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1.
J Virol ; 96(8): e0201321, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1779314

ABSTRACT

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.


Subject(s)
Lactams , Leucine , Nitriles , Peptide Hydrolases , Proline , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/drug therapy , Humans , Lactams/chemistry , Lactams/metabolism , Leucine/chemistry , Leucine/metabolism , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/enzymology , Nitriles/chemistry , Nitriles/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Proline/chemistry , Proline/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , SARS Virus/chemistry , SARS Virus/enzymology , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology
2.
J Virol ; 96(8): e0003722, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1779311

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an enormous threat to economic activity and public health worldwide. Previous studies have shown that the nonstructural protein 5 (nsp5, also called 3C-like protease) of alpha- and deltacoronaviruses cleaves Q231 of the NF-κB essential modulator (NEMO), a key kinase in the RIG-I-like receptor pathway, to inhibit type I interferon (IFN) production. In this study, we found that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleaved NEMO at multiple sites (E152, Q205, and Q231). Notably, SARS-CoV-2 nsp5 exhibited a stronger ability to cleave NEMO than SARS-CoV nsp5. Sequence and structural alignments suggested that an S/A polymorphism at position 46 of nsp5 in SARS-CoV versus SARS-CoV-2 may be responsible for this difference. Mutagenesis experiments showed that SARS-CoV-2 nsp5 (S46A) exhibited poorer cleavage of NEMO than SARS-CoV-2 nsp5 wild type (WT), while SARS-CoV nsp5 (A46S) showed enhanced NEMO cleavage compared with the WT protein. Purified recombinant SARS-CoV-2 nsp5 WT and SARS-CoV nsp5 (A46S) proteins exhibited higher hydrolysis efficiencies than SARS-CoV-2 nsp5 (S46A) and SARS-CoV nsp5 WT proteins in vitro. Furthermore, SARS-CoV-2 nsp5 exhibited stronger inhibition of Sendai virus (SEV)-induced interferon beta (IFN-ß) production than SARS-CoV-2 nsp5 (S46A), while introduction of the A46S substitution in SARS-CoV nsp5 enhanced suppression of SEV-induced IFN-ß production. Taken together, these data show that S46 is associated with the catalytic activity and IFN antagonism by SARS-CoV-2 nsp5. IMPORTANCE The nsp5-encoded 3C-like protease is the main coronavirus protease, playing a vital role in viral replication and immune evasion by cleaving viral polyproteins and host immune-related molecules. We showed that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleave the NEMO at multiple sites (E152, Q205, and Q231). This specificity differs from NEMO cleavage by alpha- and deltacoronaviruses, demonstrating the distinct substrate recognition of SARS-CoV-2 and SARS-CoV nsp5. Compared with SARS-CoV nsp5, SARS-CoV-2 nsp5 encodes S instead of A at position 46. This substitution is associated with stronger catalytic activity, enhanced cleavage of NEMO, and increased interferon antagonism of SARS-CoV-2 nsp5. These data provide new insights into the pathogenesis and transmission of SARS-CoV-2.


Subject(s)
Coronavirus 3C Proteases , Interferon Type I , SARS Virus , SARS-CoV-2 , Antiviral Agents , COVID-19/immunology , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Humans , Immune Evasion/genetics , Interferon Type I/antagonists & inhibitors , Interferon Type I/metabolism , SARS Virus/enzymology , SARS Virus/genetics , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Virus Replication/genetics
3.
Int J Biol Macromol ; 193(Pt B): 2113-2120, 2021 Dec 15.
Article in English | MEDLINE | ID: covidwho-1509846

ABSTRACT

Three dimensional structures of (chymo)trypsin-like proteinase (3CLpro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val86Leu, Lys88Arg, Phe134His, and Asn180Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CLpro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val35Thr, Ser46Ala, Asn65Ser, Ala94Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CLpro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CLpro(s) can use for functional regulation an autolytic loop and the unique Asp33-Asn95 region (the Asp33-Asn95 Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CLpro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.


