Identification of chikungunya virus nsP2 protease inhibitors using structure-base approaches

The nsP2 protease of chikungunya virus (CHIKV) is one of the essential components of viral replication and it plays a crucial role in the cleavage of polyprotein precursors for the viral replication process. Therefore, it is gaining attention as a potential drug design target against CHIKV. Based on the recently determined crystal structure of the nsP2 protease of CHIKV, this study identified potential inhibitors of the virus using structure-based approaches with a combination of molecular docking, virtual screening and molecular dynamics (MD) simulations. The top hit compounds from database searching, using the NCI Diversity Set II, with targeting at five potential binding sites of the nsP2 protease, were identified by blind dockings and focused dockings. These complexes were then subjected to MD simulations to investigate the stability and flexibility of the complexes and to gain a more detailed insight into the interactions between the compounds and the enzyme. The hydrogen bonds and hydrophobic contacts were characterized for the complexes. Through structural alignment, the catalytic residues Cys1013 and His1083 were identified in the N-terminal region of the nsP2 protease. The absolute binding free energies were estimated by the linear interaction energy approach and compared with the binding affinities predicted with docking. The results provide valuable information for the development of inhibitors for CHIKV.     

Discovery of selective dengue virus inhibitors using combination of molecular fingerprint-based virtual screening protocols,structure-based pharmacophore model development,molecular dynamics simulations and in vitro studies

Dengue virus is a major issue of tropical and sub-tropical regions. The proliferation of virus results in immense number of deaths each year because of unavailability of on-shelf drugs. This issue necessitates the design of novel anti-Dengue drugs. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of Dengue virus. Keeping in mind the worsening situation regarding Dengue virus, approximately eighteen million drug-like compounds from the ZINC small molecule database have been screened against Nonstructural Protein 3 (NS3) previously by our group. In this study, in order to investigate the effect of extended time of molecular dynamics (MD) simulations on structural and dynamical profiles of used complexes, simulation run time is increased from 50-ns to 100-ns for the each system. In addition, a well-known Dengue virus inhibitor (MB21) from literature is used as reference structure (positive control) to compare the proposed molecules. Post-processing MD analyses including Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations were conducted to predict binding free energies of inhibitors from derived trajectory frames of MD simulations. Identified compounds are further directed to Quantum-Polarized Ligand Docking (QPLD), molecular fingerprint-based virtual screening of another small molecule database (Otava Drug Like small molecule database), and Structure-based Pharmacophore Modeling (E-Pharmacophore). Finally, cell proliferation and cytotoxicity tests as well as pre- and post-treatment on HUH7 cells infected with DENV2 NGC strain are applied for four identified hit molecules (ZINC36681949, ZINC44921800, ZINC95518765 and ZINC39500661) to check whether these drugs inhibit DENV2 from entry and/or exit pathways. Based on cell-based Dengue quantification assays, there is no effect seen on pre-treatment of cells with these compounds indicating that the early infection processes of virus is not affected. In contrast, the post-treatment of cells with these compounds after Dengue virus infection has resulted in a significant 1 log PFU/ml reduction of the virus infectious titre.     

Study of the interaction of Huperzia saururus Lycopodium alkaloids with the acetylcholinesterase enzyme

In the present study, we describe and compare the binding modes of three Lycopodium alkaloids (sauroine, 6-hydroxylycopodine and sauroxine; isolated from Huperzia saururus) and huperzine A with the enzyme acetylcholinesterase. Refinement and rescoring of the docking poses (obtained with different programs) with an all atom force field helped to improve the quality of the protein–ligand complexes. Molecular dynamics simulations were performed to investigate the complexes and the alkaloid's binding modes. The combination of the latter two methodologies indicated that binding in the active site is favored for the active compounds. On the other hand, similar binding energies in both the active and the peripheral sites were obtained for sauroine, thus explaining its experimentally determined lack of activity. MM-GBSA predicted the order of binding energies in agreement with the experimental IC₅₀ values.     

