Multi-domain proteins sample a broad energetic landscape as they undergo conformational transition between different functional states. As a number of these proteins form important drug targets, it is imperative to consider their intermediate conformational states for a successful drug-design strategy. We introduce a new docking protocol pertaining to these proteins, termed as extended-ensemble docking. In its application to multidrug ABC-transporter P-glycoprotein (Pgp), extensive non-equilibrium steered molecular dynamics simulations, with different combinations of reaction coordinates capturing the alternate-access mechanism of Pgp, are first used to construct the transition cycle of the transporter. The conformational ensemble obtained along the minimum-work path, covering the entire transition between the inward- and outward-facing states of the transporter, is used to seed high-throughput docking calculations of a set of known substrates, modulators and inhibitors of the protein. Hierarchical-clustering of individual binding modes of the different compounds show all ligands preferably binding to the large central cavity of the protein, formed at the apex of the transmembrane domain (TMD), whereas only small binding populations are observed in the previously described R- and H-sites present in the individual TMD leaflets. The central cavity is further clustered into two major subsites, preferably binding high-affinity inhibitors/transported substrates or low-affinity modulators, respectively, corresponding to the biochemically characterized high- and low-affinity binding sites in Pgp. We propose further optimization strategy for developing more potent inhibitor of Pgp, based on increasing its specificity to the conformational ensemble of the protein instead of using a single protein structure, as well as its selectivity for the high-affinity binding site. In contrast to earlier in-silico studies using static structures of Pgp, our results show good correlation with other experimental studies, pointing to the importance of incorporating global conformational flexibility of multi-domain proteins in future drug-discovery endeavors.

Although several families of compounds have been identified as scaffolds for inhibitors of the CYP1 family, the isoform selectivity determining structural features has not been fully clarified at the molecular interaction level. We studied the CYP1 isoform selectivity for stilbenoid inhibitors using integrated induced fit docking and molecular dynamics simulations. The hydrophobic interactions with the specific phenylalanine residues in the F helix are correlated with inhibitory potency in the CYP1 family. Through this study, we found that the adaptable, small, and semirigid ligand is a promising starting point for the development of isoform-selective inhibitors and investigation of selectivity-determining features.

Double electron–electron resonance and computer simulations are used to describe conformational dynamics in the ATP-binding cassette transporter Pgp, which has an important role in the clearance of xenobiotics and cancer resistance to chemotherapy. Despite numerous biochemical and structural studies, it remains unclear how ATP-binding cassette (ABC) transporters convert energy from ATP hydrolysis into the translocation of substrates across cellular membranes. Now, Hassane Mchaourab and colleagues have combined double electron–electron resonance (DEER) and computer simulations to describe a two-stroke ATP hydrolysis cycle that leads the mammalian ABC transporter Pgp from an inward- to an outward-facing conformation. The results have implications for basic cellular processes such as the clearance of xenobiotics as well as clinical issues such as cancer resistance to chemotherapy. ATP binding cassette (ABC) transporters of the exporter class harness the energy of ATP hydrolysis in the nucleotide-binding domains (NBDs) to power the energetically uphill efflux of substrates by a dedicated transmembrane domain (TMD)1,2,3,4. Although numerous investigations have described the mechanism of ATP hydrolysis and defined the architecture of ABC exporters, a detailed structural dynamic understanding of the transduction of ATP energy to the work of substrate translocation remains elusive. Here we used double electron–electron resonance5,6 and molecular dynamics simulations to describe the ATP- and substrate-coupled conformational cycle of the mouse ABC efflux transporter P-glycoprotein (Pgp; also known as ABCB1), which has a central role in the clearance of xenobiotics and in cancer resistance to chemotherapy7. Pairs of spin labels were introduced at residues selected to track the putative inward-facing to outward-facing transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a fully atomistic model of the outward-facing conformation in membranes, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport and reveals mechanistic divergence within the efflux class of ABC transporters.

Significance Photosystem II (PSII) is a multisubunit membrane protein complex that catalyzes light-driven oxidation of water, supplying energy to nearly all life on Earth. Because of the challenging chemistry it performs, PSII undergoes frequent damage, prompting an intricate cycle of disassembly, repair, and reassembly. Psb28, a protein that binds transiently to the RC47 intermediate complex, protects PSII from light-induced damage. The location of Psb28 in PSII has not been determined. In this study, we used chemical cross-linking, mass spectrometry, and protein docking to demonstrate that Psb28 binds to cytochrome b 559 . Our results allow us to propose a mechanism by which Psb28 exerts its protective effect on the vulnerable RC47 complex.

