Computer-Aided Screening for Potential Coronavirus 3-Chymotrypsin-like Protease (3CLpro) Inhibitory Peptides from Putative Hemp Seed Trypsinized Peptidome.

To control the COVID-19 pandemic, antivirals that specifically target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently required. The 3-chymotrypsin-like protease (3CLpro) is a promising drug target since it functions as a catalytic dyad in hydrolyzing polyprotein during the viral life cycle. Bioactive peptides, especially food-derived peptides, have a variety of functional activities, including antiviral activity, and also have a potential therapeutic effect against COVID-19. In this study, the hemp seed trypsinized peptidome was subjected to computer-aided screening against the 3CLpro of SARS-CoV-2. Using predictive trypsinized products of the five major proteins in hemp seed (i.e., edestin 1, edestin 2, edestin 3, albumin, and vicilin), the putative hydrolyzed peptidome was established and used as the input dataset. To select the Cannabis sativa antiviral peptides (csAVPs), a predictive bioinformatic analysis was performed by three webserver screening programs: iAMPpred, AVPpred, and Meta-iAVP. The amino acid composition profile comparison was performed by COPid to screen for the non-toxic and non-allergenic candidates, ToxinPred and AllerTOP and AllergenFP, respectively. GalaxyPepDock and HPEPDOCK were employed to perform the molecular docking of all selected csAVPs to the 3CLpro of SARS-CoV-2. Only the top docking-scored candidate (csAVP4) was further analyzed by molecular dynamics simulation for 150 nanoseconds. Molecular docking and molecular dynamics revealed the potential ability and stability of csAVP4 to inhibit the 3CLpro catalytic domain with hydrogen bond formation in domain 2 with short bonding distances. In addition, these top ten candidate bioactive peptides contained hydrophilic amino acid residues and exhibited a positive net charge. We hope that our results may guide the future development of alternative therapeutics against COVID-19.

Structure-Based Virtual Screening and Functional Validation of Potential Hit Molecules Targeting the SARS-CoV-2 Main Protease.

The recent global health emergency caused by the coronavirus disease 2019 (COVID-19) pandemic has taken a heavy toll, both in terms of lives and economies. Vaccines against the disease have been developed, but the efficiency of vaccination campaigns worldwide has been variable due to challenges regarding production, logistics, distribution and vaccine hesitancy. Furthermore, vaccines are less effective against new variants of the SARS-CoV-2 virus and vaccination-induced immunity fades over time. These challenges and the vaccines' ineffectiveness for the infected population necessitate improved treatment options, including the inhibition of the SARS-CoV-2 main protease (Mpro). Drug repurposing to achieve inhibition could provide an immediate solution for disease management. Here, we used structure-based virtual screening (SBVS) to identify natural products (from NP-lib) and FDA-approved drugs (from e-Drug3D-lib and Drugs-lib) which bind to the Mpro active site with high-affinity and therefore could be designated as potential inhibitors. We prioritized nine candidate inhibitors (e-Drug3D-lib: Ciclesonide, Losartan and Telmisartan; Drugs-lib: Flezelastine, Hesperidin and Niceverine; NP-lib: three natural products) and predicted their half maximum inhibitory concentration using DeepPurpose, a deep learning tool for drug-target interactions. Finally, we experimentally validated Losartan and two of the natural products as in vitro Mpro inhibitors, using a bioluminescence resonance energy transfer (BRET)-based Mpro sensor. Our study suggests that existing drugs and natural products could be explored for the treatment of COVID-19.

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.

Structural Basis of Main Proteases of Coronavirus Bound to Drug Candidate PF-07304814.

New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.

Phenylethanoid glycosides as a possible COVID-19 protease inhibitor: a virtual screening approach.

From the beginning of pandemic, more than 240 million people have been infected with a death rate higher than 2%. Indeed, the current exit strategy involving the spreading of vaccines must be combined with progress in effective treatment development. This scenario is sadly supported by the vaccine's immune activation time and the inequalities in the global immunization schedule. Bringing the crises under control means providing the world population with accessible and impactful new therapeutics. We screened a natural product library that contains a unique collection of 2370 natural products into the binding site of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). According to the docking score and to the interaction at the active site, three phenylethanoid glycosides (forsythiaside A, isoacteoside, and verbascoside) were selected. In order to provide better insight into the atomistic interaction and test the impact of the three selected compounds at the binding site, we resorted to a half microsecond-long molecular dynamics simulation. As a result, we are showing that forsythiaside A is the most stable molecule and it is likely to possess the highest inhibitory effect against SARS-CoV-2 Mpro. Phenylethanoid glycosides also have been reported to have both protease and kinase activity. This kinase inhibitory activity is very beneficial in fighting viruses inside the body as kinases are required for viral entry, metabolism, and/or reproduction. The dual activity (kinase/protease) of phenylethanoid glycosides makes them very promising anit-COVID-19 agents.