Virtual screening of eighteen million compounds against dengue virus: Combined molecular docking and molecular dynamics simulations study

Dengue virus is a major issue of tropical and sub-tropical regions. Dengue virus has been the cause behind the major alarming epidemics in the history with mass causalities from the decades. Unavailability of on-shelf drugs for the prevention of further proliferation of virus inside the human body results in immense number of deaths each year. This issue necessitates the design of novel anti-dengue drug. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of dengue virus. Therefore, approximately eighteen million compounds from the ZINC database have been virtually screened against nonstructural protein 3 (NS3). The incremental construction algorithm of Glide docking program has been used with its features high throughput virtual screening (HTVS), standard precision (SP), extra precision (XP) and in combination of Prime module, induced fit docking (IFD) approach has also been applied. Five top-ranked compounds were then selected from the IFD results with better predicted binding energies with the catalytic triad residues (His51, Asp75, and Ser135) that may act as potential inhibitors for the underlying target protease enzyme. The top-ranked compounds ZINC95518765, ZINC44921800, ZINC71917414, ZINC39500661, ZINC36681949 have shown the predicted binding energies of −7.55, −7.36, −8.04, −8.41, −9.18 kcal/mol, respectively, forming binding interactions with three catalytically important amino acids. Top-docking poses of compounds are then used in molecular dynamics (MD) simulations. In computational studies, our proposed compounds confirm promising results against all the four serotypes of dengue virus, strengthening the opportunity of these compounds to work as potential on-shelf drugs against dengue virus. Further experimentation on the proposed compounds can result in development of strong inhibitors.     

A method for including protein flexibility in protein-ligand docking: improving tools for database mining and virtual screening

Second-generation methods for docking ligands into their biological receptors, such as FLOG, provide for flexibility of the ligand but not of the receptor. Molecular dynamics based methods, such as free energy perturbation, account for flexibility, solvent effects, etc., but are very time consuming. We combined the use of statistical analysis of conformational samples from short-run protein molecular dynamics with grid-based docking protocols and demonstrated improved performance in two test cases. Our statistical analysis explores the importance of the average strength of a potential interaction with the biological target and optionally applies a weighting depending on the variability in the strength of the interaction seen during dynamics simulation. Using these methods, we improved the num-top-ranked 10% of a database of drug-like molecules, in searches based on the three-dimensional structure of the protein. These methods are able to match the ability of manual docking to assess likely inactivity on steric grounds and indeed to rank order ligands from a homologous series of cyclooxygenase-2 inhibitors with good correlation to their true activity. Furthermore, these methods reduce the need for human intervention in setting up molecular docking experiments.     

Computational determination of binding structures and free energies of glucose 6-phosphate dehydrogenase with novel steroid inhibitors

Glucose 6-phosphate dehydrogenase (G6PD), the first and the rate-limiting enzyme in the pentose phosphate pathway (PPP), catalyzes the oxidation of G6P to 6-phosphogluconolactone and the reduction of NADP⁺ to NADPH. Its key role in cancer promotes the development of a potent and selective inhibitor that might increase cancer cell death when combined with radiotherapy. In the present study, we investigated the detailed binding modes and binding free energies for G6PD interacting with a promising series of recently developed inhibitors, i.e., the steroid derivatives, by performing molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. The docking indicates that the inhibitors occupy the binding sites of both G6P and NADP⁺. The calculated binding free energies on the basis of the MD-simulated enzyme–inhibitor complexes are in good agreement with the experimental activity data for all of the examined inhibitors. The valuable insights into the detailed enzyme–inhibitor binding including the important intermolecular interactions, e.g., the hydrogen bond interaction and the hydrophobic interaction, have been provided. The computational results provide new insights into future rational design of more potent inhibitors of G6PD as a treatment for cancer.     

