrinking dynamics is modified from our results in our previous model. Detailof our simulation results and difference from our previous model will be shown in thesession.
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)

P5-30On Time Responsive DNA Analog Computing Devices
Kazuya Yanagibashi 1

Satoshi Kobayashi 1
kobayashi.satoshi@uec.ac.jp

Ken Komiya 2
1
2
3

Kenzo Fujimoto 3

Department of Communication Engineering and Informatics, Graduate School of Informatics
and Engineering, The University of Electro -Communications
Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate
School of Science and Engineering, Tokyo In stitute of Technology
Department of Materials Science, Graduate School of Materials Science, Japan Advanced
Institute of Science and Technology

Keywords: DNA computing, Molecular robotics, DNA analog circuit
Since Adleman's seminal work on a DNA implementation of an algorithm for solving a directed
Hamiltonian path problem, it is well recognized that the property that nucleic acid hybridization
based on Watson-Crick complementarity enables us to construct chemical reaction systems withcomputational function ([1]). There has been many research results on the design andimplementation of logic circuits using nucleic acid strands ([2] [3], etc.). In particular, Qian’s
work on seesaw gates made it possible to largely scale up the size of circuits ([3]). In most of theprevious works, however, the proposed circuits are not time-responsive, where we say that a circuit
system is time-responsive if, when inputs to the circuit change after the initial computation, theoutput is re-computed to reflect the new inputs ([4]). Although time-responsive circuits were
proposed in some previous works, they deal with only discrete systems ([4] [5], etc.).
In this poster presentation, we will propose a new approach to the implementation of time
responsive DNA analog computing elements. Distinct features of the proposed devices is that theyutilize kinetic relationship of concentrations of input and output molecules at steady state and thatthe change of the concentration of the input molecule will cause the change of the concentration of
the output molecule according to the kinetic relationship. Therefore, the proposed analog device istime-responsive.
[1] L. Adleman, Molecular Computation of Solutions to Combinatorial Problems. Science 266,
pp.1021-1024, 1994.
[2] M. N. Stojanovic and D. Stefanovic, A Deoxyribozyme based Molecular Automaton. Nature
Biotechnology, 21(9):1069-1075,
2003.
[3] L. Qian, E. Winfree, Scaling Up Digital Circuit Computation with DNA Strand Displacement
Cascades, Science, 332:1196-1201, 2011.
[4] SE.Chiniforooshan, D.Doty, L.Kari, S.Seki, Scalable, time-responsive, digital, energy-efficientmolecular circuits using DNA strand displacement. Proc. of International Conference on DNA
Computing and Molecular programming, DNA16, In Lecture Notes in Computer Science, LNCS,
6518, Y.Sakakibara, Y.MI, Eds., 25-36, 2011.
[5] A. J. Genot , J. Bath , and A. J. Turberfield, Reversible Logic Circuits Made of DNA. J. Am. Chem.
Soc., 133 (50):20080-20083, 2011.

P5-31
Optimization and pharmacological characterization ofiCell cardiomyocytes for electrophysiological assessment
in a multielectrode extracellular recording
Kohei Sawada 1
k-sawada@hhc.eisai.co.jp

Atsuko Ojima 1
a-ojima@hhc.eisai.co.jp

Tomohiko Taniguchi 1

Mai Uesugi 1

t2-hihara@hhc.eisai.co.jp

m-uesugi@hhc.eisai.co.jp

Takashi Yoshinaga 1

Norimasa Miyamoto 1

t2-yoshinaga@hhc.eisai.co.jp

n-miyamoto@hhc.eisai.co.jp

1

Eisai Product Creation Systems, Eisai Co., Ltd., Tokodai 5-1-3, Tsukubashi,
Ibaraki 300-2635, Japan