C60 fullerene against SARS-CoV-2 coronavirus: an in silico insight.

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.

GC-MS, LC-MS/MS, Docking and Molecular Dynamics Approaches to Identify Potential SARS-CoV-2 3-Chymotrypsin-Like Protease Inhibitors from Zingiber officinale Roscoe.

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.

Repurposing clinically approved drugs for COVID-19 treatment targeting SARS-CoV-2 papain-like protease.

COVID-19 is a disease caused by SARS-CoV-2, which has led to more than 4 million deaths worldwide. As a result, there is a worldwide effort to develop specific drugs for targeting COVID-19. Papain-like protease (PLpro) is an attractive drug target because it has multiple essential functions involved in processing viral proteins, including viral genome replication and removal of post-translational ubiquitination modifications. Here, we established two assays for screening PLpro inhibitors according to protease and anti-ISGylation activities, respectively. Application of the two screening techniques to the library of clinically approved drugs led to the discovery of tanshinone IIA sulfonate sodium and chloroxine with their IC50 values of lower than 10 µM. These two compounds were found to directly interact with PLpro and their molecular mechanisms of binding were illustrated by docking and molecular dynamics simulations. The results highlight the usefulness of the two developed screening techniques for locating PLpro inhibitors.

Blue Biotechnology: Computational Screening of Sarcophyton Cembranoid Diterpenes for SARS-CoV-2 Main Protease Inhibition.

The coronavirus pandemic has affected more than 150 million people, while over 3.25 million people have died from the coronavirus disease 2019 (COVID-19). As there are no established therapies for COVID-19 treatment, drugs that inhibit viral replication are a promising target; specifically, the main protease (Mpro) that process CoV-encoded polyproteins serves as an Achilles heel for assembly of replication-transcription machinery as well as down-stream viral replication. In the search for potential antiviral drugs that target Mpro, a series of cembranoid diterpenes from the biologically active soft-coral genus Sarcophyton have been examined as SARS-CoV-2 Mpro inhibitors. Over 360 metabolites from the genus were screened using molecular docking calculations. Promising diterpenes were further characterized by molecular dynamics (MD) simulations based on molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. According to in silico calculations, five cembranoid diterpenes manifested adequate binding affinities as Mpro inhibitors with ΔGbinding < -33.0 kcal/mol. Binding energy and structural analyses of the most potent Sarcophyton inhibitor, bislatumlide A (340), was compared to darunavir, an HIV protease inhibitor that has been recently subjected to clinical-trial as an anti-COVID-19 drug. In silico analysis indicates that 340 has a higher binding affinity against Mpro than darunavir with ΔGbinding values of -43.8 and -34.8 kcal/mol, respectively throughout 100 ns MD simulations. Drug-likeness calculations revealed robust bioavailability and protein-protein interactions were identified for 340; biochemical signaling genes included ACE, MAPK14 and ESR1 as identified based on a STRING database. Pathway enrichment analysis combined with reactome mining revealed that 340 has the capability to re-modulate the p38 MAPK pathway hijacked by SARS-CoV-2 and antagonize injurious effects. These findings justify further in vivo and in vitro testing of 340 as an antiviral agent against SARS-CoV-2.

Molecular docking of potential SARS-CoV-2 papain-like protease inhibitors.

SARS-CoV-2 papain-like protease is considered as an important potential target for anti-SARS-CoV-2 drug discovery due to its crucial roles in viral spread and innate immunity. Here, we have utilized an in silico molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 papain-like protease, by screening 21 antiviral, antifungal and anticancer compounds. Among them, Neobavaisoflavone has the highest binding energy for SARS-CoV-2 papain-like protease. These molecules could bind near the SARS-CoV-2 papain-like protease crucial catalytic triad, ubiquitination and ISGylation residues: Trp106, Asn109, Cys111, Met208, Lys232, Pro247, Tyr268, Gln269, His272, Asp286 and Thr301. Because blocking the papain-like protease is an important strategy in fighting against viruses, these compounds might be promising candidates for therapeutic intervention against COVID-19.

The main protease and RNA-dependent RNA polymerase are two prime targets for SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented global health crisis. It is particularly urgent to develop clinically effective therapies to contain the pandemic. The main protease (Mpro) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process and viral genome replication and transcription, respectively, are two attractive drug targets for SARS-CoV-2. This review summarizes up-to-date progress in the structural and pharmacological aspects of those two key targets above. Different classes of inhibitors individually targeting Mpro and RdRP are discussed, which could promote drug development to treat SARS-CoV-2 infection.

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.

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.