Molecular modeling study of CP-690550 derivatives as JAK3 kinase inhibitors through combined 3D-QSAR,molecular docking,and dynamics simulation techniques

To develop more potent JAK3 kinase inhibitors, a series of CP-690550 derivatives were investigated using combined molecular modeling techniques, such as 3D-QSAR, molecular docking and molecular dynamics (MD). The leave-one-out correlation (q²) and non-cross-validated correlation coefficient (r²) of the best CoMFA model are 0.715 and 0.992, respectively. The q² and r² values of the best CoMSIA model are 0.739 and 0.995, respectively. The steric, electrostatic, and hydrophobic fields played important roles in determining the inhibitory activity of CP-690550 derivatives. Some new JAK3 kinase inhibitors were designed. Some of them have better inhibitory activity than the most potent Tofacitinib (CP-690550). Molecular docking was used to identify some key amino acid residues at the active site of JAK3 protein. 10 ns MD simulations were successfully performed to confirm the detailed binding mode and validate the rationality of docking results. The calculation of the binding free energies by MMPBSA method gives a good correlation with the predicted biological activity. To our knowledge, this is the first report on MD simulations and free energy calculations for this series of compounds. The combination results of this study will be valuable for the development of potent and novel JAK3 kinase inhibitors.     

Protein-ligand recognition using spherical harmonic molecular surfaces: towards a fast and efficient filter for large virtual throughput screening

Molecular surfaces are important because surface-shape complementarity is often a necessary condition in protein-ligand interactions and docking studies. We have previously described a fast and efficient method to obtain triangulated surface-meshes by topologically mapping ellipsoids on molecular surfaces. In this paper, we present an extension of our work to spherical harmonic surfaces in order to approximate molecular surfaces of both ligands and receptor-cavities and to easily check the surface-shape complementarity. The method consists of (1) finding lobes and holes on both ligand and cavity surfaces using contour maps of radius functions with spherical harmonic expansions, (2) superposing the surfaces around a given binding site by minimizing the distance between their respective expansion coefficients. This docking procedure capabilities was demonstrated by application to 35 protein-ligand complexes of known crystal structures. The method can also be easily and efficiently used as a filter to detect in a large conformational sampling the possible conformations presenting good complementarity with the receptor site, and being, therefore, good candidates for further more elaborate docking studies. This "virtual screening" was demonstrated on the platelet thrombin receptor.     

Binding mechanism of CDK5 with roscovitine derivatives based on molecular dynamics simulations and MM/PBSA methods

Roscovitine derivatives are potent inhibitors of cyclin-dependent kinase 5 (CDK5), but they exhibit different activities, which has not been understood clearly up to now. On the other hand, the task of drug design is difficult because of the fuzzy binding mechanism. In this context, the methods of molecular docking, molecular dynamics (MD) simulation, and binding free energy analysis are applied to investigate and reveal the detailed binding mechanism of four roscovitine derivatives with CDK5. The electrostatic and van der Waals interactions of the four inhibitors with CDK5 are analyzed and discussed. The calculated binding free energies in terms of MM-PBSA method are consistent with experimental ranking of inhibitor effectiveness for the four inhibitors. The hydrogen bonds of the inhibitors with Cys83 and Lys33 can stabilize the inhibitors in binding sites. The van der Waals interactions, especially the pivotal contacts with Ile10 and Leu133 have larger contributions to the binding free energy and play critical roles in distinguishing the variant bioactivity of four inhibitors. In terms of binding mechanism of the four inhibitors with CDK5 and energy contribution of fragments of each inhibitor, two new CDK5 inhibitors are designed and have stronger inhibitory potency.     

Molecular docking and molecular dynamics simulation studies of GPR40 receptor–agonist interactions

In order to explore the agonistic activity of small-molecule agonists to GPR40, AutoDock and GROMACS software were used for docking and molecular dynamics studies. A molecular docking of eight structurally diverse agonists (six carboxylic acids (CAs) agonist, and two non-carboxylic acids (non-CAs) agonist) was performed and the differences in their binding modes were investigated. Moreover, a good linear relationship based on the predicted binding affinities (pK_i) determined by using AutoDock and experimental activity values (pEC50) was obtained. Then, the 10 ns molecular dynamics (MD) simulations of three obtained ligand–receptor complexes embedded into the phospholipid bilayer were carried out. The position fluctuations of the ligands located inside the transmembrane domain were explored, and the stable binding modes of the three studied agonists were determined. Furthermore, the residue-based decomposition of interaction energies in three systems identified several critical residues for ligand binding.     

