Deciphering the molecular choreography of Janus kinase 2 inhibition via Gaussian accelerated molecular dynamics simulations: a dynamic odyssey.

The Janus kinases (JAK) are crucial targets in drug development for several diseases. However, accounting for the impact of possible structural rearrangements on the binding of different kinase inhibitors is complicated by the extensive conformational variability of their catalytic kinase domain (KD). The dynamic KD contains mainly four prominent mobile structural motifs: the phosphate-binding loop (P-loop), the αC-helix within the N-lobe, the Asp-Phe-Gly (DFG) motif, and the activation loop (A-loop) within the C-lobe. These distinct structural orientations imply a complex signal transmission path for regulating the A-loop's flexibility and conformational preference for optimal JAK function. Nevertheless, the precise dynamical features of the JAK induced by different types of inhibitors still remain elusive. We performed comparative, microsecond-long, Gaussian accelerated molecular dynamics simulations in triplicate of three phosphorylated JAK2 systems: the KD alone, type-I ATP-competitive inhibitor (CI) bound KD in the catalytically active DFG-in conformation, and the type-II inhibitor (AI) bound KD in the catalytically inactive DFG-out conformation. Our results indicate significant conformational variations observed in the A-loop and αC helix motions upon inhibitor binding. Our studies also reveal that the DFG-out inactive conformation is characterized by the closed A-loop rearrangement, open catalytic cleft of N and C-lobe, the outward movement of the αC helix, and open P-loop states. Moreover, the outward positioning of the αC helix impacts the hallmark salt bridge formation between Lys882 and Glu898 in an inactive conformation. Finally, we compared their ligand binding poses and free energy by the MM/PBSA approach. The free energy calculations suggested that the AI's binding affinity is higher than CI against JAK2 due to an increased favorable contribution from the total non-polar interactions and the involvement of the αC helix. Overall, our study provides the structural and energetic insights crucial for developing more promising type I/II JAK2 inhibitors for treating JAK-related diseases.

Microsecond dynamics of H10N7 influenza neuraminidase reveals the plasticity of loop regions and drug resistance due to the R292K mutation.

At the beginning of the last century, multiple pandemics caused by influenza (flu) viruses severely impacted public health. Despite the development of vaccinations and antiviral medications to prevent and control impending flu outbreaks, unforeseen novel strains and continuously evolving old strains continue to represent a serious threat to human life. Therefore, the recently identified H10N7, for which not much data is available for rational structure-based drug design, needs to be further explored. Here, we investigated the structural dynamics of neuraminidase N7 upon binding of inhibitors, and the drug resistance mechanisms against the oseltamivir (OTV) and laninamivir (LNV) antivirals due to the crucial R292K mutation on the N7 using the computational microscope, molecular dynamics (MD) simulations. In this study, each system underwent long 2 × 1 µs MD simulations to answer the conformational changes and drug resistance mechanisms. These long time-scale dynamics simulations and free energy landscapes demonstrated that the mutant systems showed a high degree of conformational variation compared to their wildtype (WT) counterparts, and the LNV-bound mutant exhibited an extended 150-loop conformation. Further, the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculation and MM/GBSA free energy decomposition were used to characterize the binding of OTV and LNV with WT, and R292K mutated N7, revealing the R292K mutation as drug-resistant, facilitated by a decline in binding interaction and a reduction in the dehydration penalty. Due to the broader binding pocket cavity of the smaller K292 mutant residue relative to the wildtype, the drug carboxylate to K292 hydrogen bonding was lost, and the area surrounding the K292 residue was more accessible to water molecules. This implies that drug resistance could be reduced by strengthening the hydrogen bond contacts between N7 inhibitors and altered N7, creating inhibitors that can form a hydrogen bond to the mutant K292, or preserving the closed cavity conformations.

Conformational preferences of triantennary and tetraantennary hybrid N-glycans in aqueous solution: Insights from 20 µs long atomistic molecular dynamic simulations.

