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1.
Virus Res ; 307: 198618, 2022 01 02.
Article in English | MEDLINE | ID: covidwho-1504602

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
2.
Nat Neurosci ; 24(11): 1522-1533, 2021 11.
Article in English | MEDLINE | ID: covidwho-1500484

ABSTRACT

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Coronavirus 3C Proteases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microvessels/metabolism , SARS-CoV-2/metabolism , Animals , Blood-Brain Barrier/pathology , Brain/pathology , Chlorocebus aethiops , Coronavirus 3C Proteases/genetics , Cricetinae , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mesocricetus , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microvessels/pathology , SARS-CoV-2/genetics , Vero Cells
3.
Nat Neurosci ; 24(11): 1522-1533, 2021 11.
Article in English | MEDLINE | ID: covidwho-1483143

ABSTRACT

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Coronavirus 3C Proteases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microvessels/metabolism , SARS-CoV-2/metabolism , Animals , Blood-Brain Barrier/pathology , Brain/pathology , Chlorocebus aethiops , Coronavirus 3C Proteases/genetics , Cricetinae , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mesocricetus , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microvessels/pathology , SARS-CoV-2/genetics , Vero Cells
4.
mBio ; 12(5): e0233521, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1430167

ABSTRACT

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus 3C Proteases/metabolism , DEAD Box Protein 58/metabolism , Receptors, Immunologic/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , A549 Cells , Adaptor Proteins, Signal Transducing/genetics , Animals , Caco-2 Cells , Coronavirus 3C Proteases/genetics , DEAD Box Protein 58/genetics , Enzyme-Linked Immunosorbent Assay , HCT116 Cells , HEK293 Cells , Humans , Immunity, Innate/genetics , Immunity, Innate/physiology , Immunoblotting , Interferon Type I/metabolism , Mice , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology , Ubiquitination , Virus Replication/genetics , Virus Replication/physiology
5.
Int J Mol Sci ; 22(18)2021 Sep 11.
Article in English | MEDLINE | ID: covidwho-1409703

ABSTRACT

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Molecular Dynamics Simulation , Mutation, Missense , SARS-CoV-2 , Alanine , Amino Acid Substitution , COVID-19/enzymology , COVID-19/genetics , Catalytic Domain/genetics , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Humans , SARS-CoV-2/enzymology , SARS-CoV-2/genetics
6.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: covidwho-1370748

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K i] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (K i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Drug Discovery/methods , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Animals , COVID-19/drug therapy , COVID-19/virology , Cells, Cultured , Coronavirus 3C Proteases/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Genetic Engineering , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , SARS-CoV-2/metabolism , Structure-Activity Relationship , Virus Replication
7.
J Gen Virol ; 102(3)2021 03.
Article in English | MEDLINE | ID: covidwho-1369236

ABSTRACT

Coronavirus protease nsp5 (Mpro, 3CLpro) remains a primary target for coronavirus therapeutics due to its indispensable and conserved role in the proteolytic processing of the viral replicase polyproteins. In this review, we discuss the diversity of known coronaviruses, the role of nsp5 in coronavirus biology, and the structure and function of this protease across the diversity of known coronaviruses, and evaluate past and present efforts to develop inhibitors to the nsp5 protease with a particular emphasis on new and mostly unexplored potential targets of inhibition. With the recent emergence of pandemic SARS-CoV-2, this review provides novel and potentially innovative strategies and directions to develop effective therapeutics against the coronavirus protease nsp5.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Protease Inhibitors/therapeutic use , Amino Acid Sequence , COVID-19/virology , Coronavirus/enzymology , Coronavirus/metabolism , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Humans , Phylogeny , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism
8.
FASEB J ; 35(8): e21774, 2021 08.
Article in English | MEDLINE | ID: covidwho-1331587

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/metabolism , Dimethyl Sulfoxide/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Viral/drug effects , SARS-CoV-2/enzymology , Computer Simulation , Coronavirus 3C Proteases/genetics , Humans , Microfluidic Analytical Techniques , Peptides/metabolism , Protein Stability
9.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: covidwho-1271852

ABSTRACT

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.


