Multiple protonation states in ligand-free SARS-CoV-2 main protease revealed by large-scale quantum molecular dynamics simulations.

The main protease (Mpro) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) catalyzes the cleavage of polyproteins for viral replication. Here, large-scale quantum molecular dynamics and metadynamics simulations for ligand-free Mpro were performed, where all the atoms were treated quantum-mechanically, focusing on elucidation of the controversial active-site protonation state. The simulations clarified that the interconverting multiple protonation states exist in unliganded Mpro, and the catalytically relevant ion-pair state is more stable than the neutral state, which is consistent with neutron crystallography. The results highlight the importance of the ion-pair state for repurposing or discovering antiviral drugs that target Mpro.

Asymmetric dynamics of dimeric SARS-CoV-2 and SARS-CoV main proteases in an apo form: Molecular dynamics study on fluctuations of active site, catalytic dyad, and hydration water.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely spread around the world. It is necessary to examine the viral proteins that play a notorious role in the invasion of our body. The main protease (3CLpro) facilitates the maturation of the coronavirus. It is thought that the dimerization of 3CLpro leads to its catalytic activity; the detailed mechanism has, however, not been suggested. Furthermore, the structural differences between the predecessor SARS-CoV 3CLpro and SARS-CoV-2 3CLpro have not been fully understood. Here, we show the structural and dynamical differences between the two main proteases, and demonstrate the relationship between the dimerization and the activity via atomistic molecular dynamics simulations. Simulating monomeric and dimeric 3CLpro systems for each protease, we show that (i) global dynamics between the two different proteases are not conserved, (ii) the dimerization stabilizes the catalytic dyad and hydration water molecules behind the dyad, and (iii) the substrate-binding site (active site) and hydration water molecules in each protomer fluctuate asymmetrically. We then speculate the roles of hydration water molecules in their catalytic activity.

Flexibility and Cell Permeability of Cyclic Ras-Inhibitor Peptides Revealed by the Coupled Nosé-Hoover Equation.

Quantifying the cell permeability of cyclic peptides is crucial for their rational drug design. However, the reasons remain unclear why a minor chemical modification, such as the difference between Ras inhibitors cyclorasin 9A5 and 9A54, can substantially change a peptide's permeability. To address this question, we performed enhanced sampling simulations of these two 11-mer peptides using the coupled Nosé-Hoover equation (cNH) we recently developed. The present cNH simulations realized temperature fluctuations over a wide range (240-600 K) in a dynamic manner, allowing structural samplings that were well validated by nuclear Overhauser effect measurements. The derived structural ensembles were comprehensively analyzed by all-atom structural clustering, mapping the derived clusters onto principal components (PCs) that characterize the cyclic structure, and calculating cluster-dependent geometric and chemical properties. The planar-open conformation was dominant in aqueous solvent, owing to inclusion of the Trp side chain in the main-chain ring, while the compact-closed conformation, which favors cell permeation due to its compactness and high polarity, was also accessible. Conformation-dependent cell permeability was observed in one of the derived PCs, demonstrating that decreased cell permeability in 9A54 is due to the high free energy barrier separating the two conformations. The origin of the change in free energy surface was determined to be loss of flexibility in the modified residues 2-3, resulting from the increased bulkiness of their side chains. The derived molecular mechanism of cell permeability highlights the significance of complete structural dynamics surveys for accelerating drug development with cyclic peptides.

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations.

We have performed multicanonical molecular dynamics (McMD) based dynamic docking simulations to study and compare the binding mechanism between two medium-sized inhibitors (ABT-737 and WEHI-539) that bind to the cryptic site of Bcl-xL, by exhaustively sampling the conformational and configurational space. Cryptic sites are binding pockets that are transiently formed in the apo state or are induced upon ligand binding. Bcl-xL, a pro-survival protein involved in cancer progression, is known to have a cryptic site, whereby the shape of the pocket depends on which ligand is bound to it. Starting from the apo-structure, we have performed two independent McMD-based dynamic docking simulations for each ligand, and were able to obtain near-native complex structures in both cases. In addition, we have also studied their interactions along their respective binding pathways by using path sampling simulations, which showed that the ligands form stable binding configurations via predominantly hydrophobic interactions. Although the protein started from the apo state, both ligands modulated the pocket in different ways, shifting the conformational preference of the sub-pockets of Bcl-xL. We demonstrate that McMD-based dynamic docking is a powerful tool that can be effectively used to study binding mechanisms involving a cryptic site, where ligand binding requires a large conformational change in the protein to occur.

