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1.
Biol Pharm Bull ; 47(5): 967-977, 2024.
Article in English | MEDLINE | ID: mdl-38763751

ABSTRACT

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.


Subject(s)
Alanine , Catalytic Domain , Coronavirus 3C Proteases , Drug Resistance, Viral , Molecular Dynamics Simulation , SARS-CoV-2 , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , SARS-CoV-2/drug effects , Alanine/genetics , Drug Resistance, Viral/genetics , Humans , Mutation , COVID-19 Drug Treatment , Protease Inhibitors/pharmacology , Indazoles , Triazines , Triazoles
2.
FEBS J ; 291(1): 61-69, 2024 01.
Article in English | MEDLINE | ID: mdl-37843490

ABSTRACT

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/genetics , Viral Nonstructural Proteins/genetics , Peptides/genetics , Coronavirus 3C Proteases/genetics , Protease Inhibitors/chemistry , Antiviral Agents/pharmacology , Peptide Hydrolases
3.
Commun Biol ; 6(1): 1159, 2023 11 13.
Article in English | MEDLINE | ID: mdl-37957287

ABSTRACT

A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MProWT) from precursor polyproteins. Upon expression, a model precursor of MProWT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MProM) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MProWT. MProM and the catalytic domain (MPro1-199) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MProM and MPro1-199, respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/genetics , Mutation , Coronavirus 3C Proteases/genetics
4.
Nature ; 622(7982): 376-382, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37696289

ABSTRACT

Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.


Subject(s)
Antiviral Agents , Drug Resistance, Viral , SARS-CoV-2 , Humans , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/virology , Lactams , Leucine , Nitriles , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , Binding Sites/drug effects , Binding Sites/genetics , Mutation , Substrate Specificity , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Virus Replication/drug effects , Drug Design , Proline
5.
Biochemistry ; 62(3): 747-758, 2023 02 07.
Article in English | MEDLINE | ID: mdl-36656653

ABSTRACT

The main protease of SARS-CoV-2 (Mpro) plays a critical role in viral replication; although it is relatively conserved, Mpro has nevertheless evolved over the course of the COVID-19 pandemic. Here, we examine phenotypic changes in clinically observed variants of Mpro, relative to the originally reported wild-type enzyme. Using atomistic molecular dynamics simulations, we examine effects of mutation on protein structure and dynamics. In addition to basic structural properties such as variation in surface area and torsion angles, we use protein structure networks and active site networks to evaluate functionally relevant characters related to global cohesion and active site constraint. Substitution analysis shows a continuing trend toward more hydrophobic residues that are dependent on the location of the residue in primary, secondary, tertiary, and quaternary structures. Phylogenetic analysis provides additional evidence for the impact of selective pressure on mutation of Mpro. Overall, these analyses suggest evolutionary adaptation of Mpro toward more hydrophobicity and a less-constrained active site in response to the selective pressures of a novel host environment.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Evolution, Molecular , SARS-CoV-2 , Humans , Antiviral Agents/pharmacology , COVID-19/genetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Phylogeny , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Coronavirus 3C Proteases/genetics
6.
J Biol Chem ; 299(2): 102790, 2023 02.
Article in English | MEDLINE | ID: mdl-36509143

ABSTRACT

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.


Subject(s)
Biocatalysis , Coronavirus 3C Proteases , Histidine , SARS-CoV-2 , Humans , Histidine/genetics , Histidine/metabolism , Hydrogen-Ion Concentration , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Catalytic Domain , Kinetics , Amino Acid Substitution
7.
J Mol Biol ; 434(24): 167876, 2022 12 30.
Article in English | MEDLINE | ID: mdl-36334779

ABSTRACT

We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (Tm) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [(-6)MProH41A and (-6*)MProH41A, respectively], and its corresponding mature MProH41A were systematically examined. While the H41A mutation exerts negligible effect on Tm and dimer dissociation constant (Kdimer) of MProH41A, relative to the wild type MPro, both miniprecursors show a 4-5 °C decrease in Tm and > 85-fold increase in Kdimer as compared to MProH41A. The Kd for the binding of the covalent inhibitor GC373 to (-6*)MProH41A increases ∼12-fold, relative to MProH41A, concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers' oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1-2 and 302-306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.