In the current study, we have investigated the conformational dynamics of a triantennary (N-glycan1) and tetraantennary (N-glycan2) hybrid N-glycans found on the surface of the HIV glycoprotein using 20 µs long all-atom molecular dynamics (MD) simulations. The main objective of the present study is to elucidate the influence of adding a complex branch on the overall glycan structural dynamics. Our investigation suggests that the average RMSD value increases when a complex branch is added to N-glycan1. However, the RMSD distribution is relatively wider in the case of N-glycan1 compared to N-glycan2, which indicates that multiple complex branches restrict the conformational variability of glycans. A similar observation is obtained from the principal component analysis of both glycans. All the puckering states (4C1 to 1C4) of each monosaccharide except mannose are sampled in our simulations, although the 4C1 chair form is energetically more favorable than 1C4. In N-glycan1, the 1-6 linkage in the mannose branch [Man(9)-α(1-6)-Man(5)] stays in the gauche-gauche cluster, whereas it moves towards trans-gauche in N-glycan2. For both glycans, mannose branches are more flexible than the complex branches, and adding a complex branch does not influence the dynamics of the mannose branches. We have noticed that the end-to-end distance of the complex branch shortens by ∼ 10 Å in the presence of another complex branch. This suggests that in the presence of an additional complex branch, the other complex branch adopts a close folded structure. All these conformational changes involve the selective formation of inter-residue and water-mediated hydrogen-bond networks.Communicated by Ramaswamy H. Sarma.

Computational studies indicated the effectiveness of human metabolites against SARS-Cov-2 main protease.

To fight against the devastating coronavirus disease 2019 (COVID-19), identifying robust anti-SARS-CoV-2 therapeutics from all possible directions is necessary. To contribute to this effort, we selected a human metabolites database containing waters and lipid-soluble metabolites to screen against the 3-chymotrypsin-like proteases (3CLpro) protein of SARS-CoV-2. The top 8 hits from virtual screening displayed a docking score varying between ~ - 11 and ~ - 14 kcal/mol. Molecular dynamics simulations complement the virtual screening study in conjunction with the molecular mechanics generalized Born surface area (MM/GBSA) scheme. Our analyses revealed that (HMDB0132640) has the best glide docking score, - 14.06 kcal/mol, and MM-GBSA binding free energy, - 18.08 kcal/mol. The other three lead molecules are also selected along with the top molecule through a critical inspection of their pharmacokinetic properties. HMDB0132640 displayed a better binding affinity than the other three compounds (HMDB0127868, HMDB0134119, and HMDB0125821) due to increased favorable contributions from the intermolecular electrostatic and van der Waals interactions. Further, we have investigated the ligand-induced structural dynamics of the main protease. Overall, we have identified new compounds that can serve as potential leads for developing novel antiviral drugs against SARS-CoV-2 and elucidated molecular mechanisms of their binding to the main protease. Identification of probable hits from human metabolites against SARS-CoV-2 using integrated computational approaches-Missed against MS.

A plausible contributor to multiple sclerosis; presentation of antigenic myelin protein epitopes by major histocompatibility complexes.

BACKGROUND: Multiple sclerosis (MS) can be induced upon successful presentation of myelin antigens by MHC I/II. Antigenic similarity between the myelin and viral proteins may worsen the immunological responses. METHODOLOGY: Antigenic regions within myelin proteins; PLP1, MBP, MOG, and MAG were analyzed using SVMTrip and EMBOSS. Homology search identified sequence similarity between the predicted host epitopes and viral proteins. NetMHCpan predicted MHC I/II binding followed by peptide-protein docking through the HPEPDOCK server. Thereafter we analyzed conformational flexibility and stability of 15 protein-peptide complexes based on high docking scores. The binding free energy was calculated using conventional (MD) and Gaussian accelerated molecular dynamics simulation. RESULTS: PLP1, MBP, MAG and MOG contained numerous antigenic epitopes. MBP and MOG epitopes had sequence similarity to HHV-6 BALF5; EBNA1 and CMV glycoprotein M (gM), and EBV LMP2B, gp350/220; HHV-8 ORFs respectively. Many herpes virus proteins like tegument, envelope glycoproteins, and ORFs of EBV, CMV, HHV-6, and HHV-8 demonstrated sequence similarity with MAG and PLP1. Some antigenic peptides were also linear B-cell epitopes and influenced cytokine production by T-cell. MHC I allele HLA-B*57:01 bound to PLP1 peptide and HLA-A*68:02 bound to a MAG peptide strongly. MHC II alleles HLA-DRB1*04:05 and HLA-DR1*01:01 associated with MAG- and MOG-derived peptides, respectively, demonstrating high HPEPDOCK scores. MD simulations established stable binding of certain peptides with the MHC namely HLA-B*51:01-MBP(DYKSAHKGFKGVDAQGTLSKIFKL), HLA-B*57:01-PLP1(PDKFVGITYALTVVWLLVFACSAVPVYIYF), HLA-DR1*01:01-MOG(VEDPFYWVSPGVLVLLAVLPVLLLQITVGLVFLCLQYR) and HLA-DRB1*04:05-MAG(TWVQVSLLHFVPTREA). CONCLUSIONS: Cross-reactivity between self-antigens and pathogen derived immunodominant epitopes may induce MS. Our study supported the role of specific MHC alleles as a contributing MS risk factor.