Keywords: iPS, cardiomyocyte, safety pharmacology
Human iPS cell derived cardiomyocytes (hiPS-CM) have become a valuable tool for safetyassessment in drug discovery. In order to translate the results obtained in these cells into in vivo
effects in humans, basic physiological or pharmacological properties of these cells should be wellcharacterized. Present studies examined the effects of temperature, cell density and representativeion channel modulators with MED64 in iCell cardiomyocytes.
When the experimental temperature was changed from 35 to 38.5ºC, the corrected field potential
duration (FPDc) decreased from 370 to 285 msec (24 msec/ºC) almost linearly, but beating rateincreased from 42 to 66 bpm at 37ºC and did not increase further even at 38.5ºC.
The cell density of plating affected the response to Iks blockers. The concentration-response
curves for 293B and HMR1556 shifted to the right when cell densities were decreased from 30000,
15000, 7500, and 3750 cells/well. In lower density conditions, cell sizes enlarged and expressionof the KCNQ1 gene decreased. The hERG channel blocker, E4031, consistently prolonged the
FPDc at concentrations higher than 3 nM regardless of the cell density.
Various ion channel modulators indicated pharmacological characteristics of hiPS-CM.
These results indicate that the basic information on experimental conditions and pharmacologicaleffects are very important for translation of the data.

P5-32Fracture of Biomaterials
Hiroyuki Mayama
mayama@asahikawa-med.ac.jp

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

Keywords: Surface energy, Elastics, Young’s modulus, Fracture
Living cell is consisted of various biomaterials such as lipids, microtubule, DNA, RNA, and etc.,
and spontaneously forms various morphology after cellular differentiation. To understand thereason why the various morphology are formed, it is necessary to understand the mechanicalproperties of biomaterials as elastics. Here, we present the fracture phenomena of microtubule [1],which is an example to evaluate the mechanical strength of biomaterials.
The experimental strategy is as follows. First, we fixed a microtubule on the surface of a PDMS
substrate. Next, we stretch the substrate by suitable stretching rate and observe the microtubule todetermine the fracture conditions of microtubule. Based on the fracture conditions, we evaluate theYoung’s modulus of a microtubule. We found that the fracture happens when deformation energyof a microtubule is equal to surface energy of two fracture surfaces as

E = (1 / 2 )EYoung ε 2V = 2γS

where EYoung is the Young’s modulus of a microtubule, V is the volume of the microtubule before
fracture, S is the area of a fracture surface, and ε is strain. As a result, we obtain that themechanical strength of a bare microtubule is 10 MPa, which is a good agreement with previous
studies.
Relation to this, we also present blooming phenomena of chocolate, the fracture phenomena of fatty
acids [2].
[1] Kabir, A., Inoue, D., Hamano, Y., Mayama, H., Sada, K., Kakugo, A., Biomolecular Motor
Modulates Mechanical Properties of Microtubule, Biomolecules, 15:1797-1805, 2014.
[2] Mayama, H. Blooming theory of tristearin, Soft Matter, 5:856-859, 2009.

P5-33Characterization of synaptic transmission induced
synchronized population bursts of the iPSC-derived
neurons
Norimasa Miyamoto 1
n-miyamoto@hhc.eisai.co.jp

Atsuko Ojima 1
a-ojima@ hhc.eisai.co.jp

Kohei Sawada 1
k-sawada @ hhc.eisai.co.jp

1

Global Cardiovascular Assessment, Biopharmaceutical Assessments Core Function Unit, Eisai
Product Creation Systems (Eisai Co., Ltd.), 5-1-3
Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan

