O3-1

Free Energy Perturbation Method in GENESIS
Hiraku Oshima 1

hiraku.oshima@riken.jp

123Yuji Sugita 1,2,3

sugita@riken.jp

Laboratory for Biomolecular Function Simulation, RIKEN Center for Biosystems Dynamics
Research, Integrated Innovation Building 7F, 6-7-1 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan
Theoretical Molecular Science Laboratory, RIKEN Cluster for Pioneering Research, 2 -1
Hirosawa, Wako-shi, Saitama 351-0198, Japan
Computational Biophysics Research Team, RIKEN Center for Computational Science,
Integrated Innovation Building 7F, 6-7-1 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo
650-0047, Japan

Keywords: Molecular dynamics, GENESIS, Free energy perturbation, Protein-ligand binding freeenergy
Accurate predictions of protein-ligand binding affinities have been a central challenge inin-silico drug design to reduce the total cost and time required for drug development . The
free energy perturbation (FEP) method based on the all-atom molecular dynamics (MD) simulationis one of the most essential tools to predict the binding affinity and solubility of ligands with highaccuracy. We implemented the FEP method to MD software “GENESIS” [1]. FEP in GENESIS canpredict relative and absolute binding affinities as well as relative and absolute solvation freeenergies with high accuracy [2,3]. We also proposed a modified FEP scheme by introducing non uniform scaling parameters into Hamiltonian [4]. Modified FEP greatly improves the computationalperformance, which is marked for large biomolecular systems. Here we introduce the FEP functions
in GENESIS and discuss about the applicability of modified FEP to drug discovery on
supercomputer “Fugaku”.

[1] https://www.r-ccs.riken.jp/labs/cbrt/[2] Oshima, H.; Re, S.; Sugita, Y.; J. Chem. Inf. Model., 2020, 60, 5382-5394.
[3] Kim, S.; Oshima, H.; Zhang, H.; Kern, N. R.; Re, S.; Lee, J.; Roux, B.; Sugita, Y.; Jiang, W.;
Im, W.; J. Chem. Theory Comput., 2020, 16, 7207-7218.
[4] Oshima, H.; Sugita, Y.; J. Chem. Inf. Model., 2022, 62, 2846–2856.

O3-2

Lipid composition is critical for accurate membrane
permeability prediction of large cyclic peptides bymolecular dynamics simulations

1

Masatake Sugita 1

Takuya Fujie 1

Keisuke Yanagisawa 1

sugita@bi.c.titech.ac.jp

fujie@bi.c.titech.ac.jp

yanagisawa@c.titech.ac.jp

Masahito Ohue 1

Yutaka Akiyama 1

ohue@c.titech.ac.jp

akiyama@c.titech.ac.jp

Department of Computer Science, School of Computing, Tokyo Institute of Technology, W8-76, 212-1 Ookayama, Meguro-ku, Tokyo, 152-8550, Japan

Keywords: Cyclic peptides, Membrane permeability, Molecular dynamics
Poor membrane permeability is the biggest bottleneck hindering successful drug discovery basedon cyclic peptides. Therefore, the development of computational methods that can predict membrane
permeability and support elucidation of the membrane permeation mechanism of drug candidate
peptides is eagerly awaited. In this study, we developed a protocol to simulate the behavior inmembrane permeation steps and estimate the membrane permeability, especially for large cyclic
peptides which are prone to achieve high affinity with the target.[1] This protocol requires the useof more realistic membrane model than a single-lipid phospholipid bilayer. To select a membranemodel, we first analyzed the effect of cholesterol concentration in the model membrane on thepotential of mean force and hydrogen bonding networks along the direction perpendicular to themembrane surface as predicted by molecular dynamics simulations using cyclosporine A. Theseresults suggest that a membrane model containing 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine and 40 or 50 mol% cholesterol was suitable for predicting the permeation process.
To validate the efficiency of our protocol the membrane permeability of 18 10-residue peptides werepredicted. The total calculation in this study took approximately 860 thousand GPU hour using
NVIDIA P100 GPUs. Correlation coefficients of R > 0.8 between the experimental and calculated
permeability values were obtained. The results of this study demonstrate that the lipid membrane is
not just a medium, but also among the main factors determining the membrane permeability ofmolecules. The computational protocol proposed in this study and the findings obtained on the effectof membrane model composition will contribute to build a schematic view on the membrane
permeation process. Furthermore, the results of this study will eventually aid the elucidation ofdesign rules for peptide drugs with high membrane permeability.
[1] Sugita, M.; Fujie, T.; Yanagisawa, K.; Ohue, M.; Akiyama, Y. Lipid composition is critical for
accurate membrane permeability prediction of cyclic peptides by molecular dynamics simulations.
Journal of Chemical Information and Modeling. Under review