Molecular Interaction Mechanism of a 14-3-3 Protein with a Phosphorylated Peptide Elucidated by Enhanced Conformational Sampling.

Enhanced conformational sampling, a genetic-algorithm-guided multidimensional virtual-system coupled molecular dynamics, can provide equilibrated conformational distributions of a receptor protein and a flexible ligand at room temperature. The distributions provide not only the most stable but also semistable complex structures and propose a ligand-receptor binding process. This method was applied to a system consisting of a receptor protein, 14-3-3ε, and a flexible peptide, phosphorylated myeloid leukemia factor 1 (pMLF1). The results present comprehensive binding pathways of pMLF1 to 14-3-3ε. We identified four thermodynamically stable clusters of MLF1 on the 14-3-3ε surface and free-energy barriers among some clusters. The most stable cluster includes two high-density spots connected by a narrow corridor. When pMLF1 passes the corridor, a salt-bridge relay (switching) related to the phosphorylated residue of pMLF1 occurs. Conformations in one high-density spot are similar to the experimentally determined complex structure. Three-dimensional distributions of residues in the intermolecular interface rationally explain the binding constant changes resulting from the alanine mutation experiment for the residues. We also performed a simulation of nonphosphorylated peptide and 14-3-3ε, which demonstrated that the complex structure was unstable, suggesting that phosphorylation of the peptide is crucially important for binding to 14-3-3ε.

A2B adenosine receptor inhibition by the dihydropyridine calcium channel blocker nifedipine involves colonic fluid secretion.

The adenosine A2B receptor is a critical protein in intestinal water secretion. In the present study, we screened compound libraries to identify inhibitors of the A2B receptor and evaluated their effect on adenosine-induced intestinal fluid secretion. The screening identified the dihydropyridine calcium antagonists nifedipine and nisoldipine. Their respective affinities for the A2B receptor (Ki value) were 886 and 1,399 nM. Nifedipine and nisoldipine, but not amlodipine or nitrendipine, inhibited both calcium mobilization and adenosine-induced cAMP accumulation in cell lines. Moreover, adenosine injection into the lumen significantly increased fluid volume in the colonic loop of wild-type mice but not A2B receptor-deficient mice. PSB-1115, a selective A2B receptor antagonist, and nifedipine prevented elevated adenosine-stimulated fluid secretion in mice. Our results may provide useful insights into the structure-activity relationship of dihydropyridines for A2B receptor. As colonic fluid secretion by adenosine seems to rely predominantly on the A2B receptor, nifedipine could be a therapeutic candidate for diarrhoea-related diseases.

Sertraline, chlorprothixene, and chlorpromazine characteristically interact with the REST-binding site of the corepressor mSin3, showing medulloblastoma cell growth inhibitory activities.

Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington's disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.

Variation of free-energy landscape of the p53 C-terminal domain induced by acetylation: Enhanced conformational sampling.

The C-terminal domain (CTD) of tumor suppressor protein p53 is an intrinsically disordered region that binds to various partner proteins, where lysine of CTD is acetylated/nonacetylated and histidine neutralized/non-neutralized. Because of the flexibility of the unbound CTD, a free-energy landscape (FEL) is a useful quantity for determining its statistical properties. We conducted enhanced conformational sampling of CTD in the unbound state via virtual system coupled multicanonical molecular dynamics, in which the lysine was acetylated or nonacetylated and histidine was charged or neutralized. The fragments were expressed by an all-atom model and were immersed in an explicit solvent. The acetylation and charge-neutralization varied FEL greatly, which might be convenient to exert a hub property. The acetylation slightly enhanced alpha-helix structures that are more compact than sheet/loop conformations. The charge-neutralization produced hairpins. Additionally, circular dichroism experiments confirmed the computational results. We propose possible binding mechanisms of CTD to partners by investigating FEL. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Structure-Based Development of a Protein-Protein Interaction Inhibitor Targeting Tumor Necrosis Factor Receptor-Associated Factor 6.

