Molecular Dynamics Simulations to Determine the Structure and Dynamics of Hepatitis B Virus Capsid Bound to a Novel Anti-viral Drug.

Hepatitis B virus (HBV) chronically infects millions of people worldwide and is a major cause of serious liver diseases, including liver cirrhosis and liver cancer. In our previous study, in silico screening was used to isolate new anti-viral compounds predicted to bind to the HBV capsid. Four of the isolated compounds have been reported to suppress the cellular multiplication of HBV experimentally. In the present study, molecular dynamics simulations of the HBV capsid were performed under rotational symmetry boundary conditions, to clarify how the structure and dynamics of the capsid are affected at the atomic level by the binding of one of the isolated compounds, C13. Two simulations of the free HBV capsid, two further simulations of the capsid-C13 complex, and one simulation of the capsid-AT-130 complex were performed. For statistical confidence, each set of simulations was repeated by five times, changing the simulation conditions. C13 continued to bind at the predicted binding site during the simulations, supporting the hypothesis that C13 is a capsid-binding compound. The structure and dynamics of the HBV capsid were greatly influenced by the binding and release of C13, and these effects were essentially identical to those seen for AT-130, indicating that C13 likely inhibits the function of the HBV capsid.

Principal component analysis-based unsupervised feature extraction applied to in silico drug discovery for posttraumatic stress disorder-mediated heart disease.

BACKGROUND: Feature extraction (FE) is difficult, particularly if there are more features than samples, as small sample numbers often result in biased outcomes or overfitting. Furthermore, multiple sample classes often complicate FE because evaluating performance, which is usual in supervised FE, is generally harder than the two-class problem. Developing sample classification independent unsupervised methods would solve many of these problems. RESULTS: Two principal component analysis (PCA)-based FE, specifically, variational Bayes PCA (VBPCA) was extended to perform unsupervised FE, and together with conventional PCA (CPCA)-based unsupervised FE, were tested as sample classification independent unsupervised FE methods. VBPCA- and CPCA-based unsupervised FE both performed well when applied to simulated data, and a posttraumatic stress disorder (PTSD)-mediated heart disease data set that had multiple categorical class observations in mRNA/microRNA expression of stressed mouse heart. A critical set of PTSD miRNAs/mRNAs were identified that show aberrant expression between treatment and control samples, and significant, negative correlation with one another. Moreover, greater stability and biological feasibility than conventional supervised FE was also demonstrated. Based on the results obtained, in silico drug discovery was performed as translational validation of the methods. CONCLUSIONS: Our two proposed unsupervised FE methods (CPCA- and VBPCA-based) worked well on simulated data, and outperformed two conventional supervised FE methods on a real data set. Thus, these two methods have suggested equivalence for FE on categorical multiclass data sets, with potential translational utility for in silico drug discovery.

Discovering novel direct acting antiviral agents for HBV using in silico screening.

The treatments for chronic hepatitis B (CHB) are interferon and nucleoside analogues reverse transcriptase (RT) inhibitors. Because both treatments are less than ideal, we conducted to identify novel anti-viral agents for HBV-reverse transcriptase (HBV-RT). We determined the ligand-binding site of the HBV-RT by conducting a homological search of the amino acid sequence and then we also determined not only structural arrangement of the target protein but the target protein-binding site of the ligand using known protein-ligand complexes in registered in the protein data bank (PDB). Finally we simulated binding between the ligand candidates and the HBV-RT and evaluated the degree of binding (in silico screening). PXB cells derived from human-mouse chimeric mouse liver, infected with HBV were administrated with the candidates, and HBVDNA in the culture medium was monitored by realtime qPCR. Among compounds from the AKosSamples database, twelve candidates that can inhibit RT were also identified, two of which seem to have the potential to control HBV replication in vitro.

Development of novel hepatitis B virus capsid inhibitor using in silico screening.

Antiviral therapy for chronic hepatitis B that uses nucleos(t)ide analogue is considered effective. However, most drugs of this class frequently result in viral relapse after cessation of therapy as well as the emergence of resistance, thereby limiting their clinical use. In order to increase the therapeutic efficiency of chronic hepatitis B treatments, it is important to survey novel (chemical) reagents targeting other stages of the viral replication process. The aim of this study was to identify novel capsid inhibitor candidates using in silico screening. We discovered four such candidates that decreased the levels of HBV DNA and HBsAg in vitro. These four capsid inhibitor candidates did not induce cell toxicity even at high concentrations. Results from docking simulation showed that the candidates bounded with high affinity with the capsid protein hydrophobic binding site. Identifying direct acting HBV core protein inhibitors increases the likelihood that novel medicines can be developed that allows the combination of novel anti-viral drugs and nucleos(t)ide analogue or interferon for HBV treatment.

