Formulation of next-generation polyvalent vaccine candidates against three important poxviruses by targeting DNA-dependent RNA polymerase using an integrated immunoinformatics and molecular modeling approach.

BACKGROUND: Poxviruses comprise a group of large double-stranded DNA viruses and are known to cause diseases in humans, livestock animals, and other animal species. The Mpox virus (MPXV; formerly Monkeypox), variola virus (VARV), and volepox virus (VPXV) are among the prevalent poxviruses of the Orthopoxviridae genera. The ongoing Mpox infectious disease pandemic caused by the Mpox virus has had a major impact on public health across the globe. To date, only limited repurposed antivirals and vaccines are available for the effective treatment of Mpox and other poxviruses that cause contagious diseases. METHODS: The present study was conducted with the primary goal of formulating multi-epitope vaccines against three evolutionary closed poxviruses i.e., MPXV, VARV, and VPXV using an integrated immunoinformatics and molecular modeling approach. DNA-dependent RNA polymerase (DdRp), a potential vaccine target of poxviruses, has been used to determine immunodominant B and T-cell epitopes followed by interactions analysis with Toll-like receptor 2 at the atomic level. RESULTS: Three multi-epitope vaccine constructs, namely DdRp_MPXV (V1), DdRp_VARV (V2), and DdRp_VPXV (V3) were designed. These vaccine constructs were found to be antigenic, non-allergenic, non-toxic, and soluble with desired physicochemical properties. Protein-protein docking and interaction profiling analysis depicts a strong binding pattern between the targeted immune receptor TLR2 and the structural models of the designed vaccine constructs, and manifested a number of biochemical bonds (hydrogen bonds, salt bridges, and non-bonded contacts). State-of-the-art all-atoms molecular dynamics simulations revealed highly stable interactions of vaccine constructs with TLR2 at the atomic level throughout the simulations on 300 nanoseconds. Additionally, the outcome of the immune simulation analysis suggested that designed vaccines have the potential to induce protective immunity against targeted poxviruses. CONCLUSIONS: Taken together, formulated next-generation polyvalent vaccines were found to have good efficacy against closely related poxviruses (MPXV, VARV, and VPXV) as demonstrated by our extensive immunoinformatics and molecular modeling evaluations; however, further experimental investigations are still needed.

Amyloid Accelerator Polyphosphate Implicated as the Mystery Density in α-Synuclein Fibrils.

Aberrant aggregation of α-Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived α-Synuclein fibrils, which contain a non-proteinaceous, "mystery density" at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates α-Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in α-Synuclein fibrils. Here we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the two critical lysine residues K43 and K45 leads to a loss of all previously reported effects of polyP binding on α-Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo α-Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues.

A review on structure-function mechanism and signaling pathway of serine/threonine protein PIM kinases as a therapeutic target.

The proviral integration for the Moloney murine leukemia virus (PIM) kinases, belonging to serine/threonine kinase family, have been found to be overexpressed in various types of cancers, such as prostate, breast, colon, endometrial, gastric, and pancreatic cancer. The three isoforms PIM kinases i.e., PIM1, PIM2, and PIM3 share a high degree of sequence and structural similarity and phosphorylate substrates controlling tumorigenic phenotypes like proliferation and cell survival. Targeting short-lived PIM kinases presents an intriguing strategy as in vivo knock-down studies result in non-lethal phenotypes, indicating that clinical inhibition of PIM might have fewer adverse effects. The ATP binding site (hinge region) possesses distinctive attributes, which led to the development of novel small molecule scaffolds that target either one or all three PIM isoforms. Machine learning and structure-based approaches have been at the forefront of developing novel and effective chemical therapeutics against PIM in preclinical and clinical settings, and none have yet received approval for cancer treatment. The stability of PIM isoforms is maintained by PIM kinase activity, which leads to resistance against PIM inhibitors and chemotherapy; thus, to overcome such effects, PIM proteolysis targeting chimeras (PROTACs) are now being developed that specifically degrade PIM proteins. In this review, we recapitulate an overview of the oncogenic functions of PIM kinases, their structure, function, and crucial signaling network in different types of cancer, and the potential of pharmacological small-molecule inhibitors. Further, our comprehensive review also provides valuable insights for developing novel antitumor drugs that specifically target PIM kinases in the future. In conclusion, we provide insights into the benefits of degrading PIM kinases as opposed to blocking their catalytic activity to address the oncogenic potential of PIM kinases.