Molecular docking/dynamics studies of Aurora A kinase inhibitors

The binding modes of a known 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole, quinazoline, pyrimidine and indolinone series of Aurora A kinase inhibitors have been studied using molecular docking and molecular dynamics (MD) simulations. Crystallographic bound compound 8 was precisely predicted by our docking procedure as evident from 0.43 Å root mean square (rms) deviations. In addition compound 25 (AZ_68) has been successfully cross-docked within the Aurora A kinase active site, which was pre-organized for inhibitor 8. We found four key sites (A: solvent-exposed front pocket, B: hinge region, C: selectivity pocket and D: solvent-exposed phosphate binding region) of the Aurora A kinase contributing towards the binding of these compounds. We suggest that the small hydrophobic substituents at C-6 position of pyrrolopyrazole nucleus (in compounds 1–8); C-6 and C-7 positions of the quinazoline moiety (in compounds 9–23); C-2 position of the quinazoline and C-4 position of the pyrimidine (in compound 25) could be more effective and selective through increased hydrophobic contacts and selectivity pocket interactions with these modifications of Aurora A kinase inhibitors. Five representative complexes were subjected to 1000 ps of MD simulation to determine the stability of the predicted binding conformations. The low value of the root mean square deviations (ranging from 0.725 to 1.820 Å) between the starting complex structure and the energy minimized final average complex structure suggests that the Glide Extra Precision (XP) derived docked complexes are in a state of near equilibrium. The structure-based drug design strategy described in this study will be highly useful for the development of new inhibitors with high potency and selectivity.     

Binding modes of CCR5-targetting HIV entry inhibitors: partial and full antagonists

Since the CC-chemokine receptor 5 (CCR5) was identified as a major co-receptor for human immunodeficiency virus type 1 (HIV-1) entry into a host cell, CCR5-targetting HIV entry inhibitors have been developed and some of them are currently in clinical trials. Most of these inhibitors also inhibit the physiological chemokine reaction function of CCR5, which is so far considered to be safe to patients based on the observation that individuals that naturally lack CCR5 do not show apparent health problems. Nevertheless, to minimize the toxicity and side effects, it would be ideal to preserve the chemokine receptor activity. In this work, we simulated the flexible docking of two small molecule inhibitors to CCR5 in a solvated phospholipid bilayer environment. One of the inhibitors, aplaviroc has a unique feature of preserving two of the natural chemokine ligands binding to CCR5 and subsequent activation whereas the other one, SCH-C fully blocks chemokine-CCR5 interactions. Our results revealed significantly different binding modes of these two inhibitors although both established extensive interaction networks with CCR5. Comparison of the different binding modes suggests that avoiding the deep insertion of inhibitors into the transmembrane helix bundle may be able to preserve chemokine-CCR5 interactions. These results could help design HIV co-receptor activity-specific inhibitors.     

Predicting the structures of complexes between phosphoinositide 3-kinase (PI3K) and romidepsin-related compounds for the drug design of PI3K/histone deacetylase dual inhibitors using computational docking and the ligand-based drug design approach

Predictions of the three-dimensional (3D) structures of the complexes between phosphoinositide 3-kinase (PI3K) and two inhibitors were conducted using computational docking and the ligand-based drug design approach. The obtained structures were refined by structural optimizations and molecular dynamics (MD) simulations. The ligands were located deep inside the ligand binding pocket of the p110α subunit of PI3K, and the hydrogen bond formations and hydrophobic effects of the surrounding amino acids were predicted. Although rough structures were obtained for the PI3K–inhibitor complexes before the MD simulations, the refinement of the structures by these simulations clarified the hydrogen bonding patterns of the complexes.     