TINAGL1 and B3GALNT1 are potential therapy target genes to suppress metastasis in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) remains lethal despite the development of numerous drug therapy technologies. About 85% to 90% of lung cancers are NSCLC and the 5-year survival rate is at best still below 50%. Thus, it is important to find drugable target genes for NSCLC to develop an effective therapy for NSCLC. RESULTS: Integrated analysis of publically available gene expression and promoter methylation patterns of two highly aggressive NSCLC cell lines generated by in vivo selection was performed. We selected eleven critical genes that may mediate metastasis using recently proposed principal component analysis based unsupervised feature extraction. The eleven selected genes were significantly related to cancer diagnosis. The tertiary protein structure of the selected genes was inferred by Full Automatic Modeling System, a profile-based protein structure inference software, to determine protein functions and to specify genes that could be potential drug targets. CONCLUSIONS: We identified eleven potentially critical genes that may mediate NSCLC metastasis using bioinformatic analysis of publically available data sets. These genes are potential target genes for the therapy of NSCLC. Among the eleven genes, TINAGL1 and B3GALNT1 are possible candidates for drug compounds that inhibit their gene expression.

Community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions.

Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side-chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies.

Method for predicting homology modeling accuracy from amino acid sequence alignment: the power function.

We have devised a power function (PF) that can predict the accuracy of a three-dimensional (3D) structure model of a protein using only amino acid sequence alignments. This Power Function (PF) consists of three parts; (1) the length of a model, (2) a homology identity percent value and (3) the agreement rate between PSI-PRED secondary structure prediction and the secondary structure judgment of a reference protein. The PF value is mathematically computed from the execution process of homology search tools, such as FASTA or various BLAST programs, to obtain the amino acid sequence alignments. There is a high correlation between the global distance test-total score (GDT_TS) value of the protein model based upon the PF score and the GDT_TS(MAX) value used as an index of protein modeling accuracy in the international contest Critical Assessment of Techniques for Protein Structure Prediction (CASP). Accordingly, the PF method is valuable for constructing a highly accurate model without wasteful calculations of homology modeling that is normally performed by an iterative method to move the main chain and side chains in the modeling process. Moreover, a model with higher accuracy can be obtained by combining the models ordered by the PF score with models sorted by the size of the CIRCLE score. The CIRCLE software is a 3D-1D program, in which energetic stabilization is estimated based upon the experimental environment of each amino acid residue in the protein solution or protein crystals.

HUMAN FAMSD-BASE: high quality protein structure model database for the human genome using the FAMSD homology modeling method.

Almost all proteins express their biological functions through the structural conformation of their specific amino acid sequences. Therefore, acquiring the three-dimensional structures of proteins is very important to elucidate the role of a particular protein. We had built protein structure model databases, which is called RIKEN FAMSBASE (http://famshelp.gsc.riken.jp/famsbase/). The RIKEN FAMSBASE is a genome-wide protein structure model database that contains a large number of protein models from many organisms. The HUMAN FAMSBASE that is one part of the RIKEN FAMSBASE contains many protein models for human genes, which are significant in the pharmaceutical and medicinal fields. We have now implemented an update of the human protein modeling database consisting of 242918 constructed models against the number of 20743 human protein sequences with an improved modeling method called Full Automatic protein Modeling System Developed (FAMSD). The results of our benchmark test of the FAMSD method indicated that it has an excellent capability to pack amino acid side-chains with correct torsion angles in addition to the main-chain, while avoiding the formation of atom-atom collisions that are not found in experimental structures. This new protein structure model database for human genes, which is named HUMAN FAMSD-BASE, is open to the public as a component part of the RIKEN FAMSBASE at http://mammalia.gsc.riken.jp/human_famsd/. A significant improvement of the HUMAN FAMSD-BASE in comparison with the preceding HUMAN FAMSBASE was verified in the benchmark test of this paper. The HUMAN FAMSD-BASE will have an important impact on the progress of biological science.

Author Correction: Development of a novel anti-hepatitis B virus agent via Sp1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of a novel anti-hepatitis B virus agent via Sp1.

