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1.
The main protease (Mpro, 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 Ki values with the Mpro 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 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, 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 Mpro, 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.  相似文献   

2.
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 (Mpro or 3CLpro) 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 Mpro 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 Mpro. 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 Mpro. The identified compound showed a set of drug-like properties and preferentially binds to the active site of SARS-CoV-2 Mpro. All-atom molecular dynamics (MD) simulations were performed to evaluate the conformational dynamics, stability and interaction mechanism of Mpro with ZINC02123811. MD simulation results indicated that Mpro 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 Mpro to address COVID-19.  相似文献   

3.
4.
The Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel lineage B betacoroanvirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant mortality, morbidity, and socioeconomic disruptions worldwide. Effective antivirals are urgently needed for COVID-19. The main protease (Mpro) of SARS-CoV-2 is an attractive antiviral target because of its essential role in the cleavage of the viral polypeptide. In this study, we performed an in silico structure-based screening of a large chemical library to identify potential SARS-CoV-2 Mpro inhibitors. Among 8,820 compounds in the library, our screening identified trichostatin A, a histone deacetylase inhibitor and an antifungal compound, as an inhibitor of SARS-CoV-2 Mpro activity and replication. The half maximal effective concentration of trichostatin A against SARS-CoV-2 replication was 1.5 to 2.7µM, which was markedly below its 50% effective cytotoxic concentration (75.7µM) and peak serum concentration (132µM). Further drug compound optimization to develop more stable analogues with longer half-lives should be performed. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of SARS-CoV-2.  相似文献   

5.

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 (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro 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 Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro 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 Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

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6.
SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro 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 Mpro 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 Mpro. 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 Mpro 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.  相似文献   

7.
3CLpro 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. 3CLpro 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 3CLpro were identified in this study. Changes in the 3CLpro 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 3CLpro 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  相似文献   

8.
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.  相似文献   

9.
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 (3CLpro) 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 3CLpro 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 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.  相似文献   

10.
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 (Mpro) 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 Mpro 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 Mpro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro 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 Mpro 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.  相似文献   

11.
SARS-CoV-2 main protease (Mpro) 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 Mpro 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 Mpro (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.  相似文献   

12.
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 (Mpro, also called 3CLpro) 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 Mpro, 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 Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.  相似文献   

13.
An epidemic caused by COVID-19 in China turned into pandemic within a short duration affecting countries worldwide. Researchers and companies around the world are working on all the possible strategies to develop a curative or preventive strategy for the same, which includes vaccine development, drug repurposing, plasma therapy, and drug discovery based on Artificial intelligence. Therapeutic approaches based on Computational biology and Machine-learning algorithms are specially considered, with a view that these could provide a fast and accurate outcome in the present scenario. As an effort towards developing possible therapeutics for COVID-19, we have used machine-learning algorithms for the generation of alignment kernels from diverse viral sequences of Covid-19 reported from India, China, Italy and USA. Using these diverse sequences we have identified the conserved motifs and subsequently a peptide library was designed against them. Of these, 4 peptides have shown strong binding affinity against the main protease of SARS-CoV-2 (Mpro) and also maintained their stability and specificity under physiological conditions as observed through MD Simulations. Our data suggest that these evolutionary peptides against COVID-19 if found effective may provide cross-protection against diverse Covid-19 variants.  相似文献   