Subject(s)
Coronavirus 3C Proteases , Mutation, Missense , SARS Virus , SARS-CoV-2 , Amino Acid Substitution , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Humans , Protein Domains , SARS Virus/enzymology , SARS Virus/genetics , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Species Specificity , Structure-Activity Relationship
4.
Nat Commun ; 12(1): 6055, 2021 10 18.
Article in English | MEDLINE | ID: covidwho-1475294

ABSTRACT

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/administration & dosage , Indoles/administration & dosage , Leucine/administration & dosage , Pyrrolidinones/administration & dosage , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/adverse effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacokinetics , Alanine/administration & dosage , Alanine/adverse effects , Alanine/analogs & derivatives , Alanine/pharmacokinetics , Animals , COVID-19/virology , Chlorocebus aethiops , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/enzymology , Coronavirus Protease Inhibitors/adverse effects , Coronavirus Protease Inhibitors/pharmacokinetics , Disease Models, Animal , Drug Design , Drug Synergism , Drug Therapy, Combination , HeLa Cells , Humans , Indoles/adverse effects , Indoles/pharmacokinetics , Infusions, Intravenous , Leucine/adverse effects , Leucine/pharmacokinetics , Mice , Pyrrolidinones/adverse effects , Pyrrolidinones/pharmacokinetics , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Vero Cells
5.
Proteomics ; 21(2): e2000246, 2021 01.
Article in English | MEDLINE | ID: covidwho-1384281

ABSTRACT

The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PLpro ) protease and the so-called main protease (Mpro ), a chymotrypsin-like cysteine protease, also named 3CLpro or non-structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 Mpro , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/metabolism , Coronavirus NL63, Human/enzymology , Peptide Fragments/analysis , SARS Virus/enzymology , SARS-CoV-2/enzymology , Viral Proteins/metabolism , COVID-19/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/virology , Epithelial Cells/metabolism , Epithelial Cells/virology , Eukaryotic Initiation Factor-4G/metabolism , Host-Pathogen Interactions , Humans , Lung/metabolism , Lung/virology , Substrate Specificity
6.
Bioorg Med Chem ; 46: 116301, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1333256

ABSTRACT

Severe Acute Respiratory Syndrome (SARS) is a severe febrile respiratory disease caused by the beta genus of human coronavirus, known as SARS-CoV. Last year, 2019-n-CoV (COVID-19) was a global threat for everyone caused by the outbreak of SARS-CoV-2. 3CLpro, chymotrypsin-like protease, is a major cysteine protease that substantially contributes throughout the viral life cycle of SARS-CoV and SARS-CoV-2. It is a prospective target for the development of SARS-CoV inhibitors by applying a repurposing strategy. This review focuses on a detailed overview of the chemical synthesis and computational chemistry perspectives of peptidomimetic inhibitors (PIs) and small-molecule inhibitors (SMIs) targeting viral proteinase discovered from 2004 to 2020. The PIs and SMIs are one of the primary therapeutic inventions for SARS-CoV. The journey of different analogues towards the evolution of SARS-CoV 3CLpro inhibitors and complete synthetic preparation of nineteen derivatives of PIs and ten derivatives of SMIs and their computational chemistry perspectives were reviewed. From each class of derivatives, we have identified and highlighted the most compelling PIs and SMIs for SARS-CoV 3CLpro. The protein-ligand interaction of 29 inhibitors were also studied that involved with the 3CLpro inhibition, and the frequent amino acid residues of the protease were also analyzed that are responsible for the interactions with the inhibitors. This work will provide an initiative to encourage further research for the development of effective and drug-like 3CLpro inhibitors against coronaviruses in the near future.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Peptidomimetics/pharmacology , SARS Virus/drug effects , Animals , Antiviral Agents/chemical synthesis , Cell Line, Tumor , Cysteine Proteinase Inhibitors/chemical synthesis , Humans , Peptidomimetics/chemical synthesis , SARS Virus/enzymology , SARS-CoV-2/enzymology
7.
Proteins ; 89(9): 1216-1225, 2021 09.
Article in English | MEDLINE | ID: covidwho-1293319

ABSTRACT

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.


Subject(s)
Coronavirus 3C Proteases/chemistry , SARS Virus/enzymology , SARS-CoV-2/enzymology , Apoenzymes/antagonists & inhibitors , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Models, Molecular , Protease Inhibitors/pharmacology , Protein Multimerization/drug effects , Protein Structure, Quaternary/drug effects
8.
J Med Virol ; 93(3): 1581-1588, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196480