Nucleos(t)ide analog (NA) therapy has proven effective in treating chronic hepatitis B. However, NAs frequently result in viral relapse after the cessation of therapy. This is because NAs cannot fully eliminate the viral episomal covalently closed circular DNA (cccDNA) in the nucleus. In this study, we identified small molecular compounds that control host factors related to viral replication using in silico screening with simulated annealing based on bioinformatics for protein-ligand flexible docking. Twelve chemical compound candidates for alpha-glucosidase (AG) inhibitors were identified from a library of chemical compounds and used to treat fresh human hepatocytes infected with HBV. They were then monitored for their anti-viral effects. HBV replication was inhibited by one candidate (1-[3-(4-tert-butylcyclohexyl)oxy-2-hydroxypropyl]-2,2,6,6-tetramethylpiperidin-4-ol) in a dose-dependent manner. This compound significantly reduced ccc DNA production, compared to Entecavir (p < 0.05), and had a lower anti-AG effect. Gene expression analysis and structural analysis of this compound showed that its inhibitive effect on HBV was via interaction with Sp1. The nuclear transcription factor Sp1 acts on multiple regions of HBV to suppress HBV replication. Identifying candidates that control nuclear transcription factors facilitate the development of novel therapies. Drugs with a mechanism different from NA are promising for the elimination of HBV.

FAMSD: A powerful protein modeling platform that combines alignment methods, homology modeling, 3D structure quality estimation and molecular dynamics.

The prediction of a protein three-dimensional (3D) structure is one of the most important challenges in computational structural biology. We have developed an automatic protein 3D structure prediction method called FAMSD. FAMSD is based on a comparative modeling method which consists of the following four steps: (1) generating and selecting sequence alignments between target and template proteins; (2) constructing 3D structure models based on each selected alignment; (3) selecting the best 3D structure model and (4) refining the selected model. In the FAMSD method, sequence alignment programs such as a series of BLAST programs, SP3 and SPARKS2 programs, the homology modeling program FAMS (Full Automatic Modeling System), the model quality estimation program CIRCLE and the molecular dynamics program APRICOT were used in combination to construct high quality protein models. To assess the FAMSD method we have participated in the 8th Critical Assessment of Techniques for Protein Structure Prediction (CASP8) experiment. The results of our original assessment indicate that the FAMSD method offers excellent capability in packing side-chains with the correct torsion angles while avoiding the formation of atom-atom collisions. Since side-chain packing plays a significant role in defining the biological function of proteins, this method is a valuable resource in biological, pharmaceutical and medicinal research efforts.

A prospective compound screening contest identified broader inhibitors for Sirtuin 1.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

The SKE-DOCK server and human teams based on a combined method of shape complementarity and free energy estimation.

We participated in rounds 6-12 of the critical assessment of predicted interaction (CAPRI) contest as the SKE-DOCK server and human teams. The SKE-DOCK server is based on simple geometry docking and a knowledge base scoring function. The procedure is summarized in the following three steps: (1) protein docking according to shape complementarity, (2) evaluating complex models, and (3) repacking side-chain of models. The SKE-DOCK server did not make use of biological information. On the other hand, the human team tried various intervention approaches. In this article, we describe in detail the processes of the SKE-DOCK server, together with results and reasons for success and failure. Good predicted models were obtained for target 25 by both the SKE-DOCK server and human teams. When the modeled receptor proteins were superimposed on the experimental structures, the smallest Ligand-rmsd values corresponding to the rmsd between the model and experimental structures were 3.307 and 3.324 A, respectively. Moreover, the two teams obtained 4 and 2 acceptable models for target 25. The overall result for both the SKE-DOCK server and human teams was medium accuracy for one (Target 25) out of nine targets.

Fams-ace: a combined method to select the best model after remodeling all server models.

During Critical Assessment of Protein Structure Prediction (CASP7, Pacific Grove, CA, 2006), fams-ace was entered in the 3D coordinate prediction category as a human expert group. The procedure can be summarized by the following three steps. (1) All the server models were refined and rebuilt utilizing our homology modeling method. (2) Representative structures were selected from each server, according to a model quality evaluation, based on a 3D1D profile score (like Verify3D). (3) The top five models were selected and submitted in the order of the consensus-based score (like 3D-Jury). Fams-ace is a fully automated server and does not require human intervention. In this article, we introduce the methodology of fams-ace and discuss the successes and failures of this approach during CASP7. In addition, we discuss possible improvements for the next CASP.

