Binding Mechanism of Riboswitch to Natural Ligand Elucidated by McMD-Based Dynamic Docking Simulations.

Flavin mononucleotide riboswitches are common among many pathogenic bacteria and are therefore considered to be an attractive target for antibiotics development. The riboswitch binds riboflavin (RBF, also known as vitamin B2), and although an experimental structure of their complex has been solved with the ligand bound deep inside the RNA molecule in a seemingly unreachable state, the binding mechanism between these molecules is not yet known. We have therefore used our Multicanonical Molecular Dynamics (McMD)-based dynamic docking protocol to analyze their binding mechanism by simulating the binding process between the riboswitch aptamer domain and the RBF, starting from the apo state of the riboswitch. Here, the refinement stage was crucial to identify the native binding configuration, as several other binding configurations were also found by McMD-based docking simulations. RBF initially binds the interface between P4 and P6 including U61 and G62, which forms a gateway where the ligand lingers until this gateway opens sufficiently to allow the ligand to pass through and slip into the hidden binding site including A48, A49, and A85.

A cutoff-based method with charge-distribution-data driven pair potentials for efficiently estimating electrostatic interactions in molecular systems.

We introduce a simple cutoff-based method for precise electrostatic energy calculations in the molecular dynamics (MD) simulations of point-particle systems. Our method employs a theoretically derived smooth pair potential function to define electrostatic energy, offering stability and computational efficiency in MD simulations. Instead of imposing specific physical conditions, such as dielectric environments or charge neutrality, we focus on the relationship represented by a single summation formula of charge-weighted pair potentials. This approach allows an accurate energy approximation for each particle, enabling a straightforward error analysis. The resulting particle-dependent pair potential captures the charge distribution information, making it suitable for heterogeneous systems and ensuring an enhanced accuracy through distant information inclusion. Numerical investigations of the Madelung constants of crystalline systems validate the method's accuracy.

Binding free-energy landscapes of small molecule binder and non-binder to FMN riboswitch: All-atom molecular dynamics.

A small and flexible molecule, ribocil A (non-binder) or B (binder), binds to the deep pocket of the aptamer domain of the FMN riboswitch, which is an RNA molecule. This binding was studied by mD-VcMD, which is a generalized-ensemble simulation method. Ribocil A and B are structurally similar because they are optical isomers to each other. In the initial conformation of simulation, the ligands and the aptamer were completely dissociated in explicit solvent. The aptamer-ribocil B binding was stronger than the aptamer-ribocil A binding, which agrees with experiments. The computed free-energy landscape for the aptamer-ribocil B binding was funnel-like, whereas that for the aptamer-ribocil A binding was rugged. When passing through the gate (named "front gate") of the binding pocket, each ligand interacted with bases of the riboswitch by non-native π-π stackings, and the stackings restrained the ligand's orientation to be advantageous to reach the binding site smoothly. When the ligands reached the binding site in the pocket, the non-native stackings were replaced by the native stackings. The ligand's orientation restriction is discussed referring to a selection mechanism reported in an earlier work on a drug-GPCR interaction. The present simulation showed another pathway leading the ligands to the binding site. The gate ("rear gate") for this pathway was located completely opposite to the front gate on the aptamer's surface. However, the approach from the rear gate required overcoming a free-energy barrier regarding ligand's rotation before reaching the binding site.

Computer simulation of molecular recognition in biomolecular system: from in silico screening to generalized ensembles.

Prediction of ligand-receptor complex structure is important in both the basic science and the industry such as drug discovery. We report various computation molecular docking methods: fundamental in silico (virtual) screening, ensemble docking, enhanced sampling (generalized ensemble) methods, and other methods to improve the accuracy of the complex structure. We explain not only the merits of these methods but also their limits of application and discuss some interaction terms which are not considered in the in silico methods. In silico screening and ensemble docking are useful when one focuses on obtaining the native complex structure (the most thermodynamically stable complex). Generalized ensemble method provides a free-energy landscape, which shows the distribution of the most stable complex structure and semi-stable ones in a conformational space. Also, barriers separating those stable structures are identified. A researcher should select one of the methods according to the research aim and depending on complexity of the molecular system to be studied.

Investigation on substrate specificity and catalytic activity of serine protease neuropsin.

Neuropsin is one of serine proteases mainly found at the hippocampus and the amygdala, where it contributes to the long-term potentiation and memory acquisition by rebuilding of synaptic connections. Despite of the importance of neuropsin, the substrate specificity and regulation mechanisms of neuropsin have been unclear. Thus, we investigated the substrate specificity and the catalytic activity of neuropsin by the protein-ligand docking and molecular dynamics (MD) simulations and succeeded to reproduce the trend of the experimental results. Our study revealed that the substrate specificity and the activity of neuropsin depended on multiple factors: the substrate charge, the substrate orientation, the hydrogen bond network within the catalytic triad and the substrate, and the formation of the oxyanion hole. The apo neuropsin was not reactive without proper alignment of catalytic triad. The substrate binding induced the reactive alignment of catalytic triad. Then the substrate-neuropsin interaction forms the oxyanion hole that stabilizes the transition state and reduces the free-energy barrier of the following scission reaction.