Subject(s)
Coronavirus 3C Proteases , Coronavirus Protease Inhibitors , SARS-CoV-2 , Humans , Catalytic Domain , Crystallography, X-Ray , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Protein Stability , Mutation , Coronavirus Protease Inhibitors/chemistry
8.
J Infect Public Health ; 15(11): 1326-1349, 2022 Nov.
Article in English | MEDLINE | ID: mdl-36288640

ABSTRACT

The first infection case of new coronavirus was reported at the end of 2019 and after then, the cases are reported in all nations across the world in a very short period. Further, the regular news of mutations in the virus has made life restricted with appropriate behavior. To date, a new strain (Omicron and its new subvariant Omicron XE) has brought fear amongst us due to a higher trajectory of increase in the number of cases. The researchers thus started giving attention to this viral infection and discovering drug-like candidates to cure the infections. Finding a drug for any viral infection is not an easy task and takes plenty of time. Therefore, computational chemistry/bioinformatics is followed to get promising molecules against viral infection. Molecular dynamics (MD) simulations are being explored to get drug candidates in a short period. The molecules are screened via molecular docking, which provides preliminary information which can be further verified by molecular dynamics (MD) simulations. To understand the change in structure, MD simulations generated several trajectories such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonding, and radius of gyration for the main protease (Mpro) of the new coronavirus (nCoV) in the presence of small molecules. Additionally, change in free energy for the formation of complex of Mpro of nCoV with the small molecule can be determined by applying molecular mechanics with generalized born and surface area solvation (MM-GBSA). Thus, the promising molecules can be further explored for clinical trials to combat coronavirus disease-19 (COVID-19).


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Drug Discovery , Humans , Computational Biology , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , COVID-19/genetics , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/virology , COVID-19 Drug Treatment , Molecular Docking Simulation , Molecular Dynamics Simulation
9.
J Virol ; 96(17): e0090722, 2022 09 14.
Article in English | MEDLINE | ID: mdl-36000844

ABSTRACT

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.


Subject(s)
Antiviral Agents , Coronavirus 3C Proteases , Coronavirus, Feline , Drug Resistance, Viral , Mutation , Protease Inhibitors , Animals , Antiviral Agents/pharmacology , Cats/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Coronavirus, Feline/drug effects , Coronavirus, Feline/enzymology , Coronavirus, Feline/genetics , Drug Resistance, Viral/genetics , Protease Inhibitors/pharmacology
11.
Mol Cell ; 82(13): 2385-2400.e9, 2022 07 07.
Article in English | MEDLINE | ID: mdl-35594856

ABSTRACT

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Epithelial Cells , Inflammasomes , NLR Proteins , SARS-CoV-2 , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Caspase 3/metabolism , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Epithelial Cells/metabolism , Humans , Inflammasomes/genetics , Inflammasomes/metabolism , Lung/metabolism , Lung/virology , NLR Proteins/genetics , NLR Proteins/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/metabolism , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism , Pyroptosis , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity
12.
Int J Biol Macromol ; 209(Pt A): 984-990, 2022 Jun 01.
Article in English | MEDLINE | ID: mdl-35452699

ABSTRACT

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.


Subject(s)
Coronavirus 3C Proteases , Middle East Respiratory Syndrome Coronavirus , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Middle East Respiratory Syndrome Coronavirus/enzymology , Middle East Respiratory Syndrome Coronavirus/genetics , Protein Conformation , Recombinant Proteins
13.
Phys Chem Chem Phys ; 24(16): 9141-9145, 2022 Apr 20.
Article in English | MEDLINE | ID: mdl-35411366

ABSTRACT

Dimerization of SARS-CoV-2 main protease (Mpro) is a prerequisite for its processing activity. With >2000 mutations already reported in Mpro, SARS-CoV-2 may accumulate mutations in the Mpro dimeric interface to stabilize it further. We employed high-throughput protein design strategies to design the symmetrical dimeric interface of Mpro (300 000 designs) to identify mutational hotspots that render the Mpro more stable. We found that ∼22% of designed mutations that yield stable Mpro dimers already exist in SARS-CoV-2 genomes and are currently circulating. Our multi-parametric analyses highlight potential Mpro mutations that SARS-CoV-2 may develop, providing a foundation for assessing viral adaptation and mutational surveillance.