Comparative Structural Dynamics of Isoforms of Helicobacter pylori Adhesin BabA Bound to Lewis b Hexasaccharide via Multiple Replica Molecular Dynamics Simulations.

BabA of Helicobacter pylori is the ABO blood group antigen-binding adhesin. Despite considerable diversity in the BabA sequence, it shows an extraordinary adaptation in attachment to mucosal layers. In the current study, multiple replica molecular dynamics simulations were conducted in a neutral aqueous solution to elucidate the conformational landscape of isoforms of BabA bound to Lewis b (Leb) hexasaccharide. In addition, we also investigated the underlying molecular mechanism of the BabA-glycan complexation using the MM/GBSA scheme. The conformational dynamics of Leb in the free and protein-bound states were also studied. The carbohydrate-binding site across the four isoforms was examined, and the conformational variability of several vital loops was observed. The cysteine-cysteine loops and the two diversity loops (DL1 and DL2) were identified to play an essential role in recognizing the glycan molecule. The flexible crown region of BabA was stabilized after association with Leb. The outward movement of the DL2 loop vanished upon ligand binding for the Spanish specialist strain (S381). Our study revealed that the S831 strain shows a stronger affinity to Leb than other strains due to an increased favorable intermolecular electrostatic contribution. Furthermore, we showed that the α1-2-linked fucose contributed most to the binding by forming several hydrogen bonds with key amino acids. Finally, we studied the effect of the acidic environment on the BabA-glycan complexation via constant pH MD simulations, which showed a reduction in the binding free energy in the acidic environment. Overall, our study provides a detailed understanding of the molecular mechanism of Leb recognition by four isoforms of H. pylori that may help the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa.

Identification of novel efflux pump inhibitors for Neisseria gonorrhoeae via multiple ligand-based pharmacophores, e-pharmacophore, molecular docking, density functional theory, and molecular dynamics approaches.

Neisseria gonorrhoeae have progressively developed resistance to almost all antibiotics, and it has become imperative to develop novel approaches to combat its multi-drug resistance. Overexpression of the MtrCDE, an RND family efflux pump, is one of the primary causes of antibiotic resistance in the gonococcus and is considered an important target for combating anti-microbial resistance. PaßN, D13-9001, and other EPIs are identified to target the RND efflux pumps, but due to their cytotoxicity, they have failed in clinical trials. Herein, an extensive pharmacophore-based approach was performed to identify novel EPI inhibitors with improved pharmacology/safety profiles. An integrated computational framework comprising pharmacophore generation, virtual screening using HTVS, SP and XP Glide methodology, MM-GBSA analysis, Induced fit docking, QPLD, DFT, ADMET properties calculation, Molecular Dynamics, and MM-PBSA analysis was performed. The comprehensive study leads to the identification of five non-toxic bioactive compounds, namely - ZINC000008764610, ZINC000030879142, ZINC000030879358, ZINC000253414904, and ZINC000225394671, as potential EPIs for RND efflux pump of Neisseria gonorrhoeae. The five compounds were selected based on the pharmacophore mapping, higher dock score than the known EPIs, binding stability, molecular interactions with the critical residues of MtrD protein, higher ADMET properties, non-toxicity, and free energy estimations. In summary, the analysis led to the identification of five top hits from the natural compound subset of the ZINC database that has a higher binding affinity to the MtrD and adequate physiochemical/pharmacokinetic profile that can be used for the generation of novel EPIs against Neisseria gonorrhoeae.