Classical molecular dynamics simulation identifies catechingallate as a promising antiviral polyphenol against MPOX palmitoylated surface protein.

Cumulative global prevalence of the emergent monkeypox (MPX) infection in the non-endemic countries has been professed as a global public health predicament. Lack of effective MPX-specific treatments sets the baseline for designing the current study. This research work uncovers the effective use of known antiviral polyphenols against MPX viral infection, and recognises their mode of interaction with the target F13 protein, that plays crucial role in formation of enveloped virions. Herein, we have employed state-of-the-art machine learning based AlphaFold2 to predict the three-dimensional structure of F13 followed by molecular docking and all-atoms molecular dynamics (MD) simulations to investigate the differential mode of F13-polyphenol interactions. Our extensive computational approach identifies six potent polyphenols Rutin, Epicatechingallate, Catechingallate, Quercitrin, Isoquecitrin and Hyperoside exhibiting higher binding affinity towards F13, buried inside a positively charged binding groove. Intermolecular contact analysis of the docked and MD simulated complexes divulges three important residues Asp134, Ser137 and Ser321 that are observed to be involved in ligand binding through hydrogen bonds. Our findings suggest that ligand binding induces minor conformational changes in F13 to affect the conformation of the binding site. Concomitantly, essential dynamics of the six-MD simulated complexes reveals Catechin gallate, a known antiviral agent as a promising polyphenol targeting F13 protein, dominated with a dense network of hydrophobic contacts. However, assessment of biological activities of these polyphenols need to be confirmed through in vitro and in vivo assays, which may pave the way for development of new novel antiviral drugs.

NMR insights into ß-Lactamase activity of UVI31+ Protein from Chlamydomonas reinhardtii.

ß-Lactamases (EC 3.5.2.6) confer resistance against ß-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against ß-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative ß-lactamase activity, sulbactam binding (a ß-lactam analogue) in the low µM affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known ß-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its ß-lactamase activity. Current study is the first report on ß-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for ß-lactamase activity.

Spectroscopic insights with molecular docking and molecular dynamic simulation studies of anticancer drug 5-Fluorouracil targeting human pyruvate kinase m2.

This study was conducted to test the efficacy of 5-fluorouracil (5-FU) as an anticancer drug against the human pyruvate kinase isozyme M2 (PKM2) using spectroscopic, molecular docking and molecular dynamic simulation studies. PKM2 fluorescence quenching studies in the presence of 5-FU performed at three different temperatures indicates dynamic quenching processes with single-set of binding (n ≈ 1) profile. The biomolecular quenching constants (kq) and the effective binding constants (Kb) obtained are shown to increase with temperature. The calculated enthalpy (ΔH) and entropy changes (ΔS) are estimated to be -118.06 kJ/mol and 146.14 kJ/mol/K respectively, which suggest the possible mode of interaction as electrostatic and hydrogen bonding. Further, these values were used to estimate the free energy changes (ΔG) and that increases with temperature. The negative ΔG values clearly indicates spontaneous binding process that stabilizes the complex formed between 5-FU and PKM2. Far-UV CD spectra of PKM2 in the presence of 5-FU shows decrease in α-helix contents which point towards the destabilization of secondary structure that weakens the biological activity of PKM2. The intrinsic fluorescence study and circular dichroism (CD) spectra showed minor conformational changes of PKM2 in the presence of 5-FU. Additionally, the results obtained from molecular docking and all-atom molecular dynamic simulation study supports the insight of the spectroscopic binding studies, and strengthens the dynamic stability of the complex between 5-FU and PKM2 through H-bonding. This study establishes a paradigm of 5-FU-PKM2 complexation and the efficacy of 5-FU that compromises the biological activity of the targeted PKM2.Communicated by Ramaswamy H. Sarma.

Structural insights of Labeo catla (catla) myxovirus resistance protein,GTP binding recognition and constitutive expression induced with Poly I:C.