Subject(s)
Coronavirus 3C Proteases , Protein Engineering , SARS-CoV-2 , COVID-19 , Coronavirus 3C Proteases/genetics , Dimerization , Humans , SARS-CoV-2/enzymology , SARS-CoV-2/genetics
14.
Commun Biol ; 5(1): 160, 2022 03 01.
Article in English | MEDLINE | ID: mdl-35233052

ABSTRACT

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.


Subject(s)
Coronavirus 3C Proteases/metabolism , Catalysis , Catalytic Domain , Circular Dichroism , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Models, Molecular , Mutation , Pyrrolidines/chemistry , Sulfonic Acids/chemistry , Thermodynamics
15.
STAR Protoc ; 3(1): 101158, 2022 03 18.
Article in English | MEDLINE | ID: mdl-35194584

ABSTRACT

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).


Subject(s)
Coronavirus 3C Proteases/chemistry , Models, Molecular , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/genetics , Crystallization , Crystallography, X-Ray , Humans
16.
Sci Rep ; 12(1): 2505, 2022 02 15.
Article in English | MEDLINE | ID: mdl-35169179

ABSTRACT

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Binding Sites , COVID-19/pathology , COVID-19/virology , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Docking Simulation , Nitriles/chemistry , Nitriles/metabolism , Nitriles/pharmacology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/metabolism , Quinazolines/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Virus Replication/drug effects
17.
Sci Rep ; 12(1): 1299, 2022 01 25.
Article in English | MEDLINE | ID: mdl-35079088

ABSTRACT

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Mutation, Missense , Patient Acuity , Adult , Aged , Amino Acid Substitution , COVID-19/enzymology , COVID-19/genetics , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Female , Humans , Male , Middle Aged
18.
Signal Transduct Target Ther ; 7(1): 22, 2022 01 24.
Article in English | MEDLINE | ID: mdl-35075101

ABSTRACT

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.


Subject(s)
Coronavirus 3C Proteases/genetics , Coronavirus Nucleocapsid Proteins/genetics , DEAD Box Protein 58/genetics , DNA Helicases/genetics , Poly-ADP-Ribose Binding Proteins/genetics , RNA Helicases/genetics , RNA Recognition Motif Proteins/genetics , RNA-Binding Proteins/genetics , Receptors, Immunologic/genetics , SARS-CoV-2/genetics , Stress Granules/genetics , Animals , Chlorocebus aethiops , Coronavirus 3C Proteases/immunology , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/immunology , DNA Helicases/immunology , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Immune Evasion , Phosphoproteins/genetics , Phosphoproteins/immunology , Poly I-C/pharmacology , Poly-ADP-Ribose Binding Proteins/immunology , Protein Binding , RNA Helicases/immunology , RNA Recognition Motif Proteins/immunology , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA-Binding Proteins/immunology , Receptors, Immunologic/immunology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Sendai virus/genetics , Sendai virus/immunology , Signal Transduction , Stress Granules/drug effects , Stress Granules/immunology , Stress Granules/virology , Vero Cells , Vesiculovirus/genetics , Vesiculovirus/immunology
19.
Bioengineered ; 13(2): 3350-3361, 2022 02.
Article in English | MEDLINE | ID: mdl-35048792

ABSTRACT

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (Mpro) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, Mpro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 Mpro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of Mpro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 Mpro inhibition.


Subject(s)
Antiviral Agents/pharmacology , Apigenin/pharmacology , COVID-19 Drug Treatment , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Amino Acid Sequence , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Apigenin/chemistry , Apigenin/pharmacokinetics , Bioengineering , COVID-19/virology , Computer Simulation , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacokinetics , Drug Evaluation, Preclinical , Glucosides/chemistry , Glucosides/pharmacokinetics , Glucosides/pharmacology , Humans , Molecular Docking Simulation , Phytotherapy , Protein Domains , SARS-CoV-2/genetics
20.
Virus Res ; 307: 198618, 2022 01 02.
Article in English | MEDLINE | ID: mdl-34740719

ABSTRACT

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2'O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.


Subject(s)
Coronavirus 3C Proteases/genetics , Protein Folding , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Brazil , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Genetic Variation/genetics , Genome, Viral/genetics , Humans , Phosphoproteins/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Virulence/genetics
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