Keywords: Human iPSC-derived Neurons, seizure, MEA
Many drugs have been reported to cause seizures. It has been reported that the causes ofdrug-induced seizures are GABA A antagonism, GABA B agonism, adenosine antagonism, andenhanced excitation through NMDA in the neurons. So far, there is no good in vitro assay systemfor predicting drug-induced unexpected seizure-risks. Spontaneous neuron activity recordings bymulti-electrode array (MEA) system from networks of cultured neurons could be a good risk
evaluation system for such drug-induced seizure events [1]. It was reported that long-term
electrophysiological activity and pharmacological response of human induced pluripotent stem cell(hiPSC)-derived neurons were accelerated by co-culture with rat astrocytes. [2].
In this study, we observed time course generation of population burst spikes from iCell neurons
with conditioned medium of mouse primary astrocytes by MEA system. Humoral factor(s) frommouse primary astrocytes was sufficient to generate synchronized population burst signals. GABA
antagonism enhanced and calcium channel blockers eliminated the astrocyte-induced synchronizedpopulation bursts. We concluded that the observed astrocyte-induced population bursts by MEA
system are mediated by synaptic transmission and the periodic synchronized population burst
signals could be a good prediction marker of GABA A antagonism.
[1] Biffi, E., Regalia, G., Menegon, A., Ferrigno, G., and Pedrocchi1, A ., The Influence of
Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A
Methodological Study, PLoS ONE, 8:e83899. doi:10.1371, 2013.
[2] Odawara, A., Saitoh, Y., Alhebshi, A.H., Gotoh, M., and Suzuki, I ., Long-term
electrophysiological activity and pharmacological response of a human induced pluripotent stem
cell-derived neuron and astrocyte co-culture, Biochem. Biophys. Res. Commun. 443:1176–1181,
2014.

P5-34Single-molecular Activity Measurement of Enzymes
Attached to DNA origami
Ban Okabayashi 1
okabayashi@molbot.mech.tohoku.ac.jp

Shin-ichiro M. Nomura 1
nomura@molbot.mech.tohoku.ac.jp

1

Ibuki Kawamata 1
kawamata@molbot.mech.tohoku.ac.jp

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

Tohoku University, 6-6-01, Aoba Aramaki-Aza, Aoba-ku, Sendai, Miyagi, Japan

Keywords: Molecular Robotics, DNA nanotechnology, DNA origami, enzyme
DNA is a suitable material to make various functional devices [1,2]. One of such application is ameasurement of chemical reactions at the single -molecule level. Nanostructures made by DNA origami
can be used as a platform for this purpose [3]. Here, we propose a system to restrict the reaction spaceof enzymes on DNA origami substrates. We have attached ssDNA to polymerase and nicking
endonuclease [4], and the reaction space of the enzymes is controlled by the length of the ssDNA. The
experimental results of the measurement will be reported in the poster.

[1] Murata, S., et al., New Generation Computing, 31:27-45, 2013.
[2] Hagiya, et al. Acc. Chem. Res., 47(6), 1681-1690, 2014.
[3] Endo, M., Acc. Chem. Res., 47 (6), 1645–1653, 2014.
[4] Miyazono, Y., et al., The EMBO Journal, 29: 93–106, 2010.

P5-35Making Logic Circuits with a Single Component
in a Virtual Kinematic Environment
Tetsu Narumi 1
narumi@cs.uec.ac.jp

1

Shun Kanzawa 1
k1331032@edu.cc.uec.ac.jp

Department of Communication Engineering and Informatics,
The University of Electro-Communications,
1-5-1 Chofugaoka, Chofu, Tokyo 182-8585, Japan

Keywords: Virtual kinematic environment, logic circuit, molecular robotics
The final target of molecular robotics is to achieve a small robot composed of molecules. One
need to make a variety of functional parts, such as motor, sensor, computer, and structure, using as
small number of molecules as possible since too many molecules are difficult to control in atomic
scale. It is ideal that a single component can work as computer as well as motor, s ensor, andstructure. The final goal of this research is to find such a component in a virtual kinematic
environment and to construct a preliminary robot in the environment.
In the current work, we first made a logic circuit in a virtual kinematic environment using Unity,
which is a computer-game development environment widely used recently. We used a single
component like a cube, which two faces are input, and two are outputs. As it is a mechanical logic
circuit, the input and output are expressed by the moving of the face into or out of the stable
position. In a sense, the component already include the minimum function of sensor and motor aswell as computer. By combining a lot of components, it can also form a structure of the robot.
Therefore, it is a candidate for a component of a molecular robot, though the environment isvirtual.
Second, we showed that the cubic component works to make a simple logic circuit like half adder,ring oscillator, and so on. The logical function of the cube is the combination of AND and NAND
logic elements. The first output is the AND output of two inputs, and the other face is that of
NAND. Therefore, we can theoretically make arbitrary logic circuit by connecting cubes. As it hasa certain delay inside the logic, we can make an oscillator, which works like an engine of the robot.
We plan to make a vehicle using this mechanism.
Stevens[1] showed self-replicating machine can be made by combining several cubic components
in a virtual environment. His target was self-replication and he used many different functional
cubes, such as sliding, rotating, wiring, and fusing. In our case we use a single component andself-replication is still a future target.
The problem of the current implementation is that it is too slow to achieve complicated logic
circuit which needs to construct a robot. Tens of cubes would be the limit of this Unity system. The
collision detection seems the bottleneck of the calculation. We plan to switch to a simple software
layer, such as CUDA, which is a GPU computing e nvironment. Then we could get more speed. We
already compared the calculation speed between Unity and CUDA in a simple particle collision
simulation, and found that CUDA is much faster for large number of particles. We plan to make amolecule like structure for a component, since th e collision detection becomes simpler withspheres.
[1] William M Stevens, A self-replicating programmable constructor in a kinematic simulation
environment, Robotica, 29(01):153-176, 2011.

