An insight into the simulation directed understanding of the mechanism in SARS CoV-2 N-CTD, dimer integrity, and RNA-binding: Identifying potential antiviral inhibitors.

Coronavirus 2019 is a transmissible disease and has caused havoc throughout the world. The present study identifies the novel potential antiviral inhibitors against the nucleocapsid C-terminal domain that aids in RNA-binding and replication. A total of 485,629 compounds were screened, and MD was performed. The trajectory analysis (DCCM & PCA), structural integrity, and degree of compaction depicted the protein-ligand complex stability (PDB-PISA and Rgyr). Results obtained from screening shortlists 13 compounds possessing high Docking score. Further, seven compounds had a permissible RMSD limit (3 Å), with robust RMSF. Post-MD analysis of the top two compounds (204 and 502), DCCM & PCA analysis show a positive atomic displacements correlation among residues of active sites-dimer (Chain A and Chain B) & residual clustering. The ΔGint of RNA-bound (-83.5 kcal/mol) and drug-bound N-CTD-204 (-40.8 kcal/mol) and 502(-39.7 kcal/mol) as compared to Apo (-35.95 kcal/mol) suggests stabilization of protein, with less RNA-binding possibility. The Rgyr values depict the loss of compactness on RNA-binding when compared to the drug-bound N-CTD complex. Further, overlapping the protein complexes (0 ns and 100 ns) display significant changes in RMSD of the protein (204-2.07 Å and 502-1.89 Å) as compared to the Apo (1.72 Å) and RNA-bound form (1.76 Å), suggesting strong interaction for compound 204 as compared to 502. ADMET profiling indicates that these compounds can be used for further experiments (in vitro and pre-clinical). Compound 204 could be a promising candidate for targeting the N-protein-RNA assembly and viral replication.

Computational basis of SARS-CoV 2 main protease inhibition: an insight from molecular dynamics simulation based findings.

The coronavirus disease 2019 (COVID-19) pandemic is caused by newly discovered severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). One of the striking targets amongst all the proteins in coronavirus is the main protease (Mpro), as it plays vital biological roles in replication and maturation of the virus, and hence the potential target. The aim of this study is to repurpose the Food and Drug Administration (FDA) approved molecules via computer-aided drug designing against Mpro (PDB ID: 6Y2F) of SARS CoV-2 due to its high x-ray resolution of 1.95 Å as compared to other published Mprostructures. High Through Virtual Screening (HTVS) of 2456 FDA approved drugs using structure-based docking were analyzed. Molecular Dynamics simulations were performed to check the overall structural stability (RMSD), Cα fluctuations (RMSF) and protein-ligand interactions. Further, trajectory analysis was performed to assess the binding quality by exploiting the protein-residue motion cross correlation (DCCM) and binding free energy (MM/GBSA). Tenofovir, an antiretroviral for HIV-proteases and Terlipressin, a vasoconstrictor show stable RMSD, RMSF, better MM/GBSA with good cross correlation as compared to the Apo and O6K. Moreover, the results show concurrence with Nelfinavir, Lopinavir and Ritonavir which have shown significant inhibition in in vitro studies. Therefore, we conclude that Tenofovir and Terlipresssin might also show protease inhibition but are still open to clinical validation in case of SARS-CoV 2 treatment.Communicated by Ramaswamy H. Sarma.

Virtual screening and molecular dynamics simulation study of approved drugs as a binder to the linoleic acid binding site on spike protein of SARS-CoV-2 and double mutant (E484Q and L452R).