Subject(s)
Coronavirus 3C Proteases/metabolism , Protein Processing, Post-Translational , Proteolysis , Torovirus Infections/embryology , Torovirus/enzymology , Animals , Coronavirus 3C Proteases/genetics , HEK293 Cells , Humans , Substrate Specificity , Swine , Torovirus/genetics , Torovirus Infections/genetics
10.
Biochem J ; 478(13): 2499-2515, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1291175

ABSTRACT

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Azoles/pharmacology , Chlorocebus aethiops , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Isoindoles , Leupeptins/pharmacology , Organoselenium Compounds/pharmacology , Peptidomimetics , RNA-Binding Proteins/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism
11.
ChemMedChem ; 16(15): 2339-2344, 2021 08 05.
Article in English | MEDLINE | ID: covidwho-1272172

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a global health problem. Despite the current implementation of COVID-19 vaccination schedules, identifying effective antiviral drug treatments for this disease continues to be a priority. A recent study showed that masitinib (MST), a tyrosine kinase inhibitor, blocks the proteolytic activity of SARS-CoV-2 main protease (Mpro ). Although MST is a potential candidate for COVID-19 treatment, a comprehensive analysis of its interaction with Mpro has not been done. In this work, we performed molecular dynamics simulations of the MST-Mpro complex crystal structure. The effect of the protonation states of Mpro H163 residue and MST titratable groups were studied. Furthermore, we identified the MST substituents and Mpro mutations that affect the stability of the complex. Our results provide valuable insights into the design of new MST analogs as potential treatments for COVID-19.


Subject(s)
Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/metabolism , SARS-CoV-2/enzymology , Thiazoles/metabolism , Benzamides , Catalytic Domain , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Cysteine Proteinase Inhibitors/chemistry , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Piperidines , Protein Binding , Pyridines , Static Electricity , Thiazoles/chemistry
12.
Biomed Res Int ; 2021: 6696012, 2021.
Article in English | MEDLINE | ID: covidwho-1255651

ABSTRACT

A global pandemic has emerged following the appearance of the new severe acute respiratory virus whose official name is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strongly affecting the health sector as well as the world economy. Indeed, following the emergence of this new virus, despite the existence of a few approved and known effective vaccines at the time of writing this original study, a sense of urgency has emerged worldwide to discover new technical tools and new drugs as soon as possible. In this context, many studies and researches are currently underway to develop new tools and therapies against SARS CoV-2 and other viruses, using different approaches. The 3-chymotrypsin (3CL) protease, which is directly involved in the cotranslational and posttranslational modifications of viral polyproteins essential for the existence and replication of the virus in the host, is one of the coronavirus target proteins that has been the subject of these extensive studies. Currently, the majority of these studies are aimed at repurposing already known and clinically approved drugs against this new virus, but this approach is not really successful. Recently, different studies have successfully demonstrated the effectiveness of artificial intelligence-based techniques to understand existing chemical spaces and generate new small molecules that are both effective and efficient. In this framework and for our study, we combined a generative recurrent neural network model with transfer learning methods and active learning-based algorithms to design novel small molecules capable of effectively inhibiting the 3CL protease in human cells. We then analyze these small molecules to find the correct binding site that matches the structure of the 3CL protease of our target virus as well as other analyses performed in this study. Based on these screening results, some molecules have achieved a good binding score close to -18 kcal/mol, which we can consider as good potential candidates for further synthesis and testing against SARS-CoV-2.


Subject(s)
Antiviral Agents/chemistry , Biological Products/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Neural Networks, Computer , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Antiviral Agents/classification , Antiviral Agents/pharmacology , Biological Products/classification , Biological Products/pharmacology , COVID-19/drug therapy , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Design , Gene Expression , Humans , Kinetics , Molecular Docking Simulation , Protease Inhibitors/classification , Protease Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Small Molecule Libraries/classification , Small Molecule Libraries/pharmacology , Substrate Specificity , Thermodynamics
13.
Biophys Chem ; 276: 106610, 2021 09.
Article in English | MEDLINE | ID: covidwho-1252522

ABSTRACT

In the new millennium, the outbreak of new coronavirus has happened three times: SARS-CoV, MERS-CoV, and SARS-CoV-2. Unfortunately, we still have no pharmaceutical weapons against the diseases caused by these viruses. The pandemic of SARS-CoV-2 reminds us the urgency to search new drugs with totally different mechanism that may target the weaknesses specific to coronaviruses. Herein, we disclose a computational evaluation of targeted oxidation strategy (TOS) for potential inhibition of SARS-CoV-2 by disulfiram, a 70-year-old anti-alcoholism drug, and predict a multiple-target mechanism. A preliminary list of promising TOS drug candidates targeting the two thiol proteases of SARS-CoV-2 are proposed upon virtual screening of 32,143 disulfides.


Subject(s)
Alcohol Deterrents/chemistry , Antiviral Agents/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Disulfiram/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , Alcohol Deterrents/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Disulfiram/pharmacology , Drug Repositioning , Gene Expression , Humans , Kinetics , Molecular Docking Simulation , Oxidation-Reduction , Protease Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Quantum Theory , SARS-CoV-2/enzymology , Substrate Specificity , Thermodynamics
14.
Biophys Chem ; 275: 106608, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1219972

ABSTRACT

This paper proposes natural drug candidate compounds for the treatment of coronavirus disease 2019 (COVID-19). We investigated the binding properties between the compounds in the Moringa oleifera plant and the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 using molecular docking and ab initio fragment molecular orbital calculations. Among the 12 compounds, niaziminin was found to bind the strongest to Mpro. We furthermore proposed novel compounds based on niaziminin and investigated their binding properties to Mpro. The results reveal that the introduction of a hydroxyl group into niaziminin enhances its binding affinity to Mpro. These niaziminin derivatives can be promising candidate drugs for the treatment of COVID-19.


