Identification of natural compounds as potent inhibitors of SARS-CoV-2 main protease using combined docking and molecular dynamics simulations

Coronavirus disease 2019 (COVID-19) has emerged from China and globally affected the entire population through the human-to-human transmission of a newly emerged virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genome of SARS-CoV-2 encodes several proteins that are essential for multiplication and pathogenesis. The main protease (M^pro or 3CL^pro) of SARS-CoV-2 plays a central role in its pathogenesis and thus is considered as an attractive drug target for the drug design and development of small-molecule inhibitors. We have employed an extensive structure-based high-throughput virtual screening to discover potential natural compounds from the ZINC database which could inhibit the M^pro of SARS-CoV-2. Initially, the hits were selected on the basis of their physicochemical and drug-like properties. Subsequently, the PAINS filter, estimation of binding affinities using molecular docking, and interaction analyses were performed to find safe and potential inhibitors of SARS-CoV-2 M^pro. We have identified ZINC02123811 (1-(3-(2,5,9-trimethyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)propanoyl)piperidine-4-carboxamide), a natural compound bearing appreciable affinity, efficiency, and specificity towards the binding pocket of SARS-CoV-2 M^pro. The identified compound showed a set of drug-like properties and preferentially binds to the active site of SARS-CoV-2 M^pro. All-atom molecular dynamics (MD) simulations were performed to evaluate the conformational dynamics, stability and interaction mechanism of M^pro with ZINC02123811. MD simulation results indicated that M^pro with ZINC02123811 forms a stable complex throughout the trajectory of 100 ns. These findings suggest that ZINC02123811 may be further exploited as a promising scaffold for the development of potential inhibitors of SARS-CoV-2 M^pro to address COVID-19.     

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL^pro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CL^pro activity in vitro with IC₅₀’s of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC₅₀’s of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL^pro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CL^pro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.     

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M^pro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the M^pro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the M^pro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M^pros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant M^pro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and β M^pro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.     

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (M^pro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective M^pro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative M^pro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as M^pro inhibitors. In this manner, we aimed to complement enzymatic activity assays of M^pro performed by other groups with information on ligand affinity. We have made the M^pro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of M^pro STD-NMR data, thereby accelerating ongoing drug design efforts.

     

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 (M^pro or 3CL^pro) 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 M^pro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against M^pros from multiple coronaviruses. Crystal structures of M^pros 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 M^pros from different coronaviruses. In view of the importance of M^pro 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.     

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC₅₀=1.88 ± 0.49 µM) and PL^Pro (IC₅₀=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.     

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN–nsp10) complex: implications for its role in viral genome stability and inhibitor identification

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (M^pro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.     

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 Mpro Dimer

The main protease (M^pro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug’s properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar K_i values with the M^pro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 M^pro. The comparison of a new X-ray crystal structure of M^pro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 M^pro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the M^pro, which facilitates coordination of the drug’s P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.     

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS*

All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL^pro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL^pro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL^pro is less efficient at processing a peptide substrate due to MERS-CoV 3CL^pro being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL^pro enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL^pro is a weakly associated dimer (K_d ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL^pro were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL^pro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL^pro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL^pro dimerization. Activation of MERS-CoV 3CL^pro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL^pro inhibitors as antiviral agents.     

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral M^pro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of M^pro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 M^pro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the M^pro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.     

Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design

Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M^pro), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) M^pro and a severe acute respiratory syndrome CoV (SARS-CoV) M^pro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M^pro. A monomeric form of IBV M^pro was identified for the first time in CoV M^pro structures. A comparison of these two structures to other available M^pro structures provides new insights for the design of substrate-based inhibitors targeting CoV M^pros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M^pro and was found to demonstrate in vitro inactivation of IBV M^pro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M^pro.     

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (M^pro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of M^pro (M^proC) locks M^pro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric M^pro have stalled. Isolated M^proC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of M^proC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of M^proC folding. Further, we find that a contact between a tryptophan in the M^proC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric M^pro in the early-stage viral replication of both viruses.     

Ligand-based design,synthesis, computational insights,and in vitro studies of novel N-(5-Nitrothiazol-2-yl)-carboxamido derivatives as potent inhibitors of SARS-CoV-2 main protease

The global outbreak of the COVID-19 pandemic provokes scientists to make a prompt development of new effective therapeutic interventions for the battle against SARS-CoV-2. A new series of N-(5-nitrothiazol-2-yl)-carboxamido derivatives were designed and synthesised based on the structural optimisation principle of the SARS-CoV Mpro co-crystallized WR1 inhibitor. Notably, compound 3b achieved the most promising anti-SARS-CoV-2 activity with an IC₅₀ value of 174.7 µg/mL. On the other hand, compounds 3a, 3b, and 3c showed very promising SARS-CoV-2 Mpro inhibitory effects with IC₅₀ values of 4.67, 5.12, and 11.90 µg/mL, respectively. Compound 3b docking score was very promising (−6.94 kcal/mol) and its binding mode was nearly similar to that of WR1. Besides, the molecular dynamics (MD) simulations of compound 3b showed its great stability inside the binding pocket until around 40 ns. Finally, a very promising SAR was concluded to help to design more powerful SARS-CoV-2 Mpro inhibitors shortly.     