Phosphorylation-Induced Conformational Dynamics and Inhibition of Janus Kinase 1 by Suppressors of Cytokine Signaling 1.

The dysfunction of the JAK/STAT (Janus kinase/signal transducers and activators of transcription) pathway results in several pathophysiological conditions, including autoimmune disorders. The negative feedback regulators of the JAK/STAT signaling pathway, suppressors of cytokine signaling (SOCS), act as a natural inhibitor of JAK and inhibit aberrant activity. SOCS1 is the most potent member of the SOCS family, whose kinase inhibitory region targets the substrate-binding groove of JAK with high affinity and blocks the phosphorylation of JAK kinases. Overall, we performed an aggregate of 13 µs molecular dynamics simulations on the activation loop's three different phosphorylation (double and single) states. Results from our simulations show that the single Tyr1034 phosphorylation could stabilize the JAK1/SOCS1 complex as well as the flexible activation segment. The phosphate-binding loop (P-loop) shows conformational variability at dual and single phosphorylated states. Principal component analysis and protein structure network (PSN) analysis reveal that the different phosphorylation states and SOCS1 binding induce intermediate inactive conformations of JAK1, which could be a better target for future JAK1 selective drug design. PSN analysis suggests that the com-pY1034 system is stabilized due to higher values of network hubs than the other two complex systems. Moreover, the binding free energy calculations suggest that pTyr1034 states show a higher affinity toward SOCS1 than the dual and pTyr1035 states. We believe that the mechanistic understanding of JAK1/SOCS1 complexation will aid future studies related to peptide inhibitors based on SOCS1.

Unraveling the Molecular Mechanism of Recognition of Selected Next-Generation Antirheumatoid Arthritis Inhibitors by Janus Kinase 1.

Rheumatoid arthritis (RA) is a chronic immune-related condition, primarily of joints, and is highly disabling and painful. The inhibition of Janus kinase (JAK)-related cytokine signaling pathways using small molecules is prevalent nowadays. The JAK family belongs to nonreceptor cytoplasmic protein tyrosine kinases (PTKs), including JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase 2). JAK1 has received significant attention after being identified as a promising target for developing anti-RA therapeutics. Currently, no crystal structure is available for JAK1 in complex with the next-generation anti-RA drugs. In the current study, we investigated the mechanism of binding of baricitinib, filgotinib, itacitinib, and upadacitinib to JAK1 using a combined method of molecular docking, molecular dynamics simulation, and binding free energy calculation via the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) scheme. We found that the calculated binding affinity decreases in the order upadacitinib > itacitinib > filgotinib > baricitinib. Due to the increased favorable intermolecular electrostatic contribution, upadacitinib is more selective to JAK1 compared to the other three inhibitors. The cross-correlation and principal component analyses showed that different inhibitor bindings significantly affect the binding site dynamics of JAK1. Furthermore, our studies indicated that the hydrophobic residues and hydrogen bonds from the hinge region (Glu957 and Leu959) of JAK1 played an essential role in stabilizing the inhibitors. Protein structural network analysis reveals that the total number of links and hubs in JAK1/baricitinib (354, 48) is more significant than those in apo (328, 40) and the other three complexes. The JAK1/baricitinib complex shows the highest probability of the highest-ranked community, indicating a compact network of the JAK1/baricitinib complex, consistent with its higher stability than the rest of the four systems. Overall, our study may be crucial for the rational design of JAK1-selective inhibitors with better affinity.

Decoding the Host-Parasite Protein Interactions Involved in Cerebral Malaria Through Glares of Molecular Dynamics Simulations.