The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

HDAC8 inhibitors have become an attractive treatment for cancer. This study aimed to facilitate the identification of potential chemical scaffolds for the selective inhibition of histone deacetylase 8 (HDAC8) using in silico approaches. Non-linear QSAR classification and regression models of HDAC8 inhibitors were developed with support vector machine. Mean impact value-based sequential forward feature selection and grid search strategy were used for molecular descriptor selection and parameter optimization, respectively. The generated QSAR models were validated by leave-one-out cross validation and an external test set. The best QSAR classification model yielded 84 % of accuracy on the external test prediction and Matthews correlation coefficient is 0.69. The best QSAR regression model showed low root-mean-square error (0.63) and high squared correlation coefficient (0.53) for the test set. The validated QSAR models together with various drug-like properties, molecular docking and molecular dynamics simulation were sequentially used as a multi-step query in chemical database virtual screening. Finally, two hit compounds were discovered as new structural scaffolds which can be used for further in vitro and in vivo activity analyses. The strategy used in this study could be a promising computational strategy which can be utilized for other target drug design.

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to their redox partners and integral membrane proteins and exchanging electrons. Diffusive search, recognition, binding, and unbinding of these proteins often amount to kinetic bottlenecks in cellular energy conversion, but despite the availability of structures and intense study, the physical mechanisms controlling redox partner interactions remain largely unknown. The present molecular dynamics study provides an all-atom description of the cytochrome c2-docked bc1 complex in Rhodobacter sphaeroides in terms of an ensemble of favorable docking conformations and reveals an intricate series of conformational changes that allow cytochrome c2 to recognize the bc1 complex and bind or unbind in a redox state-dependent manner. In particular, the role of electron transfer in triggering a molecular switch and in altering water-mediated interface mobility, thereby strengthening and weakening complex formation, is described. The results resolve long-standing discrepancies between structural and functional data.

P-glycoprotein (P-gp) is one of the key members of the ATP-binding cassette (ABC) transporters family and the major contributor of multidrug resistance against a broad spectrum of drugs in cancer cells. Powered by ATP, P-gp acts as an efflux pump to export a diverse set of hydrophobic molecules, including drugs, out of the cell. These hydrophobic substrates are assumed to enter the translocation pathway of P-gp either via the intracellular side of the cell or directly from the cytoplasmic leaflet of the membrane. In order to characterize the mechanism, residues facilitating drug entry, and translocation, we have taken an approach combining the strengths of molecular dynamics (MD) and molecular docking. A 100 ns MD simulation of membrane-bound inward facing P-gp with Mg2+ and ATP was performed and subsequently used in ensemble docking. Doxorubicin, one of the drug substrates of P-gp, was docked into the defined active sites of 10,000 different snapshots representing the conformational flexibility of the protein in the membrane. Clustered docked poses identified the binding sites and yielded a picture of the sequence of binding events of the substrate within the translocation pathway. Interestingly, a few of the highly populated clusters were located within the region that interacts with the cytoplasmic leaflet of the membrane supporting the recruitment of the substrate laterally and directly from the membrane. Further MD simulations and energetic calculations of representative substrate poses are then used to characterize their real-time binding behavior and dynamics within the intracellular lumen and at the cytoplasmic leaflet of the membrane. This study, for the first time, explored and utilized the dynamic ensemble of conformations of P-gp in molecular docking and dissected important and complex features of its translocation pathway.

Primary hypercholesterolemia is the root cause for major health issues like coronary heart disease and atherosclerosis. Regulating plasma cholesterol level, which is the product of biosynthesis as well as dietary intake, has become one of the major therapeutic strategies to effectively control these diseases. Human cholesterol esterase (hCEase) is an interesting target involved in the regulation of plasma cholesterol level and thus inhibition of this enzyme is highly effective in the treatment of hypercholesterolemia. This study was designed to understand the activation mechanism that enables the enzyme to accommodate long chain fatty acids and to identify the structural elements for the successful catalysis. Primarily the activation efficiencies of three different bile salts were studied and compared using molecular dynamics simulations. Based on the conformations of major surface loops, hydrogen bond interactions, and distance analyses, taurocholate was concluded as the preferred activator of the enzyme. Furthermore, the importance of two bile salt binding sites (proximal and remote) and the crucial role of \(7\upalpha \)-OH group of the bile salts in the activation of hCEase was examined and evidenced. The results of our study explain the structural insights of the activation mechanism and show the key features of the bile salts responsible for the enzyme activation which are very useful in hypolipidemic drug designing strategies.