Subject(s)
Antiviral Agents/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Moringa oleifera/chemistry , Phytochemicals/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , Thiocarbamates/chemistry , Antiviral Agents/classification , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , COVID-19/drug therapy , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Design , Drug Discovery , Gene Expression , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Phytochemicals/classification , Phytochemicals/isolation & purification , Phytochemicals/pharmacology , Protease Inhibitors/classification , Protease Inhibitors/isolation & purification , Protease Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Quantum Theory , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Structure-Activity Relationship , Thermodynamics , Thiocarbamates/classification , Thiocarbamates/isolation & purification , Thiocarbamates/pharmacology
15.
J Med Virol ; 93(5): 2722-2734, 2021 05.
Article in English | MEDLINE | ID: covidwho-1196526

ABSTRACT

The 21st century has witnessed three outbreaks of coronavirus (CoVs) infections caused by severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, spreads rapidly and since the discovery of the first COVID-19 infection in December 2019, has caused 1.2 million deaths worldwide and 226,777 deaths in the United States alone. The high amino acid similarity between SARS-CoV and SARS-CoV-2 viral proteins supports testing therapeutic molecules that were designed to treat SARS infections during the 2003 epidemic. In this review, we provide information on possible COVID-19 treatment strategies that act via inhibition of the two essential proteins of the virus, 3C-like protease (3CLpro ) or papain-like protease (PLpro ).


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Viral Proteases/drug effects , COVID-19/epidemiology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/drug effects , Coronavirus 3C Proteases/genetics , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/drug effects , Coronavirus Papain-Like Proteases/genetics , Humans , Middle East Respiratory Syndrome Coronavirus , Protease Inhibitors/therapeutic use , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
16.
Curr Top Med Chem ; 21(6): 442-460, 2021.
Article in English | MEDLINE | ID: covidwho-1183719

ABSTRACT

[Coronaviruses (CoVs) are enveloped positive-stranded RNA viruses with spike (S) protein projections that allow the virus to enter and infect host cells. The S protein is a key virulence factor determining viral pathogenesis, host tropism, and disease pathogenesis. There are currently diverse corona viruses that are known to cause disease in humans. The occurrence of Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), as fatal human CoV diseases, has induced significant interest in the medical field. The novel coronavirus disease (COVID-19) is an infectious disease caused by a novel strain of coronavirus (SAR-CoV-2). The SARS-CoV2 outbreak has been evolved in Wuhan, China, in December 2019, and identified as a pandemic in March 2020, resulting in 53.24 M cases and 1.20M deaths worldwide. SARS-CoV-2 main proteinase (MPro), a key protease of CoV-2, mediates viral replication and transcription. SARS-CoV-2 MPro has been emerged as an attractive target for SARS-CoV-2 drug design and development. Diverse scaffolds have been released targeting SARS-CoV-2 MPro. In this review, we culminate the latest published information about SARS-CoV-2 main proteinase (MPro) and reported inhibitors.


Subject(s)
Antiviral Agents/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Phytochemicals/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , Amino Acid Sequence , Antiviral Agents/classification , Antiviral Agents/pharmacology , COVID-19/drug therapy , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Discovery , Gene Expression , High-Throughput Screening Assays , Humans , Molecular Docking Simulation , Phytochemicals/classification , Phytochemicals/pharmacology , Protease Inhibitors/classification , Protease Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Structure-Activity Relationship
17.
Molecules ; 26(7)2021 Mar 30.
Article in English | MEDLINE | ID: covidwho-1160340

ABSTRACT

The main protease (Mpro) is a major protease having an important role in viral replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that caused the pandemic of 2020. Here, active Mpro was obtained as a 34.5 kDa protein by overexpression in E. coli BL21 (DE3). The optimal pH and temperature of Mpro were 7.5 and 37 °C, respectively. Mpro displayed a Km value of 16 µM with Dabcyl-KTSAVLQ↓SGFRKME-Edans. Black garlic extract and 49 polyphenols were studied for their inhibitory effects on purified Mpro. The IC50 values were 137 µg/mL for black garlic extract and 9-197 µM for 15 polyphenols. The mixtures of tannic acid with puerarin, daidzein, and/or myricetin enhanced the inhibitory effects on Mpro. The structure-activity relationship of these polyphenols revealed that the hydroxyl group in C3', C4', C5' in the B-ring, C3 in the C-ring, C7 in A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to inhibitory effects of flavonoids on Mpro.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Polyphenols/chemistry , Polyphenols/pharmacology , Protease Inhibitors/pharmacology , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Dimethyl Sulfoxide/pharmacology , Drug Synergism , Garlic/chemistry , Hydrogen-Ion Concentration , Plant Extracts/pharmacology , Plants/chemistry , Protease Inhibitors/chemistry , Structure-Activity Relationship , Temperature
18.
Biochem Biophys Res Commun ; 555: 147-153, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1157143