Binding modes of CCR5-targetting HIV entry inhibitors: Partial and full antagonists

Since the CC-chemokine receptor 5 (CCR5) was identified as a major co-receptor for human immunodeficiency virus type 1 (HIV-1) entry into a host cell, CCR5-targetting HIV entry inhibitors have been developed and some of them are currently in clinical trials. Most of these inhibitors also inhibit the physiological chemokine reaction function of CCR5, which is so far considered to be safe to patients based on the observation that individuals that naturally lack CCR5 do not show apparent health problems. Nevertheless, to minimize the toxicity and side effects, it would be ideal to preserve the chemokine receptor activity. In this work, we simulated the flexible docking of two small molecule inhibitors to CCR5 in a solvated phospholipid bilayer environment. One of the inhibitors, aplaviroc has a unique feature of preserving two of the natural chemokine ligands binding to CCR5 and subsequent activation whereas the other one, SCH-C fully blocks chemokine-CCR5 interactions. Our results revealed significantly different binding modes of these two inhibitors although both established extensive interaction networks with CCR5. Comparison of the different binding modes suggests that avoiding the deep insertion of inhibitors into the transmembrane helix bundle may be able to preserve chemokine-CCR5 interactions. These results could help design HIV co-receptor activity-specific inhibitors.     

Targeting the dengue β-OG with serotype-specific alkaloid virtual leads

The dengue envelope β-OG pocket is a crucial hinge for mediating virus-host fusion via conformational changes in the envelope to the fusion-competent form. The β-OG pocket is a small molecule target site for inhibition of virus-host fusion. As of date, the only structure of the β-OG pocket known is of serotype 2. Studies of β-OG inhibition by small molecules primarily target viral serotype 2. Envelope and β-OG sequence alignments, reveal dissimilarities across serotypes. In light of protein sequence-structure-function correlation, sequence variations suggest serotypic variations in β-OG druggability. This, together with the fact that dengue viral proteins do have serotype-specific variations of structure and function, lead to the study of the serotype-specificity of the dengue β-OG ligand binding behaviour. β-OG druggability was compared using comparative models of envelope proteins containing the β-OG pocket in four serotypes of the dengue virus. β-OG ligand binding was found to vary with respect to hydrophobicity, hydrophilicity, hydrogen bonding, van der Waals interactions with ligands and tightness of the binding site. The study also reports serotype-specific virtual leads identified from a library of 9175 alkaloids, using a consensus docking and scoring approach. The docking algorithms of Glide SP and XP, together with the Lamarckian genetic algorithm were employed for consensus docking. For consensus scoring, the Glide empirical score was employed along with the scoring function of AutoDock. A multi-dimensional lead optimisation approach was performed for optimising affinity, ligand efficiency, lipophilic ligand efficiency, ADMET and molecular torsional strains. The study proposes the serotype-specific inhibition of the β-OG for an effective inhibition of virus-host fusion, in contrast to a pan inhibitor.     

GVSS: A High Throughput Drug Discovery Service of Avian Flu and Dengue Fever for EGEE and EUAsiaGrid

In the study of Biomedicines, molecular docking simulation is a common method for predicting potential interacting complexes of small molecules in protein binding sites. However, it is a time-consuming process to search exhaustively all correct conformations of a compound. This study demonstrates how massive molecular docking benefit from state-of-the-art Grid technology. Providing intensive computing power and effective data management, the production e-infrastructure (such as EGEE and EUAsiaGrid) enables opportunities for in-silico drug discovery on the neglected and emerging diseases, for instance, avian influenza and dengue fever. In this study, Grid Application Platform (GAP) and GAP-enabled Virtual Screening Service (GVSS) were developed with the docking engine of the Autodock 3.0.5. A JAVA-based graphical user interface and the GAP allow end-users to specify target and compound library, set up docking parameters, monitor docking jobs and computing resources, visualize and refine docking results, and finally download the final results. To provide a more user-friendly Grid service, GVSS was designed for conducting large-scale molecular docking more easily.     