An iterative compound screening contest method for identifying target protein inhibitors using the tyrosine-protein kinase Yes.

We propose a new iterative screening contest method to identify target protein inhibitors. After conducting a compound screening contest in 2014, we report results acquired from a contest held in 2015 in this study. Our aims were to identify target enzyme inhibitors and to benchmark a variety of computer-aided drug discovery methods under identical experimental conditions. In both contests, we employed the tyrosine-protein kinase Yes as an example target protein. Participating groups virtually screened possible inhibitors from a library containing 2.4 million compounds. Compounds were ranked based on functional scores obtained using their respective methods, and the top 181 compounds from each group were selected. Our results from the 2015 contest show an improved hit rate when compared to results from the 2014 contest. In addition, we have successfully identified a statistically-warranted method for identifying target inhibitors. Quantitative analysis of the most successful method gave additional insights into important characteristics of the method used.

Enlarged FAMSBASE: protein 3D structure models of genome sequences for 41 species.

Enlarged FAMSBASE is a relational database of comparative protein structure models for the whole genome of 41 species, presented in the GTOP database. The models are calculated by Full Automatic Modeling System (FAMS). Enlarged FAMSBASE provides a wide range of query keys, such as name of ORF (open reading frame), ORF keywords, Protein Data Bank (PDB) ID, PDB heterogen atoms and sequence similarity. Heterogen atoms in PDB include cofactors, ligands and other factors that interact with proteins, and are a good starting point for analyzing interactions between proteins and other molecules. The data may also work as a template for drug design. The present number of ORFs with protein 3D models in FAMSBASE is 183 805, and the database includes an average of three models for each ORF. FAMSBASE is available at http://famsbase.bio.nagoya-u.ac.jp/famsbase/.

SFRP1 is a possible candidate for epigenetic therapy in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) remains a lethal disease despite many proposed treatments. Recent studies have indicated that epigenetic therapy, which targets epigenetic effects, might be a new therapeutic methodology for NSCLC. However, it is not clear which objects (e.g., genes) this treatment specifically targets. Secreted frizzled-related proteins (SFRPs) are promising candidates for epigenetic therapy in many cancers, but there have been no reports of SFRPs targeted by epigenetic therapy for NSCLC. METHODS: This study performed a meta-analysis of reprogrammed NSCLC cell lines instead of the direct examination of epigenetic therapy treatment to identify epigenetic therapy targets. In addition, mRNA expression/promoter methylation profiles were processed by recently proposed principal component analysis based unsupervised feature extraction and categorical regression analysis based feature extraction. RESULTS: The Wnt/ß-catenin signalling pathway was extensively enriched among 32 genes identified by feature extraction. Among the genes identified, SFRP1 was specifically indicated to target ß-catenin, and thus might be targeted by epigenetic therapy in NSCLC cell lines. A histone deacetylase inhibitor might reactivate SFRP1 based upon the re-analysis of a public domain data set. Numerical computation validated the binding of SFRP1 to WNT1 to suppress Wnt signalling pathway activation in NSCLC. CONCLUSIONS: The meta-analysis of reprogrammed NSCLC cell lines identified SFRP1 as a promising target of epigenetic therapy for NSCLC.

Protein structure prediction in CASP6 using CHIMERA and FAMS.

In CASP6, the CHIMERA-group predicted full-atom models of all targets using SKE-CHIMERA, a Web-user interface system for protein structure prediction that allows human intervention at necessary stages; we used a lot of information from our own data and from publicly available data. Using SKE-CHIMERA, we iterated manual step (template selection and alignment by the in-house program CHIMERA) and automatic step (three-dimensional model building by the in-house program FAMS). The official CASP6 assessment showed that CHIMERA-group was one of the most successful predictors in homology modeling, especially for FR/H (Fold Recognition/Homologous). In this article, we introduce the method of CHIMERA-group and discuss its successes and failures in CASP6.

Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

Evaluation of the third solvent clusters fitting procedure for the prediction of protein-protein interactions based on the results at the CAPRI blind docking study.

To predict protein-protein interactions, rough or coarse handling for the induced fit problem is proposed. Our method involves the overlap of two hydrophobic interactions as "third solvent clusters fitting." Predictions for binding sites and geometric centers were acceptable, but those of the binding axes were poor. In this study, only the largest benzene cluster was used for the third solvent clusters fitting. For the next CAPRI targets, we must perform protein-protein interaction analyses, which include other smaller benzene clusters.