ABSTRACT

The papain-like protease (PLpro ) is an important enzyme for coronavirus polyprotein processing, as well as for virus-host immune suppression. Previous studies reveal that a molecular analysis of PLpro indicates the catalytic activity of viral PLpro and its interactions with ubiquitin. By using sequence comparisons, molecular models, and protein-protein interaction maps, PLpro was compared in the three recorded fatal CoV epidemics, which involved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome CoV (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). The pairwise sequence comparison of SARS-CoV-2 PLpro indicated similarity percentages of 82.59% and 30.06% with SARS-CoV PLpro and MERS-CoV PLpro , respectively. In comparison with SARS-CoV PLpro , in SARS-CoV-2, the PLpro had a conserved catalytic triad of C111, H278, and D293, with a slightly lower number of polar interface residues and of hydrogen bonds, a higher number of buried interface sizes, and a lower number of residues that interact with ubiquitin and PLpro . These features might contribute to a similar or slightly lower level of deubiquitinating activity in SARS-CoV-2 PLpro. It was, however, a much higher level compared to MERS-CoV, which contained amino acid mutations and a low number of polar interfaces. SARS-CoV-2 PLpro and SARS-CoV PLpro showed almost the same catalytic site profiles, interface area compositions and polarities, suggesting a general similarity in deubiquitination activity. Compared with MERS-CoV, SARS-CoV-2 had a higher potential for binding interactions with ubiquitin. These estimated parameters contribute to the knowledge gap in understanding how the new virus interacts with the immune system.


Subject(s)
COVID-19/pathology , Coronavirus Papain-Like Proteases/metabolism , Middle East Respiratory Syndrome Coronavirus/enzymology , SARS Virus/enzymology , SARS-CoV-2/enzymology , Amino Acid Sequence , Catalytic Domain/physiology , Humans , Models, Molecular , Polyproteins/biosynthesis , Polyproteins/genetics , Sequence Alignment , Severe Acute Respiratory Syndrome/pathology , Ubiquitin/metabolism , Viral Proteins/biosynthesis , Viral Proteins/genetics
9.
Mini Rev Med Chem ; 21(20): 3191-3202, 2021.
Article in English | MEDLINE | ID: covidwho-1105938

ABSTRACT

Corona Virus Disease-2019 (COVID-19), caused by the SARS CoV-2 virus, has been announced as a pandemic by the World Health Organization. COVID-19 has affected people globally, infecting more than 39.8 million people and claiming up to 1.11 million lives, yet there is no effective treatment strategy to cure this disease. As vaccine development is a time-consuming process, currently, efforts are being made to develop alternative plans for the timely and effective management of this disease. Drug repurposing always fascinated researchers and can be utilized as the most acceptable alternative to develop the therapeutics for COVID-19 using the pre-approved drugs. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has shown resemblance with distinctive enzyme targets, such as 3CLpro/Mpro, RdRp, Cathepsin L, and TMPRSS2 present in SARS CoV and MERS CoV. Therefore, the drugs that have shown efficacy in these viruses can also be used for the treatment of COVID-19. This review focuses on why repurposing could provide a better alternative in COVID- 19 treatment. The similarity in the structure and progression of infection of SARS CoV and MERS viruses offers a direction and validation to evaluate the drugs approved for SARS and MERS against COVID-19. It has been indicated that multiple therapeutic options that demonstrate efficacy against SARS CoV 2 are available to mitigate the potential emergence of COVID-19 infection.


Subject(s)
Antiviral Agents , COVID-19/drug therapy , Coronavirus Infections/drug therapy , Drug Repositioning , Severe Acute Respiratory Syndrome/drug therapy , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/enzymology , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
10.
Biochem Biophys Res Commun ; 533(3): 467-473, 2020 Dec 10.
Article in English | MEDLINE | ID: covidwho-1064868

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested.


Subject(s)
Cysteine Endopeptidases/metabolism , Diterpenes/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Betacoronavirus/enzymology , Catalytic Domain , Coronavirus 3C Proteases , Cysteine Endopeptidases/chemistry , Diterpenes/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Molecular Docking Simulation , Protein Conformation , Protein Multimerization , SARS Virus/enzymology , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry
11.
Viruses ; 13(2)2021 01 25.
Article in English | MEDLINE | ID: covidwho-1045365

ABSTRACT

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (Mpro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 Mpro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 Mpro at 2.5 µM, which is more potent than against SAR-CoV-1 Mpro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 Mpro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.