The interactions between tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF superfamily receptors (TNFRSFs) are promising targets for rheumatoid arthritis (RA) treatment. However, due to the challenging nature of protein-protein interactions (PPIs), a potent inhibitor that surpasses the affinity of the TRAF6-TNFRSF interactions has not been developed. We developed a small-molecule PPI inhibitor of TRAF6-TNFRSF interactions using NMR and in silico techniques. The most potent compound, TRI4, exhibited an affinity higher than those of TNFRSFs and competitively inhibited a TRAF6-TNFRSF interaction. Structural characterization of the TRAF6-TRI4 complex revealed that TRI4 supplants key interactions in the TRAF6-TNFRSF interfaces. In addition, some TRAF6-TRI4 interactions extend beyond the TRAF6-TNFRSF interfaces and increase the binding affinity. Our successful development of TRI4 provides a new opportunity for RA treatment and implications for structure-guided development of PPI inhibitors.

Bongkrekic acid as a selective activator of the peroxisome proliferator-activated receptor γ (PPARγ) isoform.

Bongkrekic acid (BKA), an antibiotic isolated from Pseudomonas cocovenans, is an inhibitory molecule of adenine nucleotide translocase. Since this translocase is a core component of the mitochondrial permeability transition pore (MPTP) formed by apoptotic stimuli, BKA has been used as a tool to abrogate apoptosis. However, the other biochemical properties of BKA have not yet been resolved. Although the definition of a fatty acid is a carboxylic acid (-COOH) with a long hydrocarbon chain (tail), when focused on the chemical structure of BKA, the molecule was revealed to be a branched unsaturated tricarboxylic acid that resembled the structure of polyunsaturated fatty acids (PUFAs). Peroxisome proliferator-activated receptors (PPARs) consist of a subfamily of three isoforms: α, ß, and γ, the ligands of which include PUFAs. Using completely synthesized BKA together with simplified BKA derivatives (purity: > 98%), we herein demonstrated the utility of BKA as a selective activator of the human PPARγ isoform, which may not be associated with the anti-apoptotic nature of BKA. We also discussed the possible usefulness of BKA.

LigandBox: A database for 3D structures of chemical compounds.

A database for the 3D structures of available compounds is essential for the virtual screening by molecular docking. We have developed the LigandBox database (http://ligandbox.protein.osaka-u.ac.jp/ligandbox/) containing four million available compounds, collected from the catalogues of 37 commercial suppliers, and approved drugs and biochemical compounds taken from KEGG_DRUG, KEGG_COMPOUND and PDB databases. Each chemical compound in the database has several 3D conformers with hydrogen atoms and atomic charges, which are ready to be docked into receptors using docking programs. The 3D conformations were generated using our molecular simulation program package, myPresto. Various physical properties, such as aqueous solubility (LogS) and carcinogenicity have also been calculated to characterize the ADME-Tox properties of the compounds. The Web database provides two services for compound searches: a property/chemical ID search and a chemical structure search. The chemical structure search is performed by a descriptor search and a maximum common substructure (MCS) search combination, using our program kcombu. By specifying a query chemical structure, users can find similar compounds among the millions of compounds in the database within a few minutes. Our database is expected to assist a wide range of researchers, in the fields of medical science, chemical biology, and biochemistry, who are seeking to discover active chemical compounds by the virtual screening.

Selection of in silico drug screening results for G-protein-coupled receptors by using universal active probes.

We developed a new protocol for in silico drug screening for G-protein-coupled receptors (GPCRs) using a set of "universal active probes" (UAPs) with an ensemble docking procedure. UAPs are drug-like compounds, which are actual active compounds of a variety of known proteins. The current targets were nine human GPCRs whose three-dimensional (3D) structures are unknown, plus three GPCRs, namely ß(2)-adrenergic receptor (ADRB2), A(2A) adenosine receptor (A(2A)), and dopamine D3 receptor (D(3)), whose 3D structures are known. Homology-based models of the GPCRs were constructed based on the crystal structures with careful sequence inspection. After subsequent molecular dynamics (MD) simulation taking into account the explicit lipid membrane molecules with periodic boundary conditions, we obtained multiple model structures of the GPCRs. For each target structure, docking-screening calculations were carried out via the ensemble docking procedure, using both true active compounds of the target proteins and the UAPs with the multiple target screening (MTS) method. Consequently, the multiple model structures showed various screening results with both poor and high hit ratios, the latter of which could be identified as promising for use in in silico screening to find candidate compounds to interact with the proteins. We found that the hit ratio of true active compounds showed a positive correlation to that of the UAPs. Thus, we could retrieve appropriate target structures from the GPCR models by applying the UAPs, even if no active compound is known for the GPCRs. Namely, the screening result that showed a high hit ratio for the UAPs could be used to identify actual hit compounds for the target GPCRs.