The Myxovirus resistance (Mx) proteins are critical effectors belonging to the super-family of guanidine triphosphatase, often stimulated by type I interferon (IFN) and mediates antiviral responses to restrict the replication of numerous viral genes in fishes. In teleosts, Mx proteins display diverse and complicated antiviral activity in different species. The present investigation seeks to characterize the Mx gene from Labeo catla upon induction by double-stranded (ds) RNA, polyinosinic-polycytidylic acid, (poly I: C). Molecular modeling and all-atoms molecular dynamics (MD) simulations were employed to understand the architecture of the GTPase domain and its plausible mode of GTP recognition in Mx protein. The full-length L. catla Mx (LcMx) gene sequence (1821 bp nucleotides) encodes an open reading frame of 606 amino acids. Domain search indicated conserved tripartite domain architecture of LcMx and forms a major cluster with the Mx from other teleosts. The positively charged Arginine and polar Glutamine residues from helix 3 and 4 of stalk region LcMx aid in homo-oligomerization. MD simulation portrayed the role of conserved critical residues aid in GTP recognition by the GTPase domain which perfectly corroborates with experimental findings and prior MD studies. After injection of poly I:C, the temporal mRNA profile showed that LcMx expression was significantly elevated in the spleen, brain, kidney, liver, muscle, heart, intestine, and gill tissues. Collectively, these results suggest that the elevated expression of the major innate immune defense gene Mx was able to inhibit the poly I: C mediated virulence in fish.Communicated by Ramaswamy H. Sarma.

All-atoms molecular dynamics study to screen potent efflux pump inhibitors against KpnE protein of Klebsiella pneumoniae.

The Small Multidrug Resistance efflux pump protein KpnE, plays a pivotal role in multi-drug resistance in Klebsiella pneumoniae. Despite well-documented study of its close homolog, EmrE, from Escherichia coli, the mechanism of drug binding to KpnE remains obscure due to the absence of a high-resolution experimental structure. Herein, we exclusively elucidate its structure-function mechanism and report some of the potent inhibitors through drug repurposing. We used molecular dynamics simulation to develop a dimeric structure of KpnE and explore its dynamics in lipid-mimetic bilayers. Our study identified both semi-open and open conformations of KpnE, highlighting its importance in transport process. Electrostatic surface potential map suggests a considerable degree of similarity between KpnE and EmrE at the binding cleft, mostly occupied by negatively charged residues. We identify key amino acids Glu14, Trp63 and Tyr44, indispensable for ligand recognition. Molecular docking and binding free energy calculations recognizes potential inhibitors like acarbose, rutin and labetalol. Further validations are needed to confirm the therapeutic role of these compounds. Altogether, our membrane dynamics study uncovers the crucial charged patches, lipid-binding sites and flexible loop that could potentiate substrate recognition, transport mechanism and pave the way for development of novel inhibitors against K. pneumoniae.Communicated by Ramaswamy H. Sarma.

Identification of potential inhibitor against CTX-M-3 and CTX-M-15 proteins: an in silico and in vitro study.

Extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae infection is a serious global threat. ESBLs target 3rd generation cephalosporin antibiotics, the most commonly prescribed medicine for gram-negative bacterial infections. As bacteria are prone to develop resistance against market-available ESBL inhibitors, finding a novel and effective inhibitor has become mandatory. Among ESBL, the worldwide reported two enzymes, CTX-M-15 and CTX-M-3, are selected for the present study. CTX-M-3 protein was modeled, and two thousand phyto-compounds were virtually screened against both proteins. After filtering through docking and pharmacokinetic properties, four phyto-compounds (catechin gallate, silibinin, luteolin, uvaol) were further selected for intermolecular contact analysis and molecular dynamics (MD) simulation. MD trajectory analysis results were compared, revealing that both catechin gallate and silibinin had a stabilizing effect against both proteins. Silibinin having the lowest docking score, also displayed the lowest MIC (128 µg/mL) against the bacterial strains. Silibinin was also reported to have synergistic activity with cefotaxime and proved to have bactericidal effect. Nitrocefin assay confirmed that silibinin could inhibit beta-lactamase enzyme only in living cells, unlike clavulanic acid. Thus the present study validated the CTX-M inhibitory activity of silibinin both in silico and in vitro and suggested its promotion for further studies as a potential lead. The present study adopted a protocol through the culmination of bioinformatics and microbiological analyses, which will help future researchers identify more potential leads and design new effective drugs.Communicated by Ramaswamy H. Sarma.

Lipid and cholesterols modulate the dynamics of SARS-CoV-2 viral ion channel ORF3a and its pathogenic variants.