Subject(s)
Antiviral Agents/chemistry , Coronavirus 3C Proteases/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Amino Acid Sequence , Antiviral Agents/metabolism , Catalytic Domain , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Drug Discovery , Inhibitory Concentration 50 , Models, Molecular , Protease Inhibitors/metabolism , Protein Binding , SARS Virus/enzymology
12.
Biochimie ; 182: 177-184, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1039291

ABSTRACT

The main protease (Mpro) of SARS-CoV and SARS-CoV-2 is a key enzyme in viral replication and a promising target for the development of antiviral therapeutics. The understanding of this protein is based on a number of observations derived from earlier x-ray structures, which mostly consider substrates or ligands as the main reason behind modulation of the active site. This lead to the concept of substrate-induced subsite cooperativity as an initial attempt to explain the dual binding specificity of this enzyme in recognizing the cleavage sequences at its N- and C-termini, which are important processing steps in obtaining the mature protease. The presented hypothesis proposes that structural heterogeneity is a property of the enzyme, independent of the presence of a substrate or ligand. Indeed, the analysis of Mpro structures of SARS-CoV and SARS-CoV-2 reveals a conformational diversity for the catalytically competent state in ligand-free structures. Variation in the binding site appears to result from flexibility at residues lining the S1 subpocket and segments incorporating methionine 49 and glutamine 189. The structural evidence introduces "structure-based recognition" as a new paradigm in substrate proteolysis by Mpro. In this concept, the binding space in subpockets of the enzyme varies in a non-cooperative manner, causing distinct conformations, which recognize and process different cleavage sites, as the N- and C-termini. Insights into the recognition basis of the protease provide explanation to the ordered processing of cleavage sites. The hypothesis expands the conformational space of the enzyme and consequently opportunities for drug development and repurposing efforts.


Subject(s)
COVID-19/virology , Protein Conformation , SARS Virus/enzymology , SARS-CoV-2/enzymology , Viral Proteases/chemistry , Viral Proteases/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Catalytic Domain , Drug Design , Humans , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Substrate Specificity
13.
Sci Rep ; 10(1): 20808, 2020 11 30.
Article in English | MEDLINE | ID: covidwho-951433

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands' interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50 µM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Drug Evaluation, Preclinical , Protease Inhibitors/pharmacology , Amino Acid Sequence , Catalytic Domain/drug effects , Coronavirus 3C Proteases/metabolism , Drug Design , Drug Discovery , Humans , Models, Molecular , Molecular Docking Simulation , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
14.
Perm J ; 242020.
Article in English | MEDLINE | ID: covidwho-931342

ABSTRACT

Furin is a protease that is ubiquitous in mammalian metabolism. One of the innovations that make sudden acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) more infectious than its ancestor viruses is the addition of a furin cleavage site. Conditions associated with elevated furin levels, including diabetes, obesity, and hypertension, overlap greatly with vulnerability to the severe form of coronavirus disease 2019 (COVID-19). We suggest that diet and lifestyle modifications that reduce the associated comorbidities may prevent the development of severe COVID-19 by, in part, lowering circulating furin levels. Likewise, natural and pharmaceutical inhibitors of furin may be candidate prophylactic interventions or, if used early in the COVID-19, may prevent the development of critical symptoms.


Subject(s)
Anthrax/blood , COVID-19/blood , Diabetes Mellitus/blood , Furin/blood , Hypertension/blood , SARS Virus/metabolism , Anthrax/enzymology , COVID-19/enzymology , Diabetes Mellitus/enzymology , Humans , Hypertension/enzymology , SARS Virus/enzymology
15.
Nat Chem Biol ; 17(2): 222-228, 2021 02.
Article in English | MEDLINE | ID: covidwho-899948

ABSTRACT

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.


Subject(s)
Antiviral Agents/chemistry , COVID-19/diagnostic imaging , Coronavirus 3C Proteases/antagonists & inhibitors , Epithelial Cells/virology , Protease Inhibitors/chemistry , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Combinatorial Chemistry Techniques , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Drug Design , Epithelial Cells/ultrastructure , Fluorescent Dyes/chemistry , Gene Expression , Glutamine/chemistry , Humans , Models, Molecular , Nasopharynx/virology , Protease Inhibitors/pharmacology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2/enzymology , Substrate Specificity
16.
Bioorg Med Chem ; 28(4): 115273, 2020 02 15.
Article in English | MEDLINE | ID: covidwho-833276

ABSTRACT

An octahydroisochromene scaffold has been introduced into a known SARS 3CL protease inhibitor as a novel hydrophobic core to interact with the S2 pocket of the protease. An alkyl or aryl substituent was also introduced at the 1-position of the octahydroisochromene scaffold and expected to introduce additional interactions with the protease. Sharpless-Katsuki asymmetric epoxidation and Sharpless asymmetric dihydroxylation were employed to construct the octahydroisochromene scaffold. The introductions of the P1 site His-al and the substituent at 1-position was achieved using successive reductive amination reactions. Our initial evaluations of the diastereo-isomeric mixtures (16a-d) revealed that the octahydroisochromene moiety functions as a core hydrophobic scaffold for the S2 pocket of the protease and the substituent at the 1-position may form additional interactions with the protease. The inhibitory activities of the diastereoisomerically-pure inhibitors (3a-d) strongly suggest that a specific stereo-isomer of the octahydroisochromene scaffold, (1S, 3S) 3b, directs the P1 site imidazole, the warhead aldehyde, and substituent at the 1-position of the fused ring to their appropriate pockets in the protease.