ABSTRACT

Several existing drugs are currently being tested worldwide to treat COVID-19 patients. Recent data indicate that SARS-CoV-2 is rapidly evolving into more transmissible variants. It is therefore highly possible that SARS-CoV-2 can accumulate adaptive mutations modulating drug susceptibility and hampering viral antigenicity. Thus, it is vital to predict potential non-synonymous mutation sites and predict the evolution of protein structural modifications leading to drug tolerance. As two FDA-approved anti-hepatitis C virus (HCV) drugs, boceprevir, and telaprevir, have been shown to effectively inhibit SARS-CoV-2 by targeting the main protease (Mpro), here we used a high-throughput interface-based protein design strategy to identify mutational hotspots and potential signatures of adaptation in these drug binding sites of Mpro. Several mutants exhibited reduced binding affinity to these drugs, out of which hotspot residues having a strong tendency to undergo positive selection were identified. The data further indicated that these anti-HCV drugs have larger footprints in the mutational landscape of Mpro and hence encompass the highest potential for positive selection and adaptation. These findings are crucial in understanding the potential structural modifications in the drug binding sites of Mpro and thus its signatures of adaptation. Furthermore, the data could provide systemic strategies for robust antiviral design and discovery against COVID-19 in the future.


Subject(s)
Adaptation, Physiological/genetics , Antiviral Agents/chemistry , Coronavirus 3C Proteases/chemistry , Drug Design , Drug Resistance, Viral/genetics , Mutation , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites/drug effects , Binding Sites/genetics , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Genetic Fitness/genetics , Hepacivirus/drug effects , Hepacivirus/enzymology , Ligands , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/pharmacology , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Selection, Genetic/genetics , Structure-Activity Relationship
19.
IUBMB Life ; 73(4): 670-675, 2021 04.
Article in English | MEDLINE | ID: covidwho-1144243

ABSTRACT

Mutations in the novel coronavirus SARS-CoV2 are the major concern as they might lead to drug/vaccine resistance. In the host cell, the virus largely depends on the main protease (Mpro ) to regulate infection hence it is one of the most attractive targets for inhibitor design. However, >19,000 mutations in the Mpro have already been reported. The mutations encompassing 282 amino acid positions and these "hotspots" might change the Mpro structure, activity and potentially delay therapeutic strategies targeting Mpro . Thus, here we identified 24 mutational "coldspots" where mutations have not been observed. We compared the structure-function relationship of these coldspots with several SARS-CoV2 Mpro X-ray crystal structures. We found that three coldspot residues (Leu141, Phe185, and Gln192) help to form the active site, while seven (Gly2, Arg4, Tyr126, Lys137, Leu141, Leu286, and Leu287) contribute to dimer formation that is required for Mpro activity. The surface of the dimer interface is more resistant to mutations compared to the active site. Interestingly, most of the coldspots are found in three clusters and forms conserved patterns when compared with other coronaviruses. Importantly, several conserved coldspots are available on the surface of the active site and at the dimer interface for targeting. The identification and short list of these coldspots offers a new perspective to target the SARS-CoV2 Mpro while avoiding mutation-based drug resistance.


Subject(s)
COVID-19/metabolism , Coronavirus 3C Proteases/genetics , Mutation , SARS-CoV-2/metabolism , Antiviral Agents/pharmacology , COVID-19/virology , Coronavirus 3C Proteases/chemistry , Crystallography, X-Ray , Humans , Protein Conformation , SARS-CoV-2/drug effects
20.
Molecules ; 26(6)2021 Mar 17.
Article in English | MEDLINE | ID: covidwho-1138745

ABSTRACT

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Indoles/chemistry , Indoles/pharmacology , Leucine/chemistry , Leucine/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Binding Sites , COVID-19/drug therapy , Computer Simulation , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Databases, Protein , Diarylquinolines/chemistry , Diarylquinolines/pharmacology , Dihydropyridines/chemistry , Dihydropyridines/pharmacology , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitrobenzenes/chemistry , Nitrobenzenes/pharmacology , Nitrophenols/chemistry , Nitrophenols/pharmacology , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics
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