Binding Adaptation of GS-441524 Diversifies Macro Domains and Downregulates SARS-CoV-2 de-MARylation Capacity.

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.

Antiviral drug discovery: preparing for the next pandemic.

Clinically approved antiviral drugs are currently available for only 10 of the more than 220 viruses known to infect humans. The SARS-CoV-2 outbreak has exposed the critical need for compounds that can be rapidly mobilised for the treatment of re-emerging or emerging viral diseases, while vaccine development is underway. We review the current status of antiviral therapies focusing on RNA viruses, highlighting strategies for antiviral drug discovery and discuss the challenges, solutions and options to accelerate drug discovery efforts.

Interaction of small molecules with the SARS-CoV-2 main protease in silico and in vitro validation of potential lead compounds using an enzyme-linked immunosorbent assay.

Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic is ongoing, with no proven safe and effective vaccine to date. Further, effective therapeutic agents for COVID-19 are limited, and as a result, the identification of potential small molecule antiviral drugs is of particular importance. A critical antiviral target is the SARS-CoV-2 main protease (Mpro), and our aim was to identify lead compounds with potential inhibitory effects. We performed an initial molecular docking screen of 300 small molecules, which included phenolic compounds and fatty acids from our OliveNet™ library (224), and an additional group of curated pharmacological and dietary compounds. The prototypical α-ketoamide 13b inhibitor was used as a control to guide selection of the top 30 compounds with respect to binding affinity to the Mpro active site. Further studies and analyses including blind docking were performed to identify hypericin, cyanidin-3-O-glucoside and SRT2104 as potential leads. Molecular dynamics simulations demonstrated that hypericin (ΔG = -18.6 and -19.3 kcal/mol), cyanidin-3-O-glucoside (ΔG = -50.8 and -42.1 kcal/mol), and SRT2104 (ΔG = -8.7 and -20.6 kcal/mol), formed stable interactions with the Mpro active site. An enzyme-linked immunosorbent assay indicated that, albeit, not as potent as the covalent positive control (GC376), our leads inhibited the Mpro with activity in the micromolar range, and an order of effectiveness of hypericin and cyanidin-3-O-glucoside > SRT2104 > SRT1720. Overall, our findings, and those highlighted by others indicate that hypericin and cyanidin-3-O-glucoside are suitable candidates for progress to in vitro and in vivo antiviral studies.

Profiling SARS-CoV-2 Main Protease (MPRO) Binding to Repurposed Drugs Using Molecular Dynamics Simulations in Classical and Neural Network-Trained Force Fields.

The current COVID-19 pandemic caused by a novel coronavirus SARS-CoV-2 urgently calls for a working therapeutic. Here, we report a computation-based workflow for efficiently selecting a subset of FDA-approved drugs that can potentially bind to the SARS-CoV-2 main protease MPRO. The workflow started with docking (using Autodock Vina) each of 1615 FDA-approved drugs to the MPRO active site. This step selected 62 candidates with docking energies lower than -8.5 kcal/mol. Then, the 62 docked protein-drug complexes were subjected to 100 ns of molecular dynamics (MD) simulations in a molecular mechanics (MM) force field (CHARMM36). This step reduced the candidate pool to 26, based on the root-mean-square-deviations (RMSDs) of the drug molecules in the trajectories. Finally, we modeled the 26 drug molecules by a pseudoquantum mechanical (ANI) force field and ran 5 ns hybrid ANI/MM MD simulations of the 26 protein-drug complexes. ANI was trained by neural network models on quantum mechanical density functional theory (wB97X/6-31G(d)) data points. An RMSD cutoff winnowed down the pool to 12, and free energy analysis (MM/PBSA) produced the final selection of 9 drugs: dihydroergotamine, midostaurin, ziprasidone, etoposide, apixaban, fluorescein, tadalafil, rolapitant, and palbociclib. Of these, three are found to be active in literature reports of experimental studies. To provide physical insight into their mechanism of action, the interactions of the drug molecules with the protein are presented as 2D-interaction maps. These findings and mappings of drug-protein interactions may be potentially used to guide rational drug discovery against COVID-19.