Activation of the peroxisome proliferator-activated receptor γ (PPARγ) is important for the treatment of type 2 diabetes and obesity through the regulation of glucose metabolism and fatty acid accumulation. Hence, the discovery of novel PPARγ agonists is necessary to overcome these diseases. In this study, a newly developed approach, multi-conformation dynamic pharmacophore modeling (MCDPM), was used for screening candidate compounds that can properly bind PPARγ. Highly populated structures obtained from molecular dynamics (MD) simulations were selected by clustering analysis. Based on these structures, pharmacophore models were generated from the ligand-binding pocket and then validated to check the rationality. Consequently, two hits were retrieved as final candidates by utilizing virtual screening and molecular docking simulations. These compounds can be used in the design of novel PPARγ agonists.

BACKGROUND:Human LTA4H catalyzes the conversion of LTA4 to LTB4 and plays a key role in innate immune responses. Inhibition of this enzyme can be a valid method in the treatment of inflammatory response exhibited through LTB4.RESULTS & DISCUSSION:The quantitative structure-activity relationship (QSAR) models were developed using genetic function approximation and validated. A training set of 26 diverse compounds and their molecular descriptors were used to develop highly correlating QSAR models. A six-descriptor model explaining the biological activity of the training and test sets with correlation values of 0.846 and 0.502, respectively, was selected as the best model and used in a database screening of drug-like Maybridge database followed by molecular docking.CONCLUSION:Based on the predicted potent inhibitory activities, expected binding mode and molecular interactions at the active site of hLTA4H final leads were selected as to be utilized in designing future hLTA4H inhibitors.

The sonic hedgehog (Shh) signaling pathway is necessary for a variety of development and differentiation during embryogenesis as well as maintenance and renascence of diverse adult tissues. However, an abnormal activation of the signaling pathway is related to various cancers. In this pathway, the Shh signaling transduction is facilitated by binding of Shh to its receptor protein, Ptch. In this study, we modeled the 3D structure of functionally important key loop peptides of Ptch based on homologous proteins. Using this loop model, the molecular interactions between the structural components present in the pseudo-active site of Shh and key residues of Ptch was investigated in atomic level through molecular dynamics (MD) simulations. For the purpose of developing inhibitor candidates of the Shh signaling pathway, the Shh pseudo-active site of this interface region was selected as a target to block the direct binding between Shh and Ptch. Two different structure-based pharmacophore models were generated considering the key loop of Ptch and known inhibitor-induced conformational changes of the Shh through MD simulations. Finally two hit compounds were retrieved through a series of virtual screening combined with molecular docking simulations and we propose two hit compounds as potential inhibitory lead candidates to block the Shh signaling pathway based on their strong interactions to receptor or inhibitor induced conformations of the Shh.

Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents.

Human leukotriene A4 hydrolase (hLTA4H) is a bi-functional enzyme catalyzes the hydrolase and aminopeptidase functions upon the fatty acid and peptide substrates, respectively, utilizing the same but overlapping binding site. Particularly the hydrolase function of this enzyme catalyzes the rate-limiting step of the leukotriene (LT) cascade that converts the LTA4 to LTB4. This product is a potent pro-inflammatory activator of inflammatory responses and thus blocking this conversion provides a valuable means to design anti-inflammatory agents. Four structurally very similar chemical compounds with highly different inhibitory profile towards the hydrolase function of hLTA4H were selected from the literature. Molecular dynamics (MD) simulations of the complexes of hLTA4H with these inhibitors were performed and the results have provided valuable information explaining the reasons for the differences in their biological activities. Binding mode analysis revealed that the additional thiophene moiety of most active inhibitor helps the pyrrolidine moiety to interact the most important R563 and K565 residues. The hLTA4H complexes with the most active compound and substrate were utilized in the development of hybrid pharmacophore models. These developed pharmacophore models were used in screening chemical databases in order to identify lead candidates to design potent hLTA4H inhibitors. Final evaluation based on molecular docking and electronic parameters has identified three compounds of diverse chemical scaffolds as potential leads to be used in novel and potent hLTA4H inhibitor design.