INTRODUCTION: Binding of linoleic acid (LA) to the spike trimer stabilizes it in closed conformation hindering its binding to angiotensin-converting enzyme-2, thus decreasing infectivity. In the current study, we tend to repurpose Food and Drug Administration-approved drugs as binder to the LA binding pocket in wild and double mutant spike protein. MATERIALS AND METHODS: Approved drugs from DrugBank database (n = 2456) were prepared using Ligprep module of Schrodinger. Crystal structure of LA bound to spike trimer was retrieved (PDB: 6ZB4) and prepared using protein preparation wizard and grid was generated. A virtual screening was performed. With the help of molecular dynamics (MD) studies interaction profile of screened drugs were further evaluated. The selected hits were further evaluated for binding to the double mutant form of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). RESULTS AND DISCUSSION: Following virtual screening, a total of 26 molecules were shortlisted, which were further evaluated using 1ns MD simulation study. Four ligands showing better root mean square deviation (RMSD), RMSD to LA with interaction profile similar to LA were further evaluated using 100 ns MD simulation studies. A total of 2 hits were identified, which performed better than LA (selexipag and pralatrexate). Both these ligands were also found to bind to LA binding site of the double mutant form (E484Q and L452R); however, the binding affinity of pralatrexate was found to be better. CONCLUSION: We have identified 2 ligands (selexipag and pralatrexate) as possible stable binders to the LA binding site in spike trimer (wild and mutant form). Among them, pralatrexate has shown in vitro activity against SARS-CoV-2, validating our study results.

Indomethacin: an exploratory study of antiviral mechanism and host-pathogen interaction in COVID-19.

INTRODUCTION: COVID-19, a dreadful pandemic that has impacted human life like no other pathogenic invasion, has claimed the lives of over 100 million people. The need for effective treatment strategies is still a subject of intense research considering the rapidly evolving genome and continental diversity. Indomethacin is administered mostly as co-treatment for affected patients as a non-steroidal anti-inflammatory drug (NSAID). However, the underlying mechanism of action is unresolved. This study explores the basal mechanism of indomethacin and potency in alleviating the damage caused by SARS-CoV-2 and discusses the experimental and clinical efficacy in recent studies. AREAS COVERED: The literature search and system biology-based network formation were employed to describe the potent effects and risks associated with indomethacin in in-vitro, in-vivo, and clinical studies. This study also highlights the plausible mechanism of antiviral action of indomethacin with its apparent viral protein targets. The SARS-CoV-2 protein, the interacting host proteins, and the effect of indomethacin on this interactome as a standalone treatment or as part of a co-therapy strategy are particularly emphasized using network modeling. EXPERT OPINION: Indomethacin has demonstrated excellent clinical endpoint characteristics in several studies, and we recommend that it be utilized in the treatment of mild-to-moderate COVID patients.

Structural and conformational analysis of SARS CoV 2 N-CTD revealing monomeric and dimeric active sites during the RNA-binding and stabilization: Insights towards potential inhibitors for N-CTD.

The advent of SARS-CoV-2 has become a universal health issue with no appropriate cure available to date. The coronavirus nucleocapsid (N) protein combines viral genomic RNA into a ribonucleoprotein and protects the viral genome from the host's nucleases. Structurally, the N protein comprises two independent domains: the N-terminal domain (NTD) for RNA-binding and C-terminal domain (CTD) involved in RNA-binding, protein dimerization, and nucleocapsid stabilization. The present study explains the structural aspects associated with the involvement of nucleocapsid C-terminal domain in the subunit assembly that helps the RNA binding and further stabilizing the virus assembly by protecting RNA from the hosts exonucleases degradation. The molecular dynamics (MD) simulations of the N-CTD and RNA complex suggests two active sites (site I: a monomer) and (site II: a dimer) with structural stability (RMSD: ~2 Å), Cα fluctuations (RMSF: ~3 Å) and strong protein-ligand interactions were estimated through the SiteMap module of Schrodinger. Virtual screening of 2456 FDA-approved drugs using structure-based docking identified top two leads distinctively against Site-I (monomer): Ceftaroline fosamil (MM-GBSA = -47.12 kcal/mol) and Cefoperazone (-45.84 kcal/mol); and against Site-II (dimer): Boceprevir, (an antiviral protease inhibitor, -106.78 kcal/mol) and Ceftaroline fosamil (-99.55 kcal/mol). The DCCM and PCA of drugs Ceftaroline fosamil (PC1+PC2 = 71.9%) and Boceprevir (PC1 +PC2 = 61.6%) show significant correlated residue motions which suggests highly induced conformational changes in the N-CTD dimer. Therefore, we propose N-CTD as a druggable target with two active binding sites (monomer and dimer) involved in specific RNA binding and stability. The RNA binding site with Ceftaroline fosamil binding can prevent viral assembly and can act as an antiviral for coronavirus.