Evidence and speculations: vaccines and therapeutic options for COVID-19 pandemic.

A novel coronavirus (2019-nCov) emerged in China, at the end of December 2019 which posed an International Public Health Emergency, and later declared as a global pandemic by the World Health Organization (WHO). The International Committee on Taxonomy of Viruses (ICTV) named it SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), while the disease was named COVID-19 (Coronavirus Disease- 2019). Many questions related to the exact mode of transmission, animal origins, and antiviral therapeutics are not clear yet. Nevertheless, it is required to urgently launch a new protocol to evaluate the side effects of unapproved vaccines and antiviral therapeutics to accelerate the clinical application of new drugs. In this review, we highlight the most salient characteristics and recent findings of COVID-19 disease, molecular virology, interspecies mechanisms, and health consequences related to this disease.

Identification of Main Protease of Coronavirus SARS-CoV-2 (Mpro) Inhibitors from Melissa officinalis.

BACKGROUND: The recent outbreak of Coronavirus SARS-CoV-2 (Covid-19), which has rapidly spread around the world in about three months with tens of thousands of deaths recorded so far is a global concern. An urgent need for potential therapeutic intervention is of necessity. Mpro is an attractive druggable target for the development of anti-COVID-19 drug development. METHODS: Compounds previously characterized by Melissa officinalis were queried against the main protease of coronavirus SARS-CoV-2 using a computational approach. RESULTS: Melitric acid A and salvanolic acid A had higher affinity than lopinavir and ivermectin using both AutodockVina and XP docking algorithms. The computational approach was employed in the generation of the QSAR model using automated QSAR, and in the docking of ligands from Melissa officinalis with SARS-CoV-2 Mpro inhibitors. The best model obtained was KPLS_Radial_ 28 (R2 = 0.8548 and Q2=0.6474, which was used in predicting the bioactivity of the lead compounds. Molecular mechanics based MM-GBSA confirmed salvanolic acid A as the compound with the highest free energy and predicted bioactivity of 4.777; it interacted with His-41 of the catalytic dyad (Cys145-His41) of SARS-CoV-2 main protease (Mpro), as this may hinder the cutting of inactive viral protein into active ones capable of replication. CONCLUSION: Salvanolic acid A can be further evaluated as a potential Mpro inhibitor.

Identification of novel human USP2 inhibitor and its putative role in treatment of COVID-19 by inhibiting SARS-CoV-2 papain-like (PLpro) protease.

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.

Site mapping and small molecule blind docking reveal a possible target site on the SARS-CoV-2 main protease dimer interface.

The SARS-CoV-2 virus is causing COVID-19 resulting in an ongoing pandemic with serious health, social, and economic implications. Much research is focused in repurposing or identifying new small molecules which may interact with viral or host-cell molecular targets. An important SARS-CoV-2 target is the main protease (Mpro), and the peptidomimetic α-ketoamides represent prototypical experimental inhibitors. The protease is characterised by the dimerization of two monomers each which contains the catalytic dyad defined by Cys145 and His41 residues (active site). Dimerization yields the functional homodimer. Here, our aim was to investigate small molecules, including lopinavir and ritonavir, α-ketoamide 13b, and ebselen, for their ability to interact with the Mpro. The sirtuin 1 agonist SRT1720 was also used in our analyses. Blind docking to each monomer individually indicated preferential binding of the ligands in the active site. Site-mapping of the dimeric protease indicated a highly reactive pocket in the dimerization region at the domain III apex. Blind docking consistently indicated a strong preference of ligand binding in domain III, away from the active site. Molecular dynamics simulations indicated that ligands docked both to the active site and in the dimerization region at the apex, formed relatively stable interactions. Overall, our findings do not obviate the superior potency with respect to inhibition of protease activity of covalently-linked inhibitors such as α-ketoamide 13b in the Mpro active site. Nevertheless, along with those from others, our findings highlight the importance of further characterisation of the Mpro active site and any potential allosteric sites.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.