Cannabidiol-2',6'-dimethyl ether as an effective protector of 15-lipoxygenase-mediated low-density lipoprotein oxidation in vitro.

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein (ox-LDL), a major causal factor for atherosclerosis. Both enzymatic (15-LOX) and non-enzymatic (Cu(2+)) mechanisms have been proposed for the production of ox-LDL. We have recently reported that cannabidiol-2',6'-dimethyl ether (CBDD) is a selective and potent inhibitor of 15-LOX-catalyzed linoleic acid oxygenation (Takeda et al., Drug Metab. Dispos., 37, 1733-1737 (2009)). In the LDL, linoleic acid is present as cholesteryl linoleate, the major fatty acid esterified to cholesterol, and is susceptible to oxidative modification by 15-LOX or Cu(2+). In this investigation, we examined the efficacy of CBDD on i) 15-LOX-catalyzed oxygenation of cholesteryl linoleate, and ii) ox-LDL formation catalyzed by 15-LOX versus Cu(2+)-mediated non-enzymatic generation of this important mediator. The results obtained demonstrate that CBDD is a potent and selective inhibitor of ox-LDL formation generated by the 15-LOX pathway. These studies establish CBDD as both an important experimental tool for characterizing 15-LOX-mediated ox-LDL formation, and as a potentially useful therapeutic agent for treatment of atherosclerosis.

In silico prediction of the chemical block of human ether-a-go-go-related gene (hERG) K+ current.

A variety of compounds with different chemical properties directly interact with the cardiac repolarizing K(+) channel encoded by the human ether-a-go-go-related gene (hERG). This causes acquired forms of QT prolongation, which can result in lethal cardiac arrhythmias including torsades de pointes one of the most serious adverse effects of various therapeutic agents. Prediction of this phenomenon will improve the safety of pharmacological therapy and also facilitate the process of drug development. Here we propose a strategy for the development of an in silico system to predict the potency of chemical compounds to block hERG. The system consists of two sequential processes. The first process is a ligand-based prediction to estimate half-maximal concentrations for the block of compounds inhibiting hERG current using the relationship between chemical features and activities of compounds. The second process is a protein-based prediction that comprises homology modeling of hERG, docking simulation of chemical-channel interaction, analysis of the shape of the channel pore cavity, and Brownian dynamics simulation to estimate hERG currents in the presence and absence of chemical blockers. Since each process is a combination of various calculations, the criterion for assessment at each calculation and the strategy to integrate these steps are significant for the construction of the system to predict a chemical's block of hERG current and also to predict the risk of inducing cardiac arrhythmias from the chemical information. The principles and criteria of elemental computations along this strategy are described.

Mutational analysis of block and facilitation of HERG current by a class III anti-arrhythmic agent, nifekalant.

Chemicals and toxins are useful tools to elucidate the structure-function relationship of various proteins including ion channels. The HERG channel is blocked by many compounds and this may cause life-threatening cardiac arrhythmia. Besides block, some chemicals such as the class III anti-arrhythmic agent nifekalant stimulate HERG at low potentials by shifting its activation curve towards hyperpolarizing voltages. This is called "facilitation". Here, we report mutations and simulations analyzing the association between nifekalant and channel pore residues for block and facilitation. Alanine-scanning mutagenesis was performed in the pore region of HERG. The mutations at the base of the pore helix (T623A), the selectivity filter (V625A) and the S6 helix (G648A, Y652A and F656A) abolished and S624A attenuated both block and facilitation induced by the drug. On the other hand, the mutation of other residues caused either an increase or a decrease in nifekalant-induced facilitation without affecting block. An open-state homology model of the HERG pore suggested that T623, S624, Y652 and F656 faced the central cavity, and were positioned within geometrical range for the drug to be able to interact with all of them at the same time. Of these, S649 was the only polar residue located within possible interaction distance from the drug held in its blocking position. Further mutations and flexible-docking simulations suggest that the size, but not the polarity, of the side chain at S649 is critical for drug induced facilitation.