Fly casting with ligand sliding and orientational selection supporting complex formation of a GPCR and a middle sized flexible molecule.

A GA-guided multidimensional virtual-system coupled molecular dynamics (GA-mD-VcMD) simulation was conducted to elucidate binding mechanisms of a middle-sized flexible molecule, bosentan, to a GPCR protein, human endothelin receptor type B (hETB). GA-mD-VcMD is a generalized ensemble method that produces a free-energy landscape of the ligand-receptor binding by searching large-scale motions accompanied with stable maintenance of the fragile cell-membrane structure. All molecular components (bosentan, hETB, membrane, and solvent) were represented with an all-atom model. Then sampling was conducted from conformations where bosentan was distant from the binding site in the hETB binding pocket. The deepest basin in the resultant free-energy landscape was assigned to native-like complex conformation. The following binding mechanism was inferred. First, bosentan fluctuating randomly in solution is captured using a tip region of the flexible N-terminal tail of hETB via nonspecific attractive interactions (fly casting). Bosentan then slides occasionally from the tip to the root of the N-terminal tail (ligand-sliding). During this sliding, bosentan passes the gate of the binding pocket from outside to inside of the pocket with an accompanying rapid reduction of the molecular orientational variety of bosentan (orientational selection). Last, in the pocket, ligand-receptor attractive native contacts are formed. Eventually, the native-like complex is completed. The bosentan-captured conformations by the tip-region and root-region of the N-terminal tail correspond to two basins in the free-energy landscape. The ligand-sliding corresponds to overcoming of a free-energy barrier between the basins.

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.

Molecular Dynamics Simulation Study on Allosteric Regulation of CD44-Hyaluronan Binding as a Force Sensing Mechanism.

CD44 protein exists on surfaces of a variety of human cells, acts as a receptor for the hyaluronan (HA) molecule, and mediates cell adhesion via the HA binding in leukocyte trafficking, cell rolling, and so on. The molecular structures of both CD44 and HA are well known, and the previous work shows that the external-mechanical force induces the partially disordered (PD) conformation from the ordered (O) conformation of CD44. The PD conformation has the higher HA affinity compared to the O conformation. However, the details of force-sensing mechanics have remained unclear. This study provides new insights into allosteric regulation of HA binding by conformational shift from the O to the PD conformation of the CD44 HA binding domain by using the classical molecular dynamics simulations. The O conformation was more favorable than the PD conformation under the equilibrium state, and the O conformation showed weak HA-binding affinity. Our simulation suggests that the PD conformation induced by the external force can refold to a compact structure similar to the O conformation keeping the bound HA. This new conformation showed a higher affinity than the O and PD conformations. Our results show that the unfolding of a remote disordered region from the ligand binding site by the external force allosterically regulates the HA affinity. This study promotes understanding not only the mechanism of CD44-mediated cell rolling but also the allosteric regulation induced by the external mechanical force.

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.

Generalized-ensemble method study: A helix-mimetic compound inhibits protein-protein interaction by long-range and short-range intermolecular interactions.

A heterocyclic compound mS-11 is a helix-mimetic designed to inhibit binding of an intrinsic disordered protein neural restrictive silence factor/repressor element 1 silencing factor (NRSF/REST) to a receptor protein mSin3B. We apply a generalized ensemble method, multi-dimensional virtual-system coupled molecular dynamics developed by ourselves recently, to a system consisting of mS-11 and mSin3B, and obtain a thermally equilibrated distribution, which is comprised of the bound and unbound states extensively. The lowest free-energy position of mS-11 coincides with the NRSF/REST position in the experimentally-determined NRSF/REST-mSin3B complex. Importantly, the molecular orientation of mS-11 is ordering in a wide region around mSin3B. The resultant binding scenario is: When mS-11 is distant from the binding site of mSin3B, mS-11 descends the free-energy slope toward the binding site maintaining the molecular orientation to be advantageous for binding. Then, finally a long and flexible hydrophobic sidechain of mS-11 fits into the binding site, which is the lowest-free-energy complex structure inhibiting NRSF/REST binding to mSin3B.

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.

Difference of binding modes among three ligands to a receptor mSin3B corresponding to their inhibitory activities.

A preceding experiment suggested that a compound, which inhibits binding of the REST/NRSF segment to the cleft of a receptor protein mSin3B, can be a potential drug candidate to ameliorate many neuropathies. We have recently developed an enhanced conformational sampling method, genetic-algorithm-guided multi-dimensional virtual-system-coupled canonical molecular dynamics, and in the present study, applied it to three systems consisting of mSin3B and one of three compounds, sertraline, YN3, and acitretin. Other preceding experiments showed that only sertraline inhibits the binding of REST/NRSF to mSin3B. The current simulation study produced the spatial distribution of the compounds around mSin3B, and showed that sertraline and YN3 bound to the cleft of mSin3B with a high propensity, although acitretin did not. Further analyses of the simulation data indicated that only the sertraline-mSin3B complex produced a hydrophobic core similar to that observed in the molecular interface of the REST/NRSF-mSin3B complex: An aromatic ring of sertraline sunk deeply in the mSin3B's cleft forming a hydrophobic core contacting to hydrophobic amino-acid residues located at the bottom of the cleft. The present study proposes a step to design a compound that inhibits competitively the binding of a ligand to its receptor.