P5-36
Construction of Computational Elements
by a Swarm Network with Brownian Motion
Masashi Mori 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: Swarm network, Universal Turing machine, Brownian circuit
Designing computational devices will be of importance for molecular-sized robot systems that arecomposed of chemical or biomolecular elements, in order to be able to control these systems.
Micrometer-sized or Nanometer-sized robots can have only limited complexities, thus building theconventional types of information processing structures may be quite difficult due to their complexity.
The alternative approach to establish molecular robotics–constructing complex machines from a cloudof simple and homogeneous machines–represents a type of architecture that have been well studied in
the context of Cellular Automata (CAs). In this model identical finite automata–called cells–arranged
in a regular structure interact locally according to a set of rules.
To conduct information processing in molecular-sized robots, we have proposed the so-called Swarm
Network model and have shown it to be Turing universal through the construction of computational
elements [1]. Swarm Networks are a generalization of CA in which the neighborhoods andfunctionalities of cells are determined by the presence or absence of connections between cells. An
agent, which corresponds to a cell in a CA, can have a wide range of functionalities. One functionality
is the exertion of a force on the agent in a particular direction, but this requirement is unnatural when
implementing swarm agents by biological molecules, because it remains unclear where this force
originates from.
This presentation proposes a Swarm Network that is more realistic by assuming that agents are
subject to Brownian motion. Moreover, connections between agents can be changed dynamically,
allowing for the formation of structures and the dynamic transfer of information between agents.
These characteristics make the model mimic behavior of biological organisms more closely than its
previous incarnation. We show that this model is capable of universal computation by constructing auniversal Brownian circuit [2] based on it.
[1] Isokawa, T., Peper, F. Mitsui, M., et al., Computing by Swarm Networks, Proc. 8th Int. Conf. on
Cellular Automata for Research and Industry (ACRI '08), LNCS 5191, 50–59, 2008
[2] Peper, F., Lee, J., and Isokawa, T., Brownian Cellular Automata, Journal of Cellular Automata,5:185–206, 2010.

P5-37
Metabolomic approaches for compound annotation ofvolatile organic compounds in headspace of Allium
fistulosum using in-tube extraction devices
Miyako Kusano 1,2,* , Makoto Kobayashi 2 , Yumiko Iizuka 2,3 , Atsushi Fukushima 2 , Kazuki
Saito 2,4
kusano.miyako.fp@u.tsukuba.ac.jp, kobamako@riken.jp, yiizuka5@gmail.com,
a-fukush@psc.riken.jp, kazuk.saito@riken.jp
1

Graduate School of Life and Environmental Sciences, University of Tsukuba Tennodai,,Tsukuba, Ibaraki 305-8572, Japan
2 RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan
3 Department of Genome System Science, Graduate School of Nanobioscience, Yokohama City
University, Yokohama, Kanagawa 236-0027, Japan
4 Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Chiba 260-8675, Japan