Malaria causes millions of deaths every year. The malaria parasite spends a substantial part of its life cycle inside human erythrocytes. Inside erythrocytes, it synthesizes and displays various proteins onto the erythrocyte surface, such as Plasmodium falciparum erythrocytic membrane protein-1 (PfEMP1). This protein contains cysteine-rich interdomain region (CIDR) domains which have many subtypes based on sequence diversity and can cross-talk with host molecules. The CIDRα1.4 subtype can attach host endothelial protein C receptor (EPCR). This interaction facilitates infected erythrocyte adherence to brain endothelium and subsequent development of cerebral malaria. Through molecular dynamics simulations in conjunction with the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method, we explored the mechanism of interaction in the CIDRα1-EPCR complex. We examined the structural behavior of two CIDRα1 molecules (encoded by HB3-isolate var03-gene and IT4-isolate var07-gene) with EPCR unbound and bound (complex) forms. HB3var03CIDRα1 in apo and complexed with EPCR was comparatively more stable than IT4var07CIDRα1. Both of the complexes adopted two distinct conformational energy states. The hydrophobic residues played a crucial role in the binding of both complexes. For HB3var03CIDRα1-EPCR, the dominant energetic components were total polar interactions, while in IT4var07CIDRα1-EPCR, the primary interaction was van der Waals and nonpolar solvation energy. The study also revealed details such as correlated conformational motions and secondary structure evolution. Further, it elucidated various hotspot residues involved in protein-protein recognition. Overall, our study provides additional information on the structural behavior of CIDR molecules in unbound and receptor-bound states, which will help to design potent inhibitors.

A comparative study of structural and conformational properties of WNK kinase isoforms bound to an inhibitor: insights from molecular dynamic simulations.

The with-no-lysine (WNK) kinase causes pseudohypoaldosteronism type II, a genetic form of hypertension. Due to ∼80% similarity among four isoforms (WNK1/2/3/4) of the WNK protein family, the discovery of an ATP-competitive inhibitor renders a significant challenge. Here, we combined molecular modeling and molecular dynamics simulations to study the structural and conformational properties of the WNK kinase isoforms bound to an ATP competitive inhibitor (WNK463). We have also investigated the effect of phosphorylation on the conformational properties of each isoform. The largest deviation of Cα atoms is observed for the unphosphorylated uWNK4 complex, while the least deviation is obtained for uWNK3. The G-loop and αC-helix regions are also more flexible in uWNK4 compared to the other three unphosphorylated isoforms. However, in uWNK1, the A-loop region is the most flexible compared to other complexes. In all cases, phosphorylation stabilizes different regions of the protein-inhibitor complexes. In the case of uWNK4, relatively higher anti-correlated motions are observed compared to the other three unphosphorylated complexes. Furthermore, in the case of uWNK4, the distance between N- and C-lobes is found to be slightly higher than other complexes. This distance is reduced in all four complexes after the phosphorylation. Principal component analyses suggest that the phosphorylation leads to structural stabilization in WNK1 and WNK4, while it causes more flexibility in WNK2 and WNK3. Overall, our study provides comprehensive and comparative information on the structural dynamics of the WNK isoform family with the known competitive inhibitor that would aid in the development of a new inhibitor.Communicated by Ramaswamy H. Sarma.

Finding potential inhibitors against RNA-dependent RNA polymerase (RdRp) of bovine ephemeral fever virus (BEFV): an in-silico study.

The bovine ephemeral fever virus (BEFV) is an enzootic agent that affects millions of bovines and causes major economic losses. Though the virus is seasonally reported with a very high morbidity rate (80-100%) from African, Australian, and Asiatic continents, it remains a neglected pathogen in many of its endemic areas, with no proper therapeutic drugs or vaccines presently available for treatment. The RNA-dependent RNA polymerase (RdRp) catalyzes the viral RNA synthesis and is an appropriate candidate for antiviral drug developments. We utilized integrated computational tools to build the 3D model of BEFV-RdRp and then predicted its probable active binding sites. The virtual screening and optimization against these active sites, using several small-molecule inhibitors from a different category of Life Chemical database and FDA-approved drugs from the ZINC database, was performed. We found nine molecules that have docking scores varying between -6.84 to -10.43 kcal/mol. Furthermore, these complexes were analyzed for their conformational dynamics and thermodynamic stability using molecular dynamics simulations in conjunction with the molecular mechanics generalized Born surface area (MM-GBSA) scheme. The binding free energy calculations depict that the electrostatic interactions play a dominant role in the RdRp-inhibitor binding. The hot spot residues, such as Arg565, Asp631, Glu633, Asp740, and Glu707, were found to control the RdRp-inhibitor interaction. The ADMET analysis strongly suggests favorable pharmacokinetics of these compounds that may prove useful for treating the BEFV ailment. Overall, we anticipate that these findings would help explore and develop a wide range of anti-BEFV therapy.Communicated by Ramaswamy H. Sarma.