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (M^pro, also called 3CL^pro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native M^pro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus M^pro enzyme assays to facilitate the discovery and development of therapies targeting M^pro.     

In silico analyses of major active constituents of fingerroot (Boesenbergia rotunda) unveils inhibitory activities against SARS-CoV-2 main protease enzyme

Boesenbergia rotunda (L.) Mansf., commonly known as fingerroot is a perennial herb in the Zingiberaceae family with anticancer, anti-leptospiral, anti-inflammatory, antioxidant, antiulcer, and anti-herpes viral activities. While the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibitory activity of B. rotunda extract has been recently found, the active compounds contributing to this activity are yet unknown. The main protease (M^pro) enzyme is one of the most well established therapeutic targets among coronaviruses which plays a vital role in the maturation and cleavage of polyproteins during viral replication. The current work aims to identify active phytochemical substances from B. rotunda extract that can inhibit the replication of SARS-CoV-2 by using a combined molecular docking and dynamic simulation approaches. The virtual screening experiment revealed that fifteen molecules out of twenty-three major active compounds in the plant extract have acceptable drug-like characteristics. Alpinetin, Pinocembrin, and Pinostrobin have binding energies of ?7.51 kcal/mol, ?7.21 kcal/mol, and ?7.18 kcal/mol, respectively, and can suppress M^pro activity. The stability of the simulated complexes of the lead compounds with the drug-receptor is demonstrated by 100-ns MD simulations. The binding free energies study utilizing molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) show that the compounds and M^pro enzyme have favourable thermodynamic interactions, which are majorly driven by van der Waals forces. Thus, the selected bioactive phytochemicals from B. rotunda might be used as anti-SARS-CoV-2 candidates that target the M^pro enzyme.     

Studies of Cytotoxicity Effects,SARS-CoV-2 Main Protease Inhibition,and in Silico Interactions of Synthetic Chalcones

SARS-CoV-2 main protease (M^pro) plays an essential role in proteolysis cleavage that promotes coronavirus replication. Thus, attenuating the activity of this enzyme represents a strategy to develop antiviral agents. We report inhibitory effects against M^pro of 40 synthetic chalcones, and cytotoxicity activities, hemolysis, and in silico interactions of active compounds. Seven of them bearing a (E)-3-(furan-2-yl)-1-arylprop-2-en-1-one skeleton ( 10 , 28 , and 35–39 ) showed enzyme inhibition with IC₅₀ ranging from 13.76 and 36.13 μM. Except for 35 and 36 , other active compounds were not cytotoxic up to 150 μM against THP-1 and Vero cell lines. Compounds 10 , and 35–39 showed no hemolysis while 28 was weakly hemotoxic at 150 μM. Moreover, molecular docking showed interactions between compound 10 and M^pro (PDBID 5RG2 and 5RG3) with proximity to cys145 and His41, suggesting a covalent binding. Products of the reaction between chalcones and cyclohexanethiol indicated that this binding could be a Michael addition type.     

Investigation of changes in protein stability and substrate affinity of 3CL-protease of SARS-CoV-2 caused by mutations

3CL^pro of SARS-CoV-2 is one of the enzymes required for the replication process of the virus responsible for the COVID-19 pandemic. In this study, changes in protein stability and substrate affinity caused by mutations were investigated to stir the development of potent inhibitors. Sequence data of samples were obtained from the NCBI Virus database. Mutation analyses were performed with RDP4 and MegaX. 3CL^pro tertiary models were created using Robetta. Molecular docking for peptidomimetic substrate and inhibitor ligand was done with Autodock v4.2 and Haddock v2.4. Protein stability analysis was performed using mCSM stability and DynaMut2. Twenty-four missense mutations in 3CL^pro were identified in this study. Changes in the 3CL^pro structure induced by the mutations Met49Thr, Leu167Ser, and Val202Ala resulted in significant levels of instability (-2.029,-2.612,-2.177 kcal.mol^-1, respectively). The lowest interaction energy for substrate was -58.7 kcal.mol^-1 and -62.6 kcal.mol^-1 in wild-type and mutant, respectively. The lowest docking energy for ligand was -6.19 and -9.52 kcal.mol^-1 for wild-type and mutant, respectively. This study reports for the first time that mutations cause increased substrate affinity of 3CL^pro from SARS-CoV-2. This research provides important data for the development of potent peptidomimetic inhibitors for the treatment of COVID-19. Keywords: 3CL-protease, mutation analysis, protein stability, SARS-CoV-2 genome, substrate affinity