Keywords: Metabolomics, annotation, volatile organic compounds
Plants produce a multitude of volatile organic compounds (VOCs), which are importantcomponents of the metabolome. VOCs play a dominant role in biotic and abiotic stress responses
as well as plant-plant and plant-insect interactions. To determine what kinds of VOCs can beemitted from plants and how much their levels change, a comprehensive and a reproducible
analytical method is required. In the study, we employed one of the state-of-the-art techniques,in-tube extraction (ITEX), to conduct headspace (HS) sampling of Allium fistulosum (Japanesebunching
onions).
The
collected
HSsampleswereanalyzedbygaschromatography–time-of-flight–mass spectrometry (GC-TOF-MS) because it enables us to rapidly
identify peaks by combining our metabolomics pipeline. In parallel, we estimated how many
volatile peak s in the mass spectra overlapped among the mass spectral libraries for the tentative
identification of VOCs. Using the VOC profiling pipeline based on HS-ITEX-GC-TOF-MS, we
conducted VOC profiling of the samples that were emitted from 12 Allium cultivars. We will
present how to annotate VOCs by comparing each MS spectrum obtained by
HS-ITEX-GC-TOF-MS analysis and MS spectral database.

P5-38A Computational Framework to Analyze
Microtubule Gliding Assay
Akihiko Konagaya 1

Bulibuli Mahemuti 1

Gregory Gutmann 1

kona@dis.titech.ac.jp

bulbul.aa@m.titech.ac.jp

gutmann.g.aa@m.titech.ac.jp

Daisuke Inoue 2
daisukeinoue@mail.sci.hokudai.ac.jp
1

2
3

Akira Kakugo 23
kakugo@sci.hokudai.ac.jp

Department of Computation Intelligence and Systems Science, Tokyo Institute ofTechnology
Faculty of Science, Hokkaido University
Graduate School of chemical Sciences and Engineering, Hokkaido University

Keywords: Molecular Robotics, Microtubule Gliding Assay, Bio-imaging, Real-time
Simulation
Microtubules and motor proteins are expected to play important roles in molecularrobotics as molecular actuators and skeletons, to name a few [1,2]. Microtubule gliding
assay is a biological experiment observing the dynamics of microtubules driven by
motor proteins fixed on a glass surface when ATPs are dosed [3]. In order to understandcontrol mechanism of microtubules, we propose a computational framework to analyze
microtubule gliding assay video data and to reproduce the behavior of microtubules on
3D real-time simulation [4]. Novelty of this framework is in cooperative feedback
between bio-imaging and 3D real-time simulation to estimate control parameters of
microtubule gliding assay. Bio-imaging play an essential role to estimate control
parameters of stochastic microtubule head movement by means of microtubule tracking
analysis. 3D real-time simulation enables us to confirm the validity of the control
parameters by reproducing overall dynamics of thousands of microtubules.
[1] Murata S, Konagaya A, Kobayashi S, Saito H, Hagiya M: Molecular Robotics: A New
Paradigm for Artifacts, New Generation Computing, Ohmsha, Ltd. and Springer,
2013, .31 (1), 27-45[2] Hagiya M, Konagaya A, Kobayashi S, Saito H, Murata S, Molecular Robots with Sensors
and Intelligence, Accounts of Chemical Research, ACS, 47(6): 1681-1690, 2014.
[3] Inoue D., Kabir A.M.R., Mayama H., Gong J.P., Sada K., Kakugo A.: Growth of
ring-shaped microtubule assemblies through stepwise active self-organisation, Soft Matter
2013, 9, 7061–7068.
[4] Gutmann G, Inoue D, Kakugo A, Konagaya A: Real-time 3D Microtubule Gliding
Simulation, in Proc. of LSMS&ICSEE 2014, Shanghai, China 2014.