O3-3

Molecular Dynamics Study of
3D-DS dimerization process of Cyt c
Shimoyama Hiromitsu

Shigeta Yasuteru

simoyama@ccs.tsukuba.ac.jp

shigeta@ccs.tsukuba.ac.jp

Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8577, Japan

Keywords: Molecular Dynamics, 3D-DS, Free-energy analysis
The 3-dimensional domain swapping (3D-DS) is a phenomenon where multiple proteins form
unique complexes by donating part of their domains each other [1,2]. One of the characteristic
points of 3D-DS is that the formed 3D-DS complexes are rather stable because the inter-protein
interaction is the same as the intra-protein one in their monomer structure. Then, 3D-DS is possibly
utilized to make an artificial protein complex to regulate, combine, encourage or discourage theirfunctions and activities. 3D-DS formation occurs when denatured monomers by alcohol or salt
refold by dilution. However, its detailed formation process is not so well understood.
In this study, 3D-DS formation process of the cytochrome c (cyt c) complex as shown in Fig. 1
was studied by molecular dynamics simulations with a coarse-grained (CG) model and the smoothed
Wang-Landau (SWL) method [3]. As the distance of the two cyt c monomer plays a critical role informing their complex, the 1- (energy) and 2-dimensional SWL-MD (energy and gyration radius)
are applied to enhance the conformational sampling. In Fig. 2, obtained trajectories by theconventional CG simulation and 1D and 2D SWL simulations are mapped onto the subspace spannedby root-mean-square deviations (RMSDs) from monomer A/B structures indicating the sampling
efficiency of each method. Although 3D-DS region is the lower left corner, the conventional CG
did not
samplethiscomplex
region.canOn be
thedesigned,other hand,
1Dworkandas
2Ddesigned.
SWL simulations can sample the 3D-DS
DesiredProteinbut notstructures.
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CG modelsuperimposed with the crystal structure (see→ Complex
of example,”originally”thefunctionalproteinswould wasbe work.
figureDesired
legendProtein
in detail).
The canmodel
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complexbe designed,
but thenot work
as structure,designed. indicating the robustness ofThis
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our SWL
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The
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→ Complex of ”originally” functional proteins would be work.explained

Abstract

Abstract→ Higher order multimer, hetero multimer prediction

This study:T300
MD-simulation forSWL
dimercomplex
1D→ Higher order multimer, hetero multimer prediction
T300

3D-DSregion
SWL 2D3D-DSregion

30 102520SWL 2D

151010Fig. 1 Monomer and 3D-DS dimer
structure of cyt c, where blue and cyancolors indicate chain A and light anddeep green ones chain B, respectively.

1520
RMSDA [Å]−630 102520 10
10−5251530

202530RMSDA [Å]Population
RMSDB [Å]Electron transfer protein
cytochrome c

SWL 1DRMSDB [Å]Electron transfer protein
cytcrome c−410−310−210−115Fig. 2 Population of the samples
on the RMSD A/B plane;
Population
RMSD A/B is RMSD of the 10
monomer A/B with respect tothe 3D-DS structure (PDB ID:shows
10−63NBS).
10−5 10−4 Right
10−3 10−2panel
10−1
the most similar structure to the 3D-DS structure obtained
from MD simulation.