Efficacy and safety of steroid therapy in COVID-19: A rapid systematic review and Meta-analysis.

PURPOSE: Although the use of steroids in the management of COVID-19 has been addressed by a few systematic review and meta-analysis, however, they also used data from "SARS-CoV" and "MERS-CoV." Again, most of these studies addressed only one severity category of patients or addressed only one efficacy endpoint (mortality). In this context, we conducted this meta-analysis to evaluate the efficacy and safety of steroid therapy among all severity categories of patients with COVID-19 (mild to moderate and severe to critical category) in terms of "mortality," "requirement of mechanical ventilation," "requirement of ICU" and clinical cure parameters. METHODS: 11 databases were screened. Only randomized controlled trials (RCTs) or high quality (on the basis of risk of bias analysis) comparative-observational studies were included in the analysis. RevMan5.3 was used for the meta-analysis. RESULTS: A total of 15 studies (3 RCT and 12 comparative-observational studies) were included. In the mechanically-ventilated COVID-19 population, treatment with dexamethasone showed significant protection against mortality (single study). Among severe and critically ill combined population, steroid administration was significantly associated with lowered mortality (risk ratio [RR] 0.83 [0.76-0.910]), lowered requirement of mechanical ventilation (RR 0.59 [0.51-0.69]), decreased requirement of intensive care unit (ICU) (RR 0.62 [0.45-0.86]), lowered length of ICU stay (single-study) and decreased duration of mechanical ventilation (two-studies). In mild to moderate population, steroid treatment was associated with a higher "duration of hospital stay," while no difference was seen in other domains. In patients at risk of progression to "acute respiratory distress syndrome," steroid administration was associated with "reduced requirement of mechanical ventilation" (single-study). CONCLUSION: This study guides the use of steroid across patients with different severity categories of COVID-19. Among mechanically ventilated patients, steroid therapy may be beneficial in terms of reduced mortality. Among "severe and critical" patients; steroid therapy was associated with lowered mortality, decreased requirement of mechanical ventilation, and ICU. However, no benefit was observed in "mild to moderate" population. To conclude, among properly selected patient populations (based-upon clinical severity and biomarker status), steroid administration may prove beneficial in patients with COVID-19.

Ivermectin as a potential drug for treatment of COVID-19: an in-sync review with clinical and computational attributes.

INTRODUCTION: COVID-19 cases are on surge; however, there is no efficient treatment or vaccine that can be used for its management. Numerous clinical trials are being reviewed for use of different drugs, biologics, and vaccines in COVID-19. A much empirical approach will be to repurpose existing drugs for which pharmacokinetic and safety data are available, because this will facilitate the process of drug development. The article discusses the evidence available for the use of Ivermectin, an anti-parasitic drug with antiviral properties, in COVID-19. METHODS: A rational review of the drugs was carried out utilizing their clinically significant attributes. A more thorough understanding was met by virtual embodiment of the drug structure and realizable viral targets using artificial intelligence (AI)-based and molecular dynamics (MD)-simulation-based study. CONCLUSION: Certain studies have highlighted the significance of ivermectin in COVID-19; however, it requires evidences from more Randomised Controlled Trials (RCTs) and dose- response studies to support its use. In silico-based analysis of ivermectin's molecular interaction specificity using AI and classical mechanics simulation-based methods indicates positive interaction of ivermectin with viral protein targets, which is leading for SARS-CoV 2 N-protein NTD (nucleocapsid protein N-terminal domain).

Safety and efficacy of lopinavir/ritonavir combination in COVID-19: A systematic review, meta-analysis, and meta-regression analysis.