P5-39Hemodialysis treatment weakens the contraction force ofcentral sinoatrial nodal cell
Hiroyuki Hamada 1,2,3

Akihiro C. Yamashita 4

Masahiro Okamoto 1,2,3

hamada@brs.kyushu-u.ac.jp

yama@hosei.ac.jp

okahon@brs.kyushu-u.ac.jp

1

2

3

4

Department of Systems Life Sciences, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Synthetic Systems Biology Research Center, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Department of Chemical Science and Technology, Hosei University,
3-7-2 Kajino-machi, Koganei, Tokyo 184-8584, Japan

Keywords: Na + /Ca 2+ exchanger, Contraction force, Cardiac myocyte, Hemodialysis
In the hemodialysis treatment which is one of the renal replacement therapies, it is difficult toprovide adequate serum calcium (Ca) level to patients [1]. Pun et al. reported that patients who
were exposed to larger serum dialysate Ca gradient had a greater fall in blood pressure during
treatment and increased a risk of sudden cardiac arrest [2]. It is well -known that the blood pressure
depends on the cardiomyocyte contraction force which correlates significantly with cytosol Ca 2+
concentration (excitation-contraction coupling) [3]. However, the question of whether any effect ofhemodialysis treatment is observed on the C a dynamics in cardiac myocyte remains unclear. In thispaper, we constructed a massive integrative mathematical model for which a change of Ca 2+
concentration in interstitial fluid during treatment affects on the excitation -contraction coupling of
the central sinoatrial nodal cell. Using this model, we explored a mechanism of the fall in bloodpressure during treatment from a viewpoint of single cell biology.
Prescription of the low Ca concentration dialysate (2.5mEq/L) for hypercalcemic patient
gradually decreased Ca 2+ concentration in interstitial fluid during hemodialysis treatment.
Although the decrease in Ca 2+ concentration in interstitial fluid had little effect on amplitude in
membrane potential, the peak level of inward current on cellular membr ane increased during
treatment. A componential analysis of the inward current demonstrated that the increase in peaklevel of inward current was induced by an activation of N a + /Ca 2+ exchanger. This finding impliedthat the intracellular Ca 2+ and extracellular Na + were excessively transported to extracellular andintracellular space, respectively. As a result, the cell showed both a 2% decline in the beating
rhythm and an 8% decrease in the contraction force during treatment.
In summary, the hemodialysis treatment caused an activation of Na + /Ca 2+ exchanger andweakened the contraction force of central sinoatrial nodal cell in hypercalcemic patient. The
minimization of serum dialysate Ca gradient was shown to be useful in the prevention of a fall inblood pressure during treatment.
[1] Clinical Practice Guideline for CKD-MBD, Journal of Japanese Society for Dialysis Therapy
45: 301-356, 2012.
[2] Pun P.H., Horton J.R., and Middleton J.P., Dialysate calcium concentration and the risk of
sudden cardiac arrest in hemodialysis patients, Clin.J.Am.Soc.Nephrol. 8: 797 -803, 2013.
[3] Bers D.M., Cardiac excitation-contraction coupling, Nature 415: 198-205, 2002.

P5-40Development of a Molecular Sensor that Works on a
Liposomal Membrane

1

Koh-ichiroh Shohda 1

Akira Suyama 1

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

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, conjugate, vesicle
In the molecular robotics project, molecular robots that is based on a vesicle or a gel as a body consistof molecular sensors, molecular computers, and molecular actuators. [1] These components arecommunicated with single-stranded DNAs each other through the hybridization. We cover a
development of molecular sensor that is conjugated molecules between DNA strands and lipid. [2] A
structure of the conjugated molecules has a long hydrophobic part at the center of molecule and two
DNA strands at the both ends. The long hydrophobic parts of sensor molecules sticked in a liposomal
membrane. A complementary DNA/RNA strand that represented an exterior environmental information
was hybridized with the two DNA strands of conjugate molecules. The hybridization caused an
approach of the two molecular sensors, consequently, two short DNA strands of conjugate moleculeson the inner surface of liposomal membrane behaved as a long DNA molecule. The approached DNA
strands that showed high melting temperature caused a strand displacement between a DNA duplex thatwas labeled with a fluorescent and a quenching dye. Then, a single-stranded DNA that was labeled withthe fluorescent dye was released into an inner water pool of liposome. The released DNA can be a
trigger molecule for molecular computers and molecular actuators encapsulated in the liposome.

[1] S. Murata, A. Konagaya, S. Kobayashi, H. Saito, and M. Hagiya New Generat. Comput., 2013, 31,
27-45.
[2] P. Barton, C. A. Hunter, T. J. Potter, S. J. Webb, and N. H. Williams Angew. Chem. Int. Ed. 2002, 41,3878-3881.