Structural Fluctuations of Aromatic Residues in an Apo-Form Reveal Cryptic Binding Sites: Implications for Fragment-Based Drug Design.

Cryptic sites are binding pockets that are transiently formed in an apo form or that are induced by ligand binding. The investigation of cryptic sites is crucial for drug discovery, since these sites are ubiquitous in disease-related human proteins, and targeting them expands the number of drug targets greatly. However, although many computational studies have attempted to identify cryptic sites, the detection remains challenging. Here, we aimed to characterize and detect cryptic sites in terms of structural fluctuations in an apo form, investigating proteins each of which possesses a cryptic site. From their X-ray structures, we saw that aromatic residues tended to be found in cryptic sites. To examine structural fluctuations of the apo forms, we performed molecular dynamics (MD) simulations, producing probability distributions of the solvent-accessible surface area per aromatic residue. To detect aromatic residues in cryptic sites, we have proposed a "cryptic-site index" based on the distribution, demonstrating the performance via several measures, such as recall and specificity. Besides, we found that high-ranking aromatic residues were likely to probe concaves in a cryptic site. This implies that such fluctuations provide a profile of scaffolds of compounds with the potential to bind to a particular cryptic site.

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.

GA-guided mD-VcMD: A genetic-algorithm-guided method for multi-dimensional virtual-system coupled molecular dynamics.

We introduced a conformational sampling method in an earlier report: The multi-dimensional virtual-system coupled molecular dynamics (mD-VcMD) enhances conformational sampling of a biomolecular system by computer simulations. Herein, new sampling method, a subzone-based mD-VcMD, is presented as an extension of mD-VcMD. Then, the subzone-based method is extended further using a genetic algorithm (GA) named the GA-guided mD-VcMD. In these methods, iterative simulation runs are performed to increase the sampled region gradually. The new methods have the following benefits: (1) They are free from a production run: i.e., all snapshots from all iterations are useful for analyses. (2) They are free from fine tuning of a weight function (probability distribution function or potential of mean force). (3) A canonical ensemble (i.e., a thermally equilibrated ensemble) is generated from a simple procedure. A thermodynamic weight is assigned to each snapshot. (4) Selective sampling can be performed for particularly addressing a poorly sampled region without breaking the proportion of the canonical ensemble if the poorly sampled conformational region emerges in sampling. By applying the methods to a simple system that involves an energy barrier between potential-energy minima, we demonstrated that the new methods have considerably higher sampling efficiency than the original mD-VcMD does.

Prediction of Passive Membrane Permeability by Semi-Empirical Method Considering Viscous and Inertial Resistances and Different Rates of Conformational Change and Diffusion.

Membrane permeability is an important property of drugs in adsorption. Many prediction methods work well for small molecules, but the prediction of middle-molecule permeability is still difficult. In the present study, we modified a classical permeability model based on Fick's law to study passive membrane permeability. The model consisted of the distribution of solute from water to membrane and the diffusion of solute in each solvent. The diffusion coefficient is the inverse of the resistance, and we examined the inertial resistance in addition to the viscous resistance, the latter of which has been widely used in permeability prediction. Also, we examined three models changing the balance between the diffusion of solute in membrane and the conformational change of solute. The inertial resistance improved the prediction results in addition to the viscous resistance. The models worked well not only for small molecules but also for middle molecules, whose structures have more conformational freedom.

Free-energy landscape of molecular interactions between endothelin 1 and human endothelin type B receptor: fly-casting mechanism.

The free-energy landscape of interaction between a medium-sized peptide, endothelin 1 (ET1), and its receptor, human endothelin type B receptor (hETB), was computed using multidimensional virtual-system coupled molecular dynamics, which controls the system's motions by introducing multiple reaction coordinates. The hETB embedded in lipid bilayer was immersed in explicit solvent. All molecules were expressed as all-atom models. The resultant free-energy landscape had five ranges with decreasing ET1-hETB distance: completely dissociative, outside-gate, gate, binding pocket, and genuine-bound ranges. In the completely dissociative range, no ET1-hETB interaction appeared. In the outside-gate range, an ET1-hETB attractive interaction was the fly-casting mechanism. In the gate range, the ET1 orientational variety decreased rapidly. In the binding pocket range, ET1 was in a narrow pathway with a steep free-energy slope. In the genuine-bound range, ET1 was in a stable free-energy basin. A G-protein-coupled receptor (GPCR) might capture its ligand from a distant place.

Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate, a muscarinic receptor antagonist with anti-inflammatory activity.

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(-)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM3R). Compared to 1 and 5, (R)-(-)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(-)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(-)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.