BACKGROUND: Being protease inhibitors and owing to their efficacy in SARS-CoV, lopinavir + ritonavir (L/R) combination is being used in the management of COVID-19. In this systematic review and meta-analysis, we have evaluated the comparative safety and efficacy of L/R combination. MATERIALS AND METHODS: Comparative, observational studies and controlled clinical trials comparing L/R combination to standard of care (SOC)/control or any other antiviral agent/combinations were included. A total of 10 databases were searched to identify 13 studies that fulfilled the predefined inclusion/exclusion criteria. RESULTS: No discernible beneficial effect was seen in the L/R group in comparison to SOC/control in terms of "progression to more severe state" (4 studies, odds ratio [OR]: 1.446 [0.722-2.895]), "mortality" (3 studies, OR: 1.208 [0.563-2.592]), and "virological cure on days 7-10" (3 studies, OR: 0.777 [0.371-1.630]), while the L/R combination arm performed better than the SOC/control arm in terms of "duration of hospital stay" (3 studies, mean difference (MD): -1.466 [-2.403 to - 0.529]) and "time to virological cure" (3 studies, MD: -3.272 [-6.090 to - 0.454]). No difference in efficacy was found between L/R versus hydroxychloroquine (HCQ) and L/R versus arbidol. However, in a single randomized controlled trail (open label), chloroquine (CQ) performed better than L/R. The combination L/R with arbidol may be beneficial (in terms of virological clearance and radiological improvement); however, we need more dedicated studies. Single studies report efficacy of L/R + interferon (IFN, either alpha or 1-beta) combination. We need more studies to delineate the proper effect size. Regarding adverse effects, except occurrence of diarrhea (higher in the L/R group), safety was comparable to SOC. CONCLUSION: In our study, no difference was seen between the L/R combination and the SOC arm in terms of "progression to more severe state," "mortality," and virological cure on days 7-10;" however, some benefits in terms of "duration of hospital stay" and "time to virological cure" were seen. No significant difference in efficacy was seen when L/R was compared to arbidol and HCQ monotherapy. Except for the occurrence of diarrhea, which was higher in the L/R group, safety profile of L/R is comparable to SOC. Compared to L/R combination, CQ, L/R + arbidol, L/R + IFN-α, and L/R + IFN-1ß showed better efficacy, but the external validity of these findings is limited by limited number of studies (1 study each).

In Silico Structure-Based Repositioning of Approved Drugs for Spike Glycoprotein S2 Domain Fusion Peptide of SARS-CoV-2: Rationale from Molecular Dynamics and Binding Free Energy Calculations.

The membrane-anchored spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a pivotal role in directing the fusion of the virus particle mediated by the host cell receptor angiotensin-converting enzyme 2 (ACE-2). The fusion peptide region of the S protein S2 domain provides SARS-CoV-2 with the biological machinery needed for direct fusion to the host lipid membrane. In our present study, computer-aided drug design strategies were used for the identification of FDA-approved small molecules using the optimal structure of the S2 domain, which exhibits optimal interaction ratios, structural features, and energy variables, which were evaluated based on their performances in molecular docking, molecular dynamics simulations, molecular mechanics/generalized Born model and solvent accessibility binding free energy calculations of molecular dynamics trajectories, and statistical inferences. Among the 2,625 FDA-approved small molecules, chloramphenicol succinate, imipenem, and imidurea turned out to be the molecules that bound the best at the fusion peptide hydrophobic pocket. The principal interactions of the selected molecules suggest that the potential binding site at the fusion peptide region is centralized amid the Lys790, Thr791, Lys795, Asp808, and Gln872 residues.IMPORTANCE The present study provides the structural identification of the viable binding residues of the SARS-CoV-2 S2 fusion peptide region, which holds prime importance in the virus's host cell fusion and entry mechanism. The classical molecular mechanics simulations were set on values that mimic physiological standards for a good approximation of the dynamic behavior of selected drugs in biological systems. The drug molecules screened and analyzed here have relevant antiviral properties, which are reported here and which might hint toward their utilization in the coronavirus disease 2019 (COVID-19) pandemic owing to their attributes of binding to the fusion protein binding region shown in this study.