Molecular modelling of the differential interaction between several non-steroidal anti-inflammatory drugs and human prostaglandin endoperoxide H synthase-2 (h-PGHS-2)

The prostaglandin endoperoxide H synthase-1 (PGHS-1) and prostaglandin endoperoxide H synthase-2 (PGHS-2) are the targets of non-steroidal anti-inflammatory drugs (NSAIDs). The high degree of selectivity for inhibition of PGHS-2 shown by certain compounds appears to stem from two mechanisms (time-dependent, time-independent inhibition) by which they interact with each isoform. Molecular models of the complexes between indomethacin, fenamates, 2-phenylpropionic acids and the selective cyclooxygenase-2 (COX-2) inhibitors, with the cyclooxygenase active site of human PGHS-2 have been built by combining homology modelling, conformational searching and automated docking techniques. The stability of the resulting complexes has been assessed by molecular dynamics simulations combined with extended linear response calculations. The results allow us to identify regions of biological significance consistent with both X-ray crystallographic and kinetic results. The selective PGHS-2 inhibitors exploit the extra space of a side-pocket in the active site of PGHS-2 that is not found in PGHS-1. The results obtained point out a marked relationship between the experimental affinity and the electrostatic interaction energy alone for a series of NSAIDs. Analysis of the structural and the energetic data provides evidence supporting that network of hydrogen bonds between Tyr355, Glu524, Arg120 and Arg513 might be involved in mediating the binding of the time-dependent inhibitors of PGHS-2.     

PKR-inhibitor binds efficiently with human microtubule affinity-regulating kinase 4

MAP/microtubule affinity-regulating kinase 4 (MARK4) plays a central role in the cellular physiology, and it is inseparably linked with many human diseases including cancer, diet induced obesity, type2 diabetes and neurodegenerative disorders. Here, we studied the interaction of PKR-inhibitor with two variants of human MARK4. One variant is named as MARK4-F1 which has 59 N-terminal residues along with kinase domain while another variant is MARK4-F2 which has kinase domain only. Molecular-docking, molecular dynamics (MD) simulation and fluorescence-binding studies were undertaken to understand the role of N-terminal 59-residues in the binding of substrate/inhibitors. Molecular docking studies revealed that the PKR-inhibitor binds in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Furthermore, MD simulation showed a stable parameters for the complexes of both MARK4-F1 and MARK4-F2 to PKR-inhibitor with marginal difference in their binding affinities. A significant decrease in the fluorescence intensity of MARK4 was observed on successive addition of the PKR-inhibitor. Using fluorescence data we have calculated the binding-affinity and the number of binding site of PKR-inhibitor to the MARK4. A significantly high binding affinity was observed for the PKR-inhibitor to the MARK4 variants. However, there is no any significant difference in the binding affinity of PKR-inhibitor to the MARK4 variants was observed, indicating that 59 N-terminal residues of MARK4-F1 are not playing a crucial role in the ligand binding. The present study will provide an insights into designing of new PKR-inhibitor derivative as potent and selective therapeutic agent against many life threatening diseases which are associated with MARK4.     

Molecular docking and analysis of interactions between vascular endothelial growth factor (VEGF) and SPARC protein

The extracellular module of SPARC/osteonectin binds to vascular endothelial growth factor (VEGF) and inhibits VEGF-stimulated proliferation of endothelial cells. In an attempt to identify the binding site for SPARC on VEGF, we hypothesized that this binding site could overlap at least partially the binding site of VEGF receptor 1 (VEGFR-1), as SPARC acts by preventing VEGF-induced phosphorylation of VEGFR-1. To this end, a docking simulation was carried out using a predictive docking tool to obtain modeled structures of the VEGF-SPARC complex. The predicted structure of VEGF-SPARC complex indicates that the extracellular domain of SPARC interacts with the VEGFR-1 binding site of VEGF, and is consistent with known biochemical data. Following molecular dynamics refinement, side-chain interactions at the protein interface were identified that were predicted to contribute substantially to the free energy of binding. These provide a detailed prediction of key amino acid side-chain interactions at the protein-protein interface. To validate the model further, the identified interactions will be used for designing mutagenesis studies to investigate their effect on binding activity. This model of the VEGF-SPARC complex should provide a basis for future studies aimed at identifying inhibitors of VEGF-induced angiogenesis.