Histone deacetylases (HDACs) have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E) in HDAC10 and leucine (L) in HDAC 11) based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M) mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E) mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.

Cathepsin D is a major component of lysosomes and plays a major role in catabolism and degenerative diseases. The quantitative structure–activity relationship study was used to explore the critical chemical features of cathepsin D inhibitors. Top 10 hypotheses were built based on 36 known cathepsin D inhibitors using HypoGen/Discovery Studio v2.5. The best hypothesis Hypo1 consists of three hydrophobic, one hydrogen bond acceptor lipid, and one hydrogen bond acceptor features. The selected Hypo1 model was cross‐validated using Fischer’s randomization method to identify the strong correlation between experimental and predicted activity value as well as the test set and decoy sets used to validate its predictability. Moreover, the best hypothesis was used as a 3D query in virtual screening of Scaffold database. Subsequently, the screened hit molecules were filtered by applying Lipinski’s rule of five, absorption, distribution, metabolism, and toxicity, and molecular docking studies. Finally, 49 compounds were obtained as potent cathepsin D inhibitors based on the consensus scoring values, critical interactions with protein active site residues, and predicted activity values. Thus, we suggest that the application of Hypo1 could assist in the selection of potent cathepsin D leads from various databases. Hence, this model was used as a valuable tool to design new candidate for cathepsin D inhibitors.

Human histamine H1 receptor (HHR1) is one of the receptors through which histamine exerts its allergic reactions, and inhibition of this receptor is an important strategy in treatment of inflammatory diseases. In this study, classical and 3D quantitative structure–activity relationship (QSAR) studies were carried out using 36 inverse agonists of which 27 diverse compounds were used in training set. The MOE® and SYSTAT® programs were used in descriptor calculation and step-wise multiple regression analysis. A predictive and statistically significant QSAR model based on four descriptors was developed and cross-validated with the leave-one-out (LOO) method. The statistical data of this model were r = 0.908, F = 25.703, s = 0.445, . Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) studies were carried out using the same training set compounds. Pharmacophore-based molecular alignment was used to develop models using different fields. The best CoMFA model was the one developed using the CoMFA indicator fields (r 2 = 0.998, ), and the best CoMSIA model was the one with CoMSIA steric fields (r 2 = 0.862, ). As the models explain well the observed variance in HHR1 binding affinities in both the training and the test set, they can be used in designing future antihistamines.

Aldose reductase 2 (ALR2), which catalyzes the reduction of glucose to sorbitol using NADP as a cofactor, has been implicated in the etiology of secondary complications of diabetes. A pharmacophore model, Hypo1, was built based on 26 compounds with known ALR2-inhibiting activity values. Hypo1 contains important chemical features required for an ALR2 inhibitor, and demonstrates good predictive ability by having a high correlation coefficient (0.95) as well as the highest cost difference (128.44) and the lowest RMS deviation (1.02) among the ten pharmacophore models examined. Hypo1 was further validated by Fisher's randomization method (95%), test set (r = 0.91), and the decoy set shows the goodness of fit (0.70). Furthermore, during virtual screening, Hypo1 was used as a 3D query to screen the NCI database, and the hit leads were sorted by applying Lipinski's rule of five and ADME properties. The best-fitting leads were subjected to docking to identify a suitable orientation at the ALR2 active site. The molecule that showed the strongest interactions with the critical amino acids was used in molecular dynamics simulations to calculate its binding affinity to the candidate molecules. Thus, Hypo1 describes the key structure-activity relationship along with the estimated activities of ALR2 inhibitors. The hit molecules were searched against PubChem to find similar molecules with new scaffolds. Finally, four molecules were found to satisfy all of the chemical features and the geometric constraints of Hypo1, as well as to show good dock scores, PLPs and PMFs. Thus, we believe that Hypo1 facilitates the selection of novel scaffolds for ALR2, allowing new classes of ALR2 inhibitors to be designed.