14.
BackgroundSARS-CoV-2, an emerging strain of coronavirus, has affected millions of people from all the continents of world and received worldwide attention. This emerging health crisis calls for the urgent development of specific therapeutics against COVID-19 to potentially reduce the burden of this emerging pandemic.PurposeThis study aims to evaluate the anti-viral efficacy of natural bioactive entities against COVID-19 via molecular docking and molecular dynamics simulation.MethodsA library of 27 caffeic-acid derivatives was screened against 5 proteins of SARS-CoV-2 by using Molegro Virtual Docker 7 to obtain the binding energies and interactions between compounds and SARS-CoV-2 proteins. ADME properties and toxicity profiles were investigated via www.swissadme.ch web tools and Toxtree respectively. Molecular dynamics simulation was performed to determine the stability of the lead-protein interactions.ResultsOur obtained results has uncovered khainaoside C, 6-O-Caffeoylarbutin, khainaoside B, khainaoside C and vitexfolin A as potent modulators of COVID-19 possessing more binding energies than nelfinavir against COVID-19 Mpro, Nsp15, SARS-CoV-2 spike S2 subunit, spike open state and closed state structure respectively. While Calceolarioside B was identified as pan inhibitor, showing strong molecular interactions with all proteins except SARS-CoV-2 spike glycoprotein closed state. The results are supported by 20 ns molecular dynamics simulations of the best complexes.ConclusionThis study will hopefully pave a way for development of phytonutrients-based antiviral therapeutic for treatment or prevention of COVID-19 and further studies are recommended to evaluate the antiviral effects of these phytochemicals against SARS-CoV-2 in in vitro and in vivo models.  相似文献   

15.
Xue X  Yu H  Yang H  Xue F  Wu Z  Shen W  Li J  Zhou Z  Ding Y  Zhao Q  Zhang XC  Liao M  Bartlam M  Rao Z 《Journal of virology》2008,82(5):2515-2527
Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (Mpro), 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) Mpro and a severe acute respiratory syndrome CoV (SARS-CoV) Mpro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of Mpro. A monomeric form of IBV Mpro was identified for the first time in CoV Mpro structures. A comparison of these two structures to other available Mpro structures provides new insights for the design of substrate-based inhibitors targeting CoV Mpros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV Mpro and was found to demonstrate in vitro inactivation of IBV Mpro 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 Mpro.  相似文献   

16.
The 3C-like protease (3CLpro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CLpro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CLpro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CLpro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)–disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CLpro to SARS-CoV-2 3CLpro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CLpro seems to have increased activity relative to that of SARS-CoV 3CLpro.  相似文献   

17.
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 (Mpro) 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 Mpro (MproC) locks Mpro 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 Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC 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 Mpro in the early-stage viral replication of both viruses.  相似文献   

18.
The Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the biggest public health challenge the world has witnessed in the past decades. SARS-CoV-2 undergoes constant mutations and new variants of concerns (VOCs) with altered transmissibility, virulence, and/or susceptibility to vaccines and therapeutics continue to emerge. Detailed analysis of host factors involved in virus replication may help to identify novel treatment targets. In this study, we dissected the metabolome derived from COVID-19 patients to identify key host factors that are required for efficient SARS-CoV-2 replication. Through a series of metabolomic analyses, in vitro, and in vivo investigations, we identified ATP citrate lyase (ACLY) as a novel host factor required for efficient replication of SARS-CoV-2 wild-type and variants, including Omicron. ACLY should be further explored as a novel intervention target for COVID-19.  相似文献   

19.
Despite a huge effort by the scientific community to determine the animal reservoir of SARS-CoV-2, which led to the identification of several SARS-CoV-2-related viruses both in bats and in pangolins, the origin of SARS-CoV-2 is still not clear. Recently, Temmam et al. reported the discovery of bat coronaviruses with a high degree of genome similarity with SARS-CoV-2, especially concerning the RBDs of the S protein, which mediates the capability of such viruses to enter and therefore infect human cells through a hACE2-dependent pathway. These viruses, especially the one named BANAL-236, showed a higher affinity for the hACE2 compared to the original strain of SARS-CoV-2. In the present work, we analyse the similarities and differences between the 3CL protease (main protease, Mpro) of these newly reported viruses and SARS-CoV-2, discussing their relevance relative to the efficacy of existing therapeutic approaches against COVID-19, particularly concerning the recently approved orally available Paxlovid, and the development of future ones.  相似文献   

20.
Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.  相似文献   

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