Virtual screening and molecular dynamics study of approved drugs as inhibitors of spike protein S1 domain and ACE2 interaction in SARS-CoV-2.

BACKGROUND: The receptor binding domain (RBD) of spike protein S1 domain SARS-CoV-2 plays a key role in the interaction with ACE2, which leads to subsequent S2 domain mediated membrane fusion and incorporation of viral RNA into host cells. In this study we tend to repurpose already approved drugs as inhibitors of the interaction between S1-RBD and the ACE2 receptor. METHODS: 2456 approved drugs were screened against the RBD of S1 protein of SARS-CoV-2 (target PDB ID: 6M17). As the interacting surface between S1-RBD and ACE2 comprises of bigger region, the interacting surface was divided into 3 sites on the basis of interactions (site 1, 2 and 3) and a total of 5 grids were generated (site 1, site 2, site 3, site 1+site 2 and site 2+site 3). A virtual screening was performed using GLIDE implementing HTVS, SP and XP screening. The top hits (on the basis of docking score) were further screened for MM-GBSA. All the top hits were further evaluated in molecular dynamics studies. Performance of the virtual screening protocol was evaluated using enrichment studies. RESULT: and discussion: We performed 5 virtual screening against 5 grids generated. A total of 42 compounds were identified after virtual screening. These drugs were further assessed for their interaction dynamics in molecular dynamics simulation. On the basis of molecular dynamics studies, we come up with 10 molecules with favourable interaction profile, which also interacted with physiologically important residues (residues taking part in the interaction between S1-RBD and ACE2. These are antidiabetic (acarbose), vitamins (riboflavin and levomefolic acid), anti-platelet agents (cangrelor), aminoglycoside antibiotics (Kanamycin, amikacin) bronchodilator (fenoterol), immunomodulator (lamivudine), and anti-neoplastic agents (mitoxantrone and vidarabine). However, while considering the relative side chain fluctuations when compared to the S1-RBD: ACE2 complex riboflavin, fenoterol, cangrelor and vidarabine emerged out as molecules with prolonged relative stability. CONCLUSION: We identified 4 already approved drugs (riboflavin, fenoterol, cangrelor and vidarabine) as possible agents for repurposing as inhibitors of S1:ACE2 interaction. In-vitro validation of these findings are necessary for identification of a safe and effective inhibitor of S1: ACE2 mediated entry of SARS-CoV-2 into the host cell.

Update on the target structures of SARS-CoV-2: A systematic review.

Knowledge of structural details is very much essential from the drug-design perspective. In the systematic review, we systematically reviewed the structural basis of different target proteins of SARS-corona virus (CoV2) from a viral life cycle and from drug design perspective. We searched four literature (PubMed, EMBASE, NATURE, and Willey online library) databases and one structural database (RCSB.org) with appropriate keywords till April 18, and finally, 26 articles were included in the systematic review. The published literature mainly centered upon the structural details of "spike protein," "main protease/M Pro/3CL pro," "RNA-dependent RNA polymerase," and "nonstructural protein 15 Endoribonuclease" of SARS-CoV-2. However, inhibitor bound structures were very less. We need better structures elucidating the interactions between different targets and their inhibitors which will help us in understanding the atomic level importance of different amino acid residues in the functionality of the target structures. To summarize, we need structures with fine resolution, co-crystallized structures with biologically validated inhibitors, and functional characterization of different target proteins. Some other routes of entry of SARS-CoV-2 are also mentioned (e.g., CD147); however, these findings are not structurally validated. This review may pave way for better understanding of SARS-CoV-2 life cycle from structural biology perspective.

Virological and clinical cure in COVID-19 patients treated with hydroxychloroquine: A systematic review and meta-analysis.