Reference: [1] Margoliash, E. Lustgarten, J. J. Biol. Chem. 237, 3397–3405 (1962), [2] Hirota, S. et al.,Proc. Nat. Acad. Sci. U.S.A., 107(29), 12854–12859 (2010), [3] Shimoyama, H. et al., J. Chem. Phys., 134(2),024109 (2010), [4] Shimoyama, H. and Shigeta, Y. Life, 11(11), 1241 (2021)

O3-4

Complex model of the bile acid transporter NTCP andbinding peptides involved in the HBV infection exploredby molecular dynamics simulation
Toru Ekimoto 1

Kenichiro Yamamoto 1

Tsutomu Yamane 2

ekimoto@yokohama-cu.ac.jp

w225434g@yokohama-cu.ac.jp

tsutomu.yamane@riken.jp

1

2

Sam-Yong Park 1

Mitsunori Ikeguchi 1,2

park@yokohama-cu.ac.jp

ike@yokohama-cu.ac.jp

Graduate School of Medical Life Science, yokohama City University, 1-7-29 Suehiro-cho,
Tsurumi-ku, Yokohama 230-0045, Japan
Center for Computational Science, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 2300045, Japan

Keywords: hepatitis B virus, sodium taurocholate cotransporting polypeptide, preS1, Mycludex B,
cyclosporine A, molecular dynamics simulation
Hepatic diseases involved in the infection of hepatitis B virus (HBV) are one of the worldwide
issues of health [1]. In the cellular entry process of HBV, the N-terminal myristoylated preS1
domain (myr-preS1) of the viral large protein interacts with sodium taurocholate cotransporting
polypeptide (NTCP), i.e., NTCP acts as both a bile acid transporter and a receptor of the HBV entry[2]. Therefore, NTCP is a pharmaceutical target, and its inhibitors, such as small compounds, apeptide drug mimicking myr-preS1 called MyrcludexB, and a cyclic peptide cyclosporine A (CsA)
and CsA derivatives, have been approved and developed [1,3]. In 2022, the structure of human
NTCP has been solved for the first time using cryo-EM technique [4,5]. The structure adopts an
outward-facing state, and a hole is formed. However, in complexes with myr-preS1 or MyrcludexB, the binding peptide were unresolved, and therefore, the complex structure and interaction modes
were still unknown. Since the binding peptide is flexible and is presumed to be non-resolving due
to its dynamic binding mode, a method that is capable for searching dynamic binding modes, suchas molecular dynamics (MD) simulation, will be effective. In this study, using MD simulation,complex structure model of NTCP and binding peptides was explored. The regions of NTCP incontact with myr-preS1, Myrcludex B, and CsA were analyzed from the complex model. The NTCP
residues with high frequency of contacts were essentially identical to the regions experimentallyshown to be important for the HBV infection.
[1] Zakrzewicz, D.; Geyer, J. Multitasking Na + /taurocholate cotransporting polypeptide (NTCP) asa drug target for HBV infection: from protein engineering to drug discovery, Biomedicines, 2022,
10, 196.
[2] Yan, H.; et al., Sodium taurocholate cotransporting polypeptide is a functional receptor forhuman hepatitis B and D virus. eLife, 2012, 1, e00049.
[3] Watashi, K.; et al., Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured
hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting
polypeptide (NTCP), Hepatology, 2014, 59, 1726-1737.
[4] Park, J-H; et al., Structural insights into the HBV receptor and bile acid transporter NTCP,Nature, 2022, 606, 1027-1031.
[5] Asami, J.; et al., Structure of the bile acid transporter and HBV receptor NTCP, Nature, 2022,606, 1021-1026

O3-5
MAXS for 3D molecular visualization of human antibody
conformational changes
Takashi Matsumoto 1
t-matumo@rigaku.co.jp