Finding potent inhibitors against SARS-CoV-2 main protease through virtual screening, ADMET, and molecular dynamics simulation studies.

Currently, no antiviral drug or vaccine is available to treat COVID-19 caused by SARS-CoV-2. This underscores an urgent need for developing a drug against SARS-CoV-2. The main protease (3CLpro) of SARS-CoV-2 is considered an essential protein for maintaining the viral life cycle and, therefore, a potential target for drug development. In a recent study, 1000 potential ligands were identified for 3CLpro by screening 1.3 billion compounds from the ZINC15 library. In the current study, we have further screened these 1000 compounds using structure-based virtual screening utilizing the Schrödinger suite and identified nine compounds having a docking score of ∼ -11.0 kcal/mol or less. The top 5 hits display good pharmacological profiles revealing better absorption, proper permeability across the membrane, uniform distribution, and non-toxic. The molecular docking study is further complemented by molecular dynamics simulations of the top 5 docked complexes. The binding free energy analyses via the molecular mechanics generalized Born surface area (MM/GBSA) scheme reveals that ZINC000452260308 is the most potent (ΔGbind = -14.31 kcal/mol) inhibitor. The intermolecular van der Waals interactions mainly drive the 3CLpro-ligand association. This new compound may have great potential as a lead molecule to develop a new antiviral drug to fight against COVID-19.Communicated by Ramaswamy H. Sarma.

Mining of Ebola virus genome for the construction of multi-epitope vaccine to combat its infection.

Ebola virus is the primary causative agent of viral hemorrhagic fever that is an epidemic disease and responsible for the massive premature deaths in humans. Despite knowing the molecular mechanism of its pathogenesis, to date, no commercial or FDA approved multiepitope vaccine is available against Ebola infection. The current study focuses on designing a multi-epitope subunit vaccine for Ebola using a novel immunoinformatic approach. The best predicted antigenic epitopes of Cytotoxic-T cell (CTL), Helper-T cells (HTL), and B-cell epitopes (BCL) joined by various linkers were selected for the multi-epitope vaccine designing. For the enhanced immune response, two adjuvants were also added to the construct. Further analysis showed the vaccine to be immunogenic and non-allergenic, forming a stable and energetically favorable structure. The stability of the unbound vaccine construct and vaccine/TLR4 was elucidated via atomistic molecular dynamics simulations. The binding free energy analysis (ΔGBind = -194.2 ± 0.5 kcal/mol) via the molecular mechanics Poisson-Boltzmann docking scheme revealed a strong association and thus can initiate the maximal immune response. Next, for the optimal expression of the vaccine construct, its gene construct was cloned in the pET28a + vector system. In summary, the Ebola viral proteome was screened to identify the most potential HTLs, CTLs, and BCL epitopes. Along with various linkers and adjuvants, a multi-epitope vaccine is constructed that showed a high binding affinity with the immune receptor, TLR4. Thus, the current study provides a highly immunogenic multi-epitope subunit vaccine construct that may induce humoral and cellular immune responses against the Ebola infection.Communicated by Ramaswamy H. Sarma.

Unraveling the Molecular Mechanism of Recognition of Human Interferon-Stimulated Gene Product 15 by Coronavirus Papain-Like Proteases: A Multiscale Simulation Study.