Following the demonstration of the efficacy of hydroxychloroquine against severe acute respiratory syndrome coronavirus 2 in vitro, many trials started to evaluate its efficacy in clinical settings. However, no systematic review and meta-analysis have addressed the issue of the safety and efficacy of hydroxychloroquine (HCQ) in coronavirus disease 2019. We conducted a systematic review and meta-analysis with the objectives of evaluation of safety and efficacy of HCQ alone or in combination in terms of "time to clinical cure," "virological cure," "death or clinical worsening of disease," "radiological progression," and safety. RevMan was used for meta-analysis. We searched 16 literature databases out of which seven studies (n = 1358) were included in the systematic review. In terms of clinical cure, two studies reported possible benefit in "time to body temperature normalization" and one study reported less "cough days" in the HCQ arm. Treatment with HCQ resulted in less number of cases showing the radiological progression of lung disease (odds ratio [OR], 0.31, 95% confidence interval [CI], 0.11-0.9). No difference was observed in virological cure (OR, 2.37, 95% CI, 0.13-44.53), death or clinical worsening of disease (OR, 1.37, 95% CI, 1.37-21.97), and safety (OR, 2.19, 95% CI, 0.59-8.18), when compared with the control/conventional treatment. Five studies reported either the safety or efficacy of HCQ + azithromycin. Although seems safe and effective, more data are required for a definitive conclusion. HCQ seems to be promising in terms of less number of cases with radiological progression with a comparable safety profile to control/conventional treatment. We need more data to come to a definite conclusion.

In-silico homology assisted identification of inhibitor of RNA binding against 2019-nCoV N-protein (N terminal domain).

The N terminal domain (NTD) of Nucleocapsid protein (N protein) of coronavirus (CoV) binds to the viral (+) sense RNA and results in CoV ribonucleoprotien (CoV RNP) complex, essential for the virus replication. In this study, the RNA-binding N terminal domain (NTD) of the N protein was targeted for the identification of possible inhibitors of RNA binding. Two NTD structures of N proteins were selected (2OFZ and 1SSK, 92% homology) for virtual screening of 56,079 compounds from Asinex and Maybridge library to identify top 15 hits for each of the targets based on 'docking score'. These top-hits were further screened for MM-GBSA binding free energy, pharmacokinetic properties (QikProp) and drug-likeness (SwissADME) and subjected to molecular dynamics (MD) studies. Two suitable binders (ZINC00003118440 and ZINC0000146942) against the target 2OFZ were identified. ZINC00003118440 is a theophylline derivative under the drug class 'bronchodilators' and further screening with approved bronchodilators was also studied to identify their ability to bind to the RNA binding region on the N protein. The other identified top hit is ZINC0000146942, which is a 3,4dihydropyrimidone class molecule. Hence this study suggests two important class of compounds, theophylline and pyrimidone derivaties as possible inhibitors of RNA binding to the N terminal domain of N protein of coronavirus, thus opening new avenues for in vitro validations. Communicated by Ramaswamy H. Sarma.

Drug targets for corona virus: A systematic review.

The 2019-novel coronavirus (nCoV) is a major source of disaster in the 21th century. However, the lack of specific drugs to prevent/treat an attack is a major need at this current point of time. In this regard, we conducted a systematic review to identify major druggable targets in coronavirus (CoV). We searched PubMed and RCSB database with keywords HCoV, NCoV, corona virus, SERS-CoV, MERS-CoV, 2019-nCoV, crystal structure, X-ray crystallography structure, NMR structure, target, and drug target till Feb 3, 2020. The search identified seven major targets (spike protein, envelop protein, membrane protein, protease, nucleocapsid protein, hemagglutinin esterase, and helicase) for which drug design can be considered. There are other 16 nonstructural proteins (NSPs), which can also be considered from the drug design perspective. The major structural proteins and NSPs may serve an important role from drug design perspectives. However, the occurrence of frequent recombination events is a major deterrent factor toward the development of CoV-specific vaccines/drugs.