P5-41
Designing an Artificial Ion Channel From
DNA origami
Kazuki Hirahara 1

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

murata@molbot.mech.tohoku.ac.jp

1

Ibuki Kawamata 1

kawamata@molbot.mech.tohoku.ac.jp

Shin-ichiro M. Nomura 1

nomura@molbot.mech.tohoku.ac.jp

Molecular Robotics Laboratory, Department of Bioengineering and Robotics, Division of
Mechanical Engineering, Tohoku University, M2-519, 6-6-01, Aramaki Aza-Aoba,
Aobaku, Sendai 980-8579, Japan

Keywords: Molecular Robotics, DNA nanotechnology, Ion channel, Lipid membrane, DNAorigami
Ion channel, which electric signal transduction molecule across the cell membrane, is one of themost indispensable devices of living organisms. In natural case, the channel molecule of membraneproteins attach on cell membrane, pass ions, and regulate the ion flux. In the point of view formolecular robotics [1,2], it is quite important to construct such a channel device passing small
information molecule. Controllable synthetic channel molecules would expand possibility ofmolecular nanobiotechnology[3].
Here we report an artificial ion channel using DNA origami–a DNA self-assembly methodology
to create predesigned nano-scale structure[4]. Our DNA origami channel is constructed of two
vertically positioned parts, a flat plate part and a cylindrical tubular part. The flat planner part willplay a role to be pressed onto the membrane by inputted voltage on account of DNA
negative-charge, and will lead the cylindrical part insert into the lipid bilayer. Electrophoresis and
AFM imaging indicated that the DNA origami channel structure was constructed as designed.
Their function identification of current measuring was also accomplished by using home-madedroplet interface bilayers system [5]. The current value showed about 70pA per contact area when
the potential difference inputted 100mV across a lipid bilayer. The current-voltage correlation was
proportional in the range of -100mV to +100mV. The result supports that the designed origami
worked as voltage-independent channel. The pore was designed as in the size of 4x4 nm 2 . By
modify the gate of the tubule structure, we are now investigating the new functions of the origami
channel. The possibility of some high-selectivity function such as diodicity, specific ssDNA
transportation, and size controlled molecular transportation. These researches would show the clue
to help constructing compartmentalized molecular robot or artificial cell.
[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] Hagiya, M., Konagaya, A., Kobayashi, S., Saito, H., & Murata, S. (2014). Molecular Robots
with Sensors and Intelligence. Accounts of Chem. Res., 47, 1681-1690, 2014.
[3] Martin Langecker, Vera Arnaut, Thomas G. Martin, Jonathan List, Stephan Renner, Michael
Mayer, Hendrik Dietz, Friedrich C. Simmel. “Synthetic Lipid Membrane Channels Formed by
Designed DNA Nanostructures” Science , 338: 932-936, 2012.
[4] Paul W.K.Rothemund. “Folding DNA to Create nano scale shapes and patterns” Nature, 440:
297-302, 2006.
[5] Hagan Bayley , Brid Cronin , Andrew Heron , Matthew A. Holden, William L. Hwang , Ruhma
Syeda , James Thompson and Mark Wallace. “Droplet interface bilayer” Mol. BioSyst, 4,1191-1208, 2008.

P5-42
Effects of substrate elasticity on gene expression profiles ofhuman iPS-derived cardiomyocytes
Miki Fujizuka 1

Yuji Nakai 2

Yasunari Kanda 3

ma130084@tmd.ac.jp

yunakai@cc.hirosaki-u.ac.jp

kanda@nihs.go.jp

Shushi Nagamori 4

Yoshikatsu Kanai 4nagamori@pharma1.med.osaka-u.ac.jp

ykanai@pharma1.med.osaka-u.ac.jp

Tetsushi Furukawa 1

Junko Kurokawa 1

t_furukawa.bip@mri.tmd.ac.jp

junkokuro.bip@mri.tmd.ac.jp

1

Tokyo Medical and Dental University, Medical Research Institute,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
2
Institute for Food Sciences, Hirosaki University,
2-1-1 Yanagawa, Aomori, Aomori 038-0012, Japan
3
Division of Pharmacology, National Institute of Health Sciences,
1-18-1 Kamiyouga, Setagaya-ku, Tokyo 158-8501, Japan
4
Osaka University Graduate School of Medicine,
2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan

Keywords: human iPS cells, cardiac toxicology, hydrogel culture, motion vector analysis.
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) provides
promising possibilities to generate a new cell resource for drug safety screening as well as
patient-specific lineages for a variety of translational research. However, the suitablecardiac differentiation method has not been attained yet both for toxicological and clinicalapplications. Because previous studies showed that mechanical stimulation regulates
differentiation and maturation of stem cells, we tested the effects of substrate elasticity on
contractile behaviors of hiPSC-CMs cultured on hydrogel ranging from 12 to 50 kPa andrigid plates using a high-speed video imaging system with motion vector prediction
algorithm [1]. The motion vector prediction analysis revealed that substrate elasticity
affects velocity of contraction-relaxation without altering beating rates. The cells cultured
on 12kPa hydrogel showed the largest velocity of contraction-relaxation andpharmacological responses. In order to pursue the underlying mechanisms of the effects ofsubstrate elasticity, gene expressions in hiPSC-CMs (iCell-CM, CDI) provided from
cultured with rigid plates and 12kPa hydrogel were compared by microarray analysis(TORAY) or real-time PCR. Differentially expressed genes were identified using afold-change cut-off > 2.0 (188 up-regulated genes and 63 down-regulated genes). Gene
Ontology analysis and pathway analysis (DAVID) identified enriched functional-related
gene groups such as ion transport, response to stress and calcium regulation for
up-regulation, and cell adhesion for down-regulation. The responses of drugs will be tested.
These results suggest that appropriate mechanical force may be an important factor for
maturation of hiPSC-CMs.
This research was supported by CDI iCell grant.

[1] Hayakawa, T., Kunihiro, T., Ando, T., Kobayashi, S., Matsui, E., Yada, H., Kanda, Y., Kurokawa, J.
and Furukawa, T. , Image-based evaluation of contraction-relaxation kinetics of human-induced
pluripotent stem cell-derived cardiomyocytes: correlation and complementarity with extracellularelectrophysiology , J Mol Cell Cardiol, in press 2014.

P5-43
Building An Artificial Protein Capsid

1
2

Ali D. Malay 1

Kenji Iwasaki 2

a.malay@riken.jp

kenji@protein.osaka-u.ac.jp

Zuben Brown 2Jonathan G. Heddle 1

zubenbrown@gmail.com

heddle@riken.jp

RIKEN Heddle Initiative Unit, 2-1 Hirosawa, Wako, Saitama, Japan
Institute for Protein Research, Dept of Biological Sciences, Osaka University, Osaka, Japan

Keywords: Artifical capsid, protein engineering, symmetry, nanostructures, bionanoscience
The long-term goal of bionanoscience is to be able to programmably construct designed
nanoscale objects using biological molecules. Such objects could have a myriad of uses as
templates for inorganic materials, as interfaces between biological and silicon systems, as highly
sensitive disease detectors and as smart, robot-like drugs. Proteins and DNA are widely usedmolecules in this area but offer different possibilities and challenges. We have used proteins toconstruct a variety of nano-shapes such as self-assembled nanotubes and in particular, an artificial
virus capsid1. This capsid is an example of an engineered protein cage, which we hope will havenumerous applications, including drug delivery devices and nano reaction vessels. Our cage isbased on a modified ring-shaped TRAP protein as the starting material. Addition of a metal
catalyst to the protein leads to the efficient formation of a hollow ~21-nm diameter sphericalprotein cage. The cage displays high stability under various conditions, yet readily disassembles
under reducing conditions. Structural analyses have revealed that it is composed of 24 identical
TRAP rings arranged in a unique symmetry.
[1] Malay, A. D. et al. Gold Nanoparticle-Induced Formation of Artificial Protein Capsids. Nano
Lett. 12, 2056-2059 (2012).