Subject(s)
Benzopyrans/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS Virus/enzymology , Benzopyrans/chemical synthesis , Benzopyrans/chemistry , Coronavirus 3C Proteases/metabolism , Dose-Response Relationship, Drug , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity Relationship
17.
J Enzyme Inhib Med Chem ; 35(1): 145-151, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-826697

ABSTRACT

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3' sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , SARS Virus/drug effects , Viral Proteins/antagonists & inhibitors , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Coronavirus 3C Proteases , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Flavonoids/chemical synthesis , Flavonoids/chemistry , Humans , Molecular Structure , SARS Virus/enzymology , Structure-Activity Relationship , Viral Proteins/isolation & purification , Viral Proteins/metabolism
18.
Nat Commun ; 11(1): 4282, 2020 08 27.
Article in English | MEDLINE | ID: covidwho-733525

ABSTRACT

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus, Feline/drug effects , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , A549 Cells , Animals , Antiviral Agents/chemistry , Betacoronavirus/enzymology , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus, Feline/enzymology , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cytopathogenic Effect, Viral/drug effects , Drug Repositioning , Humans , Inhibitory Concentration 50 , Molecular Structure , Prodrugs , Protease Inhibitors/chemistry , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2 , Sulfonic Acids , Vero Cells , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects
19.
Med Chem ; 17(4): 380-395, 2021.
Article in English | MEDLINE | ID: covidwho-688767

ABSTRACT

BACKGROUND: Globally, over 4.3 million laboratory confirmed cases of COVID-19 have been reported from over 105 countries. No FDA approved antiviral is available for the treatment of this infection. Zhavoronkov et al., with their generative chemistry pipeline, have generated structures that can be potential novel drug-like inhibitors for COVID-19, provided they are validated. 3C-like protease (3CLP) is a homodimeric cysteine protease that is present in coronaviruses. Interestingly, 3CLP is 96.1% structurally similar between SARS-CoV and SARS-CoV-2. OBJECTIVE: To evaluate interaction of generated structures with 3CLP of SARS-CoV (RCSB PDB ID: 4MDS). METHODS: Crystal structure of human SARS-CoV with a non-covalent inhibitor with resolution: 1.598 Å was obtained and molecular docking was performed to evaluate the interaction with generated structures. The MM-GBSA and IFD-SP were performed to narrow down to the structures with better binding energy and IFD score. The ADME analysis was performed on top 5 hits and further MD simulation was employed for top 2 hits. RESULTS: In XP docking, IFD-SP and molecular dynamic simulation studies, the top 2 hits 32 and 61 showed interaction with key amino acid residue GLU166. Structure 61, also showed interaction with HIS164. These interactions of generated structure 32 and 61, with GLU166 and HIS164, indicate the binding of the selected drug within the close proximity of 3CLP. In the MD simulation, the protein- ligand complex of 4MDS and structure 61 was found to be more stable for 10ns. CONCLUSION: These identified structures can be further assessed for their antiviral activity to combat SARS-CoV and COVID-19.


Subject(s)
Antiviral Agents/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Antiviral Agents/metabolism , COVID-19/drug therapy , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Drug Discovery , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , SARS Virus/chemistry , SARS Virus/enzymology , SARS-CoV-2/enzymology , Small Molecule Libraries/metabolism , Structural Homology, Protein , Structure-Activity Relationship , Substrate Specificity , Thermodynamics , User-Computer Interface
20.
J Struct Biol ; 211(3): 107575, 2020 09 01.
Article in English | MEDLINE | ID: covidwho-640251

ABSTRACT

COVID-19 is a respiratory disease caused by the coronavirus SARS-CoV-2. SARS-CoV-2 has many similarities with SARS-CoV. Both viruses rely on a protease called the main protease, or Mpro, for replication. Therefore, inhibiting Mpro may be a successful strategy for treating COVID-19. Structures of the main proteases of SARS-CoV and SARS-CoV-2 with and without inhibitor N3 are available in the Protein Data Bank. