Template Entrance Channel as Possible Allosteric Inhibition and Resistance Site for Quinolines Tricyclic Derivatives in RNA Dependent RNA Polymerase of Bovine Viral Diarrhea Virus.

The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds' most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals.

Computational study of novel inhibitory molecule, 1-(4-((2S,3S)-3-amino-2-hydroxy-4-phenylbutyl)piperazin-1-yl)-3-phenylurea, with high potential to competitively block ATP binding to the RNA dependent RNA polymerase of SARS-CoV-2 virus.

For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an essential enzyme that catalyses the replication from RNA template and therefore remains an attractive therapeutic target for anti-COVID drug discovery. In the present study, we performed a comprehensive in silico screening for 16,776 potential molecules from recently established drug libraries based on two important pharmacophores (3-amino-4-phenylbutan-2-ol and piperazine). Based on initial assessment, 4042 molecules were obtained suitable as drug candidates, which were following Lipinski's rule. Molecular docking implemented for the analysis of molecular interactions narrowed this number of compounds down to 19. Subsequent to screening filtering criteria and considering the critical parameters viz. docking score and MM-GBSA binding free energy, 1-(4-((2S,3S)-3-amino-2-hydroxy-4-phenylbutyl)piperazin-1-yl)-3-phenylurea (compound 1) was accomplished to score highest in comparison to the remaining 18 shortlisted drug candidates. Notably, compound 1 displayed higher docking score (-8.069 kcal/mol) and MM-GBSA binding free energy (-49.56 kcal/mol) than the control drug, remdesivir triphosphate, the active form of remdesivir as well as adenosine triphosphate. Furthermore, a molecular dynamics simulation was carried out (100 ns), which substantiated the candidacy of compound 1 as better inhibitor. Overall, our systematic in silico study predicts the potential of compound 1 to exhibit a more favourable specific activity than remdesivir triphosphate. Hence, we suggest compound 1 as a novel potential drug candidate, which should be considered for further exploration and validation of its potential against SARS-CoV-2 in wet lab experimental studies.Communicated by Ramasawamy H. Sarma.

SARS-CoV-2 envelope protein attain Kac mediated dynamical interaction network to adopt 'histone mimic' at BRD4 interface.

Interface mimicry, achieved by recognition of host-pathogen interactions, is the basis by which pathogen proteins can hijack the host machinery. The envelope (E) protein of SARS-CoV-2 is reported to mimic the histones at the BRD4 surface via establishing the structural mimicry; however, the underlying mechanism of E protein mimicking the histones is still elusive. To explore the mimics at dynamic and structural residual network level an extensive docking, and MD simulations were carried out in a comparative manner between complexes of H3-, H4-, E-, and apo-BRD4. We identified that E peptide is able to attain an 'interaction network mimicry', as its acetylated lysine (Kac) achieves orientation and residual fingerprint similar to histones, including water-mediated interactions for both the Kac positions. We identified Y59 of E, playing an anchor role to escort lysine positioning inside the binding site. Furthermore, the binding site analysis confirms that E peptide needs a higher volume, similar to the H4-BRD4 where both the lysine's (Kac5 and Kac8) can accommodate nicely, however, the position of Kac8 is mimicked by two additional water molecules other than four water-mediated bridging's, strengthening the possibility that E peptide could hijack host BRD4 surface. These molecular insights seem pivotal for mechanistic understanding and BRD4-specific therapeutic intervention. KEY POINTSMolecular mimicry is reported in hijacking and then outcompeting the host counterparts so that pathogens can rewire their cellular function by overcoming the host defense mechanism.The molecular recognition process is the basis of molecular mimicry. The E peptide of SARS-CoV-2 is reported to mimic host histone at the BRD4 surface by utilizing its C-terminally placed acetylated lysine (Kac63) to mimic the N-terminally placed acetylated lysine Kac5GGKac8 histone (H4) by interaction network mimicry identified through microsecond molecular dynamics (MD) simulations and post-processing extensive analysis.There are two steps to mimic: firstly, tyrosine residues help E to anchor at the BRD4 surface to position Kac and increase the volume of the pocket. Secondary, after positioning of Kac, a common durable interaction network N140:Kac5; Kac5:W1; W1:Y97; W1:W2; W2:W3; W3:W4; W4:P82 is established between Kac5, with key residues P82, Y97, N140, and four water molecules through water mediate bridge. Furthermore, the second acetylated lysine Kac8 position and its interaction as polar contact with Kac5 were also mimicked by E peptide through interaction network P82:W5; W5:Kac63; W5:W6; W6:Kac63.The binding event at BRD4/BD1 seems an induced-fit mechanism as a bigger binding site volume was identified at H4-BRD4 on which E peptide attains its better stability than H3-BRD4.We identified the tyrosine residue Y59 of E that acts like an anchor on the BRD4 surface to position Kac inside the pocket and attain the interaction network by using aromatic residues of the BRD4 surface.Communicated by Ramaswamy H. Sarma.

Designing and characterization of a SARS-CoV-2 immunogen with receptor binding motif grafted on a protein scaffold: An epitope-focused vaccine approach.

The COVID-19 pandemic caused by SARS-CoV-2 has a significant burden on the economy and healthcare around the world. Vaccines are the most effective tools to fight infectious diseases by containing the spread of the disease. The current vaccines against SARS-CoV-2 are mostly based on the spike protein of SARS-CoV-2, which is large and has many immune-dominant non-neutralizing epitopes that may effectively skew the antibody response towards non-neutralizing antibodies. Here, we have explored the possibility of immune-focusing the receptor binding motif (RBM) of the spike protein of SARS-CoV-2 that induces mostly neutralizing antibodies in natural infection or in vacinees. The result shows that the scaffolded RBM can bind to Angiotensin Converting Enzyme 2 (ACE2) although with low affinity and induces a strong antibody response in mice. The immunized sera can bind both, the receptor binding domain (RBD) and the spike protein, which holds the RBM in its natural context. Sera from the immunized mice showed robust interferon γ response but poor neutralization of SARS-CoV-2 suggesting presence of a predominant T cell epitope on scaffolded RBM. Together, we provide a strategy for inducing strong antigenic T cell response which could be exploited further for future vaccine designing and development against SARS-CoV-2 infection.

Molecular Dynamics Simulations Reveal the Interaction Fingerprint of Remdesivir Triphosphate Pivotal in Allosteric Regulation of SARS-CoV-2 RdRp.

The COVID-19 pandemic has now strengthened its hold on human health and coronavirus' lethal existence does not seem to be going away soon. In this regard, the optimization of reported information for understanding the mechanistic insights that facilitate the discovery towards new therapeutics is an unmet need. Remdesivir (RDV) is established to inhibit RNA-dependent RNA polymerase (RdRp) in distinct viral families including Ebola and SARS-CoV-2. Therefore, its derivatives have the potential to become a broad-spectrum antiviral agent effective against many other RNA viruses. In this study, we performed comparative analysis of RDV, RMP (RDV monophosphate), and RTP (RDV triphosphate) to undermine the inhibition mechanism caused by RTP as it is a metabolically active form of RDV. The MD results indicated that RTP rearranges itself from its initial RMP-pose at the catalytic site towards NTP entry site, however, RMP stays at the catalytic site. The thermodynamic profiling and free-energy analysis revealed that a stable pose of RTP at NTP entrance site seems critical to modulate the inhibition as its binding strength improved more than its initial RMP-pose obtained from docking at the catalytic site. We found that RTP not only occupies the residues K545, R553, and R555, essential to escorting NTP towards the catalytic site, but also interacts with other residues D618, P620, K621, R624, K798, and R836 that contribute significantly to its stability. From the interaction fingerprinting it is revealed that the RTP interact with basic and conserved residues that are detrimental for the RdRp activity, therefore it possibly perturbed the catalytic site and blocked the NTP entrance site considerably. Overall, we are highlighting the RTP binding pose and key residues that render the SARS-CoV-2 RdRp inactive, paving crucial insights towards the discovery of potent inhibitors.

Binding mode characterization of 13b in the monomeric and dimeric states of SARS-CoV-2 main protease using molecular dynamics simulations.

The main protease, Mpro/3CLpro, plays an essential role in processing polyproteins translated from viral RNA to produce functional viral proteins and therefore serve as an attractive target for discovering COVID-19 therapeutics. The availability of both monomer and dimer crystal bound with a common ligand, '13b' (α-ketoamide inhibitor), opened up opportunities to understand the Mpro mechanism of action. A comparative analysis of both forms of Mpro was carried out to elucidate the binding site architectural differences in the presence and absence of '13b'. Molecular dynamics simulations suggest that the presence of '13b' enhances the stability of Mpro than the unbound APO form. The N- and C- terminals of both the protomers stabilize each other, and making it's interface essential for the active form of Mpro. In comparison to monomer, the relatively high affinity of '13b' is gained in dimer pocket due to the high stability of the pocket by the interaction of S1 residue of chain B with residues F140, E166 and H172 of chain A, which is absent in monomer. The comprehensive essential dynamics, protein structure network analysis and thermodynamic profiling highlight the hot-spots, pivotal in molecular recognition process at protein-ligand and protein-protein interaction levels, cross-validated through computational alanine scanning study. A comparative description of '13b' binding mechanism in both forms illustrates valuable insights into the inhibition mechanism and the selection of critical residues suitable for the structure-based approaches for the identification of more potent Mpro inhibitors.Communicated by Ramaswamy H. Sarma.

Wuhan to World: The COVID-19 Pandemic.

COVID-19 is a Severe Acute Respiratory Syndrome (SARS), caused by SARS-CoV-2, a novel virus which belongs to the family Coronaviridae. It was first reported in December 2019 in the Wuhan city of China and soon after, the virus and hence the disease got spread to the entire world. As of February 26, 2021, SARS-CoV-2 has infected ~112.20 million people and caused ~2.49 million deaths across the globe. Although the case fatality rate among SARS-CoV-2 patient is lower (~2.15%) than its earlier relatives, SARS-CoV (~9.5%) and MERS-CoV (~34.4%), the SARS-CoV-2 has been observed to be more infectious and caused higher morbidity and mortality worldwide. As of now, only the knowledge regarding potential transmission routes and the rapidly developed diagnostics has been guiding the world for managing the disease indicating an immediate need for a detailed understanding of the pathogen and the disease-biology. Over a very short period of time, researchers have generated a lot of information in unprecedented ways in the key areas, including viral entry into the host, dominant mutation, potential transmission routes, diagnostic targets and their detection assays, potential therapeutic targets and drug molecules for inhibiting viral entry and/or its replication in the host including cross-neutralizing antibodies and vaccine candidates that could help us to combat the ongoing COVID-19 pandemic. In the current review, we have summarized the available knowledge about the pathogen and the disease, COVID-19. We believe that this readily available knowledge base would serve as a valuable resource to the scientific and clinical community and may help in faster development of the solution to combat the disease.

Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library.

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Identification of potential molecules against COVID-19 main protease through structure-guided virtual screening approach.

The pandemic caused by novel coronavirus disease 2019 (COVID-19) infecting millions of populations worldwide and counting, has demanded quick and potential therapeutic strategies. Current approved drugs or molecules under clinical trials can be a good pool for repurposing through in-silico techniques to quickly identify promising drug candidates. The structural information of recently released crystal structures of main protease (Mpro) in APO and complex with inhibitors, N3, and 13b molecules was utilized to explore the binding site architecture through Molecular dynamics (MD) simulations. The stable state of Mpro was used to conduct extensive virtual screening of the aforementioned drug pool. Considering the recent success of HIV protease molecules, we also used anti-protease molecules for drug repurposing purposes. The identified top hits were further evaluated through MD simulations followed by the binding free energy calculations using MM-GBSA. Interestingly, in our screening, several promising drugs stand out as potential inhibitors of Mpro. However, based on control (N3 and 13b), we have identified six potential molecules, Leupeptin Hemisulphate, Pepstatin A, Nelfinavir, Birinapant, Lypression and Octreotide which have shown the reasonably significant MM-GBSA score. Further insight shows that the molecules form stable interactions with hot-spot residues, that are mainly conserved and can be targeted for structure- and pharmacophore-based designing. The pharmacokinetic annotations and therapeutic importance have suggested that these molecules possess drug-like properties and pave their way for in-vitro studies.Communicated by Ramaswamy H. Sarma.