The papain-like protease (PLpro) of the coronavirus (CoV) family plays an essential role in processing the viral polyprotein and immune evasion. Additional proteolytic activities of PLpro include deubiquitination and deISGylation, which can reverse the post-translational modification of cellular proteins conjugated with ubiquitin or (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). These activities regulate innate immune responses against viral infection. Thus, PLpro is a potential antiviral target. Here, we have described the structural and energetic basis of recognition of PLpro by the human ISG15 protein (hISG15) using atomistic molecular dynamics simulation across the CoV family, i.e., MERS-CoV (MCoV), SARS-CoV (SCoV), and SARS-CoV-2 (SCoV2). The cumulative simulation length for all trajectories was 32.0 µs. In the absence of the complete crystal structure of complexes, protein-protein docking was used. A mutation (R167E) was introduced across all three PLpro to study the effect of mutation on the protein-protein binding. Our study reveals that the apo-ISG15 protein remains closed while it adopts an open conformation when bound to PLpro, although the degree of openness varies across the CoV family. The binding free energy analysis suggests that hISG15 binds more strongly with SCoV2-PLpro compared to SCoV or MCoV. The intermolecular electrostatic interaction drives the hISG15-PLpro complexation. Our study showed that SCoV or MCoV-PLpro binds more strongly with the C-domain of hISG15, while SCoV2-PLpro binds more favorably the N-domain of hISG15. Overall, our study explains the molecular basis of differential deISGylating activities of PLpro among the CoV family and the specificity of SCoV2-PLpro toward hISG15.

Identification of Potential Inhibitors against Epstein-Barr Virus Nuclear Antigen 1 (EBNA1): An Insight from Docking and Molecular Dynamic Simulations.

Epstein-Barr virus (EBV), a known tumorigenic virus, is associated with various neuropathies, including multiple sclerosis (MS). However, there is no anti-EBV FDA-approved drug available in the market. Our study targeted EBV protein EBV nuclear antigen 1 (EBNA1), crucial in virus replication and expressed in all the stages of viral latencies. This dimeric protein binds to an 18 bp palindromic DNA sequence and initiates the process of viral replication. We chose phytochemicals and FDA-approved MS drugs based on literature survey followed by their evaluation efficacies as anti-EBNA1 molecules. Molecular docking revealed FDA drugs ozanimod, siponimod, teriflunomide, and phytochemicals; emodin; protoapigenone; and EGCG bound to EBNA1 with high affinities. ADMET and Lipinski's rule analysis of the phytochemicals predicted favorable druggability. We supported our assessments of pocket druggability with molecular dynamics simulations and binding affinity predictions by the molecular mechanics generalized Born surface area (MM/GBSA) method. Our results establish a stable binding for siponimod and ozanimod with EBNA1 mainly via van der Waals interactions. We identified hot spot residues like I481', K477', L582', and K586' in the binding of ligands. In particular, K477' at the amino terminal of EBNA1 is known to establish interaction with two bases at the major groove of the DNA. Siponimod bound to EBNA1 engaging K477', thus plausibly making it unavailable for DNA interaction. Computational alanine scanning further supported the significant roles of K477', I481', and K586' in the binding of ligands with EBNA1. Conclusively, the compounds showed promising results to be used against EBNA1.

Identification of Food Compounds as inhibitors of SARS-CoV-2 main protease using molecular docking and molecular dynamics simulations.

SARS-CoV-2 has rapidly emerged as a global pandemic with high infection rate. At present, there is no drug available for this deadly disease. Recently, Mpro (Main Protease) enzyme has been identified as essential proteins for the survival of this virus. In the present work, Lipinski's rules and molecular docking have been performed to identify plausible inhibitors of Mpro using food compounds. For virtual screening, a database of food compounds was downloaded and then filtered using Lipinski's rule of five. Then, molecular docking was accomplished to identify hits using Mpro protein as the target enzyme. This led to identification of a Spermidine derivative as a hit. In the next step, Spermidine derivatives were collected from PubMed and screened for their binding with Mpro protein. In addition, molecular dynamic simulations (200 ns) were executed to get additional information. Some of the compounds are found to have strong affinity for Mpro, therefore these hits could be used to develop a therapeutic agent for SARS-CoV-2.

Elucidating specificity of an allosteric inhibitor WNK476 among With-No-Lysine kinase isoforms using molecular dynamic simulations.

Specifically targeting the With-No-Lysine (WNK1) kinase, which is implicated in hypertension, renders a significant challenge in discovering competitive inhibitors due to the highly conserved ATP-binding pocket. However, an allosteric inhibitor may impart high specificity against the WNK kinase isoforms since it targets the less conserved site and can provide greater efficacy even under high physiological ATP concentration. In the current study, we have investigated the structural and energetic basis of the specificity of the allosteric inhibitor WNK476 against WNK kinase isoforms by combining molecular dynamics simulations and free energy calculations using molecular mechanics Poisson-Boltzmann surface area. Our study reveals that the conformational stabilization of αC-helix near the allosteric binding site, including conformational changes in activation and glycine-rich loop regions, favors the specificity of WNK476 toward WNK1. The MM/PBSA calculations suggest that the non-polar contribution from hydrophobic residues and polar solvation energy influences WNK/WNK476 complexation. Despite more favorable electrostatic and van der Waals interactions in WNK2/WNK476, WNK476 is more potent against WNK1 due to the lower contribution of disfavoring components-polar solvation and entropy. Further, we have identified that the hydrophobic residues of DLG, αC-helix, ß4 , and ß5 regions, and H-bond network near the ß4 strand play a critical role in the specificity of WNK476 against WNK1. Finally, our study reveals that residues Leu272 , Val281 , Phe283 , and Leu369 of WNK1 actively contribute to the overall hydrophobic interactions for WNK1/WNK476. Overall, our study might help in the rational design of novel allosteric inhibitors against hypertension.

Characterizing an allosteric inhibitor-induced inactive state in with-no-lysine kinase 1 using Gaussian accelerated molecular dynamics simulations.

The With-No-Lysine (WNK) kinase plays a significant role in controlling blood pressure and body fluid homeostasis. Consequently, WNK1 is considered a potential target for treating hypertension. However, the highly conserved ATP-binding pocket in human isoforms WNK1/2/3/4 poses an immense challenge in designing competitive inhibitors. In contrast, allosteric inhibitors that bind to a non-conserved site provide a promising approach. To better understand how the allosteric inhibitors induce an inactive state in WNK1, we have performed 1 µs long Gaussian accelerated molecular dynamics simulations (GaMD) of the apo and complex systems along with free energy calculations and structural analyses. Our results indicate that major structural variations come from the activation loop and αC-helix. Our studies suggest that the inactive state is characterized by an open catalytic cleft between the N- and C-lobe, outward movement of the αC-helix, open P-loop, distorted αC-helix, and an extended activation loop that rearranges with a vanished short helix in its N-terminal. The outward movement of the αC-helix breaks the salt-bridge between Glu268 and R348 and renders the kinase domain inactive. Overall, our study provides detailed insights into the inhibitor-induced allosteric mechanisms and may help design specific allosteric inhibitors against WNK1 for treating hypertension.

Exploring the potency of currently used drugs against HIV-1 protease of subtype D variant by using multiscale simulations.

Acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV), type 1 and 2. Further, the diversity in HIV-1 has given rise to many serotypes and recombinant strains. The currently used protease inhibitors have been developed for subtype B, although non-B subtype strains account for ∼ 90% of the global HIV infections. Subtype D is spreading rapidly and infecting a large population in North Africa and the Middle East. In the current study, molecular dynamics simulations in conjunction with the molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) scheme was used to investigate the potency of four drugs, namely atazanavir (ATV), darunavir (DRV), lopinavir (LPV) and tipranavir (TPV) against the subtype D variant. Our calculations predicted that the potency of the inhibitors decreased in the order TPV > ATV > DRV > LPV. TPV was found to be the most potent against subtype D due to an increase in van der Waals and electrostatic interactions and reduction in the desolvation energy compared to other inhibitors. This result is further supported by the hydrogen bond interactions between inhibitors and protease. Furthermore, our analyses suggested that the binding of TPV induced a more closed conformation of the flap compared to apo or other complexes. It was observed that TPV/PRD has a lower cavity volume relative to the other three complexes leading to a tighter binding. The open conformation of the flap was observed for LPV/PRD. We expect that this study might be useful for designing more potent inhibitors against HIV-1 subtype D. Communicated by Ramaswamy H. Sarma.