SARS-CoV-2 accessory protein, ORF3a is a putative ion channel which immensely contributes to viral pathogenicity by modulating host immune responses and virus-host interactions. Relatively high expression of ORF3a in diseased individuals and implication with inflammasome activation, apoptosis and autophagy inhibition, ratifies as an effective target for developing vaccines and therapeutics. Herein, we present the elusive dynamics of ORF3a-dimeric state using all-atoms molecular dynamics (MD) simulations at µ-seconds scale in a heterogeneous lipid-mimetic system in multiple replicates. Additionally, we also explore the effect of non-synonymous pathogenic mutations on ORF3a ion channel activity and viral pathogenicity in different SARS-CoV-2 variants using various structure-based protein stability (ΔΔG) tools and computational saturation mutagenesis. Our study ascertains the role of phosphatidylcholines and cholesterol in modulating the structure of ORF3a, which perturbs the size and flexibility of the polar cavity that allows permeation of large cations. Discrete trend in ion channel pore radius and area per lipid arises the premise that presence of lipids might also affect the overall conformation of ORF3a. MD structural-ensembles, in some replicates rationalize the crucial role of TM2 in maintaining the native structure of ORF3a. We also infer that loss of structural stability primarily grounds for pathogenicity in more than half of the pathogenic variants of ORF3a. Overall, the effect of mutation on alteration of ion permeability of ORF3a, proposed in this study brings mechanistic insights into variant consequences on viral membrane proteins of SARS-CoV-2, which can be utilized for the development of novel therapeutics to treat COVID-19 and other coronavirus diseases.

Computational study on Schiff base derived salicylaldehyde and furfuraldehyde derivatives as potent anti-tubercular agents: prospect to dihydropteroate synthase inhibitors.

Nowadays, bacterial multidrug resistance has become a commonplace problem in clinics due to several intrinsic factors mediated through resistance to antibacterials obtained via bacterial consortia and extrinsic factors, such as non-uniform antibacterial policy and migration of resistant bacteria through human and other routes. The development of newer, effective anti-mycobacterial candidate(s) is coveted by clinics. Hybrid molecules would be comparatively more emulating against invasive bacterial strains; nevertheless, newer antibiotics are continually added. Herein, designing and developments of two series of Schiff-based salicylaldehyde S1-S7 and furfuraldehyde F1-F7 molecules individually bearing sulfonamide group are described; and those were synthesized and their structures by spectral characterization were confirmed. Concomitantly, molecule dynamic simulations of all atoms had been performed to fathom the mechanism of the action with these leading complexes. These data imply that the synthesized Schiff-based salicylaldehyde hybrids would be promising anti-tubercular compounds, which further need potent pharmacological evaluations.Communicated by Ramaswamy H. Sarma.

Molecular docking and MD simulations reveal protease inhibitors block the catalytic residues in Prp8 intein of Aspergillus fumigatus: a potential target for antimycotics.

Resistance to azoles and amphotericin B especially in Aspergillus fumigatus is a growing concern towards the treatment of invasive fungal infection. At this critical juncture, intein splicing would be a productive, and innovative target to establish therapies against resistant strains. Intein splicing is the central event for the activation of host protein, essential for the growth and survival of various microorganisms including A. fumigatus. The splicing process is a four-step protease-like nucleophilic cascade. Thus, we hypothesise that protease inhibitors would successfully halt intein splicing and potentially restrict the growth of the aforementioned pathogen. Using Rosetta Fold and molecular dynamics simulations, we modelled Prp8 intein structure; resembling classic intein fold with horse shoe shaped splicing domain. To fully comprehend the active site of Afu Prp8 intein, C1, T62, H65, H818, N819 from intein sequences and S820, the first C-extein residue are selected. Molecular docking shows that two FDA-approved drugs, i.e. Lufotrelvir and Remdesivir triphosphate efficiently interact with Prp8 intein from the assortment of 212 protease inhibitors. MD simulation portrayed that Prp8 undergoes conformational change upon ligand binding, and inferred the molecular recognition and stability of the docked complexes. Per-residue decomposition analysis confirms the importance of F: block R802, V803, and Q807 binding pocket in intein splicing domain towards recognition of inhibitors, along with active site residues through strong hydrogen bonds and hydrophobic contacts. However, in vitro and in vivo assays are required to confirm the inhibitory action on Prp8 intein splicing; which may pave the way for the development of new antifungals for A. fumigatus.Communicated by Ramaswamy H. Sarma.

Inhibition potential of natural flavonoids against selected omicron (B.1.19) mutations in the spike receptor binding domain of SARS-CoV-2: a molecular modeling approach.

The omicron (B.1.19) variant of contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered a variant of concern (VOC) due to its increased transmissibility and highly infectious nature. The spike receptor-binding domain (RBD) is a hotspot of mutations and is regarded as a prominent target for screening drug candidates owing to its crucial role in viral entry and immune evasion. To date, no effective therapy or antivirals have been reported; therefore, there is an urgent need for rapid screening of antivirals. An extensive molecular modelling study has been performed with the primary goal to assess the inhibition potential of natural flavonoids as inhibitors against RBD from a manually curated library. Out of 40 natural flavonoids, five natural flavonoids, namely tomentin A (-8.7 kcal/mol), tomentin C (-8.6 kcal/mol), hyperoside (-8.4 kcal/mol), catechin gallate (-8.3 kcal/mol), and corylifol A (-8.2 kcal/mol), have been considered as the top-ranked compounds based on their binding affinity and molecular interaction profiling. The state-of-the-art molecular dynamics (MD) simulations of these top-ranked compounds in complex with RBD exhibited stable dynamics and structural compactness patterns on 200 nanoseconds. Additionally, complexes of these molecules demonstrated favorable free binding energies and affirmed the docking and simulation results. Moreover, the post-simulation validation of these interacted flavonoids using principal component analysis (PCA) revealed stable interaction patterns with RBD. The integrated results suggest that tomentin A, tomentin C, hyperoside, catechin gallate, and corylifol A might be effective against the emerging variants of SARS-CoV-2 and should be further evaluated using in-vitro and in-vivo experiments.Communicated by Ramaswamy H. Sarma.

Illuminating the structural basis of human neurokinin 1 receptor (NK1R) antagonism through classical all-atoms molecular dynamics simulations.

Advances in structural biology have bestowed insights into the pleiotropic effects of neurokinin 1 receptors (NK1R) in diverse patho-physiological processes, thereby highlighting the potential therapeutic value of antagonists directed against NK1R. Herein, we investigate the mode of antagonist recognition to discern the obscure atomic facets germane for the function and molecular determinants of NK1R. To commence discernment of potent antagonists and the conformational changes in NK1R, induced upon antagonist binding, state-of-the-art classical all-atoms molecular dynamics (MD) simulations in lipid mimetic bilayers have been utilized. MD simulations of structural ensembles reveals the involvement of TM5 and TM6 in tight anchoring of antagonists through a network of interhelical hydrogen-bonds, while, the extracellular loop 2 (ECL2) governs the overall size and nature of the pocket, thereby modulating NK1R. Consistent comparison between experiments and MD simulation results discerns the predominant role of TM3, TM4, and TM6 in lipid-NK1R interaction. Correlation between hydrophobic index and helicity of TM domains elucidates their importance in maintaining the structural stability in addition to regulating NK1R antagonism. Taken together, we anticipate that our computational study marks a comprehensive structural basis of NK1R antagonism in lipid bilayers, which may facilitate designing of new therapeutics against associated diseases targeting human neurokinin receptors.

Computational saturation mutagenesis to explore the effect of pathogenic mutations on extra-cellular domains of TREM2 associated with Alzheimer's and Nasu-Hakola disease.

CONTEXT: The specialised family of triggering receptors expressed on myeloid cells (TREMs) plays a pivotal role in causing neurodegenerative disorders and activating microglial anti-inflammatory responses. Nasu-Hakola disease (NHD), a rare autosomal recessive disorder, has been associated with mutations in TREM2, which is also responsible for raising the risk of Alzheimer's disease (AD). Herein, we have made an endeavour to differentiate the confirmed pathogenic variants in TREM2 extra-cellular domain (ECD) linked with NHD and AD using mutation-induced fold stability change (∆∆G), with the computation of 12distinct structure-based methods through saturation mutagenesis. Correlation analysis between relative solvent accessibility (RSA) and ∆∆G expresses the discrete distributive behaviour of mutants associated with TREM2 in AD (R2 = 0.061) and NHD (R2 = 0.601). Our findings put an emphasis on W50 and V126 as major players in maintaining V-like domain in TREM2. Interestingly, we discern that both of them interact with a common residue Y108, which is dissolved upon mutation. This Y108 could have structural or functional role for TREM2 which can be an ideal candidate for further study. Furthermore, the residual interaction network highlights the importance of R47 and R62 in maintaining the CDR loops that are crucial for ligand binding. Future studies using biophysical characterisation of ligand interactions in TREM2-ECD would be helpful for the development of novel therapeutics for AD and NHD. METHODS: ConSurf algorithm and ENDscript were used to determine the position and conservation of each residue in the wild-type ECD of TREM2. The mutation-induced fold stability change (∆∆G) of confirmed pathogenic mutants associated with NHD and AD was estimated using 12 state-of-the-art structure-based protein stability tools. Furthermore, we also computed the effect of random mutation on these sites using computational saturation mutagenesis. Linear regression analysis was performed using mutants ∆∆G and RSA through GraphPad software. In addition, a comprehensive non-bonded residual interaction network (RIN) of wild type and its mutants of TREM2-ECD was enumerated using RING3.0.

Dissecting the Molecular Basis of Host Leucine-Rich Repeat Containing 15 Mediated Interaction with Receptor Binding Domain of SARS-CoV-2 Spike Protein: A Computational Approach.

The detection of leucine-rich repeat containing 15 (LRRC15) as a connecting link with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the possibility of its involvement in differential restriction activity of SARS-CoV-2 pathways. However, the structure-function mechanism of LRRC15 involving the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and their mode of interaction is largely unknown. Using state-of-the-art AlphaFold2 and all-atom molecular dynamics simulations, our findings provide evidences of alternative binding modes of RBD with LRR units of LRRC15 having varied affinities. Contribution of both the receptor binding regions in RBD, including receptor binding motif in accommodating the LRR domain, towards the C-terminal region, emphasizes its differential role in modulating host cell receptiveness for SARS-CoV-2, the innate immune system, as well as antiviral tone. However, further experimental validations are necessary for unravelling the unknown mechanism and distinctive features of this host receptor in the COVID-19 pandemic, involving both the transmembrane as well as cytoplasmic domain.

Exploiting the potential of natural polyphenols as antivirals against monkeypox envelope protein F13 using machine learning and all-atoms MD simulations.

The re-emergence of monkeypox (MPX), in the era of COVID-19 pandemic is a new global menace. Regardless of its leniency, there are chances of MPX expediting severe health deterioration. The role of envelope protein, F13 as a critical component for production of extracellular viral particles makes it a crucial drug target. Polyphenols, exhibiting antiviral properties have been acclaimed as an effective alternative to the traditional treatment methods for management of viral diseases. To facilitate the development of potent MPX specific therapeutics, herein, we have employed state-of-the-art machine learning techniques to predict a highly accurate 3-dimensional structure of F13 as well as identify binding hotspots on the protein surface. Additionally, we have effectuated high-throughput virtual screening methodology on 57 potent natural polyphenols having antiviral activities followed by all-atoms molecular dynamics (MD) simulations, to substantiate the mode of interaction of F13 protein and polyphenol complexes. The structure-based virtual screening based on Glide SP, XP and MM/GBSA scores enables the selection of six potent polyphenols having higher binding affinity towards F13. Non-bonded contact analysis, of pre- and post- MD complexes propound the critical role of Glu143, Asp134, Asn345, Ser321 and Tyr320 residues in polyphenol recognition, which is well supported by per-residue decomposition analysis. Close-observation of the structural ensembles from MD suggests that the binding groove of F13 is mostly hydrophobic in nature. Taken together, this structure-based analysis from our study provides a lead on Myricetin, and Demethoxycurcumin, which may act as potent inhibitors of F13. In conclusion, our study provides new insights into the molecular recognition and dynamics of F13-polyphenol bound states, offering new promises for development of antivirals to combat monkeypox. However, further in vitro and in vivo experiments are necessary to validate these results.

Machine learning and classical MD simulation to identify inhibitors against the P37 envelope protein of monkeypox virus.

Monkeypox virus (MPXV) outbreak is a serious public health concern that requires international attention. P37 of MPXV plays a pivotal role in DNA replication and acts as one of the promising targets for antiviral drug design. In this study, we intent to screen potential analogs of existing FDA approved drugs of MPXV against P37 using state-of-the-art machine learning and computational biophysical techniques. AlphaFold2 guided all-atoms molecular dynamics simulations optimized P37 structure is used for molecular docking and binding free energy calculations. Similar to members of Phospholipase-D family , the predicted P37 structure also adopts a ß-α-ß-α-ß sandwich fold, harbouring strongly conserved HxKxxxxD motif. The binding pocket comprises of Tyr48, Lys86, His115, Lys117, Ser130, Asn132, Trp280, Asn240, His325, Lys327 and Tyr346 forming strong hydrogen bonds and dense hydrophobic contacts with the screened analogs and is surrounded by positively charged patches. Loops connecting the two domains and C-terminal region exhibit high degree of flexibility. In some structural ensembles, the partial disorderness in the C-terminal region is presumed to be due to its low confidence score, acquired during structure prediction. Transition from loop to ß-strands (244-254 aa) in P37-Cidofovir and its analog complexes advocates the need for further investigations. MD simulations support the accuracy of the molecular docking results, indicating the potential of analogs as potent binders of P37. Taken together, our results provide preferable understanding of molecular recognition and dynamics of ligand-bound states of P37, offering opportunities for development of new antivirals against MPXV. However, the need of in vitro and in vivo assays for confirmation of these results still persists.Communicated by Ramaswamy H. Sarma.

Diet-induced induction of hepatic serine/threonine kinase STK38 triggers proinflammation and hepatic lipid accumulation.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Although the involvement of chronic overnutrition, systemic inflammation, and insulin resistance in the development of NAFLD is well-established, however, the associations among these remain to be elucidated. Several studies have reported that chronic overnutrition, such as excessive consumption of fats (high-fat diet, HFD), can cause insulin resistance and inflammation. However, the mechanisms by which HFD exerts inflammation and thereby promotes insulin resistance and intrahepatic fat accumulation remain poorly understood. Here, we show that HFD induces the expression of hepatic serine/threonine kinase 38 (STK38), which further induces systemic inflammation leading to insulin resistance. Notably, ectopic expression of STK38 in mouse liver leads to lean NAFLD phenotype with hepatic inflammation, insulin resistance, intrahepatic lipid accumulation, and hypertriglyceridemia in mice fed on a regular chow diet. Further, depletion of hepatic STK38 in HFD-fed mice remarkably reduces proinflammation, improves hepatic insulin sensitivity, and decreases hepatic fat accumulation. Mechanistically, two critical stimuli are elicited by STK38 action. For one stimulus, STK38 binds to Tank-Binding protein Kinase 1 and induces Tank-Binding protein Kinase 1 phosphorylation to promote NF-κß nuclear translocation that mobilizes the release of proinflammatory cytokines and eventually leads to insulin resistance. The second, stimulus involves intrahepatic lipid accumulation by enhanced de novo lipogenesis via reducing the AMPK-ACC signaling axis. These findings identify STK38 as a novel nutrient-sensitive proinflammatory and lipogenic factor in maintaining hepatic energy homeostasis, and it provides a promising target for hepatic and immune health.

Structural insights into SARS-CoV-2 main protease conformational plasticity.

The spread of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants underscores the need for insights into the structural properties of its structural and non-structural proteins. The highly conserved homo-dimeric chymotrypsin-like protease (3CL MPRO ), belonging to the class of cysteine hydrolases, plays an indispensable role in processing viral polyproteins that are involved in viral replication and transcription. Studies have successfully demonstrated the role of MPRO as an attractive drug target for designing antiviral treatments because of its importance in the viral life cycle. Herein, we report the structural dynamics of six experimentally solved structures of MPRO (i.e., 6LU7, 6M03, 6WQF, 6Y2E, 6Y84, and 7BUY including both ligand-free and ligand-bound states) at different resolutions. We have employed a structure-based balanced forcefield, CHARMM36m through state-of-the-art all-atoms molecular dynamics simulations at µ-seconds scale at room temperature (303K) and pH 7.0 to explore their structure-function relationship. The helical domain-III responsible for dimerization mostly contributes to the altered conformational states and destabilization of MPRO . A keen observation of the high degree of flexibility in the P5 binding pocket adjoining domain II-III highlights the reason for observation of conformational heterogeneity among the structural ensembles of MPRO . We also observe a differential dynamics of the catalytic pocket residues His41, Cys145, and Asp187, which may lead to catalytic impairment of the monomeric proteases. Among the highly populated conformational states of the six systems, 6LU7 and 7M03 forms the most stable and compact MPRO conformation with intact catalytic site and structural integrity. Altogether, our findings from this extensive study provides a benchmark to identify physiologically relevant structures of such promising drug targets for structure-based drug design and discovery of potent drug-like compounds having clinical potential.