1

Akihito Yamano 1

yamano@rigaku.co.jp

Takashi Sato 1
satow@rigaku.ac.jp

Rigaku Corporation, 3-9-12 Matsubara-cho, Akishima-shi, Tokyo 196-8666, Japan

Keywords: solution scattering, MAXS, conformational change, flexibility
Biopharmaceuticals, especially antibody drugs, have been increasing their use and presence.
While they have the advantage of higher specificity and lesser side effects, they have thedisadvantage of expensive development and production costs. Moreover, maintaining andcontrolling their quality is more difficult since they consist of extensive protein moieties with acomplex molecular nature. The complexities come from both chemical and physical characteristics
and also their heterogeneities. The chemical heterogeneities come from spontaneous degradation;
charge variants are one of them, and biochemical heterogeneity is like sugar chain variation. Suchchemical characteristics have been well assigned and controlled; however, the rapi d expansion ofnovel modalities is becoming more challenging to develop analytical methods for novel
antibody-based drugs.
In contrast, most analytical methods for physical characterizations tolerate the varieties inthose novel modalities. However, many of those are time and sample-requiring processes.
Moreover, the results are challenging to manage and limited in use, such as monitoring theidentities because the essential information for physical properties, the three -dimensionalstructures, are unavailable. X-ray crystallography and single particle cryo-EM observation are theestablished methods to explore the tertiary structure of biomolecules. However, those results arefrom some extreme conditions, may be affected by crystallization or freezing, and areexperimentally selected from a specific group of the original ensemble. Those limitations comefrom the flexible nature of biomolecules. Especially antibodies are highly flexible, mainly between
Fab-Fc domains and essential sugar chains, making crystal lization of whole antibody molecules
unrealistic and/or limited insight from frozen snapshots.
It is undoubtedly of great help in developing novel modalities, their CMC studies, and quality
control; once we had a method to visualize the flexible biomolecu les avoiding prerequisites andspecific requirements. X-ray solution scattering experiments have been utilized to analyze
structures and conformational changes. In those experiments, scattering data within a small -angle
region are widely used and thus called SAXS (Small Angle X-ray Scattering). The SAXS
experiments often focus on macroscopic shapes and sizes of the molecules. In principle, thescattering data among the higher region contains detailed structural information; thus, we could
obtain much information; for example, q values around 0.30 to 0.65 Å -1 reflect distances between
domains, secondary structure components, and/or adjunct chemical groups in ADCs. The
experiments should give us a novel picture of complex molecular behaviors as well as
conformational changes of flexible biomolecules.
In the above background, we are substantially working on the molecular visualization of
medicinal macromolecules utilizing extensive SAXS data reaching the sub -WAXS region. We
abbreviate it as MAXS, Middle Angle X-ray Scattering. As shown in our presentation, MAXS
experiments give us novel and valuable information that could lead to the molecular design andprocessing of antibody drugs. In this presentation, as the first step of our work, we will discuss thesolution structure analysis of human IgG that revealed significant differences between the solution
and crystalline states, as well as a novel observation of its flexibility. We also discuss the effects ofthe sugar chain moiety in the Fc region on the structura l nature of human IgG.

O3-6

CrotBiopsy: New methods for evaluation of lunginflammation
YASUHIRO YOSHIDA 1

Duo WANG 1 Zirui ZENG 1 Mengyue SHEN 1

freude@med.uoeh-u.ac.jp

1

University of Occupational and Environmental Health, Japan . Iseigaoka 1-1, Yahatanishi-ku,
Kitakyushu,807-0804, Japan

Keywords: CrotBiopsy, serum, lung inflammation, bleomycin
In general, laboratory methods for assessing health effects rely heavily on blood tests. However,
the amount of sample used in the test is limited, and since it is only for the liquid factor, the dataobtained is also limited. Furthermore, there are often various limitations when collecting samples,and repeated sample collection from the same patient should be avoided. Therefore, we proposed atesting method that focuses on clots that are discarded as precipitates during serum preparation. An
advantage of using a clot biopsy is that both the protein contained in the clot and the industrial
waste discarded during serum preparation can be used.
In this study, we succeeded in detecting NF-κB as an activation marker in a mouse model of
bleomycin-induced pulmonary inflammation by preparing clot lysates. These results strongly
suggested that the clotbiopsy method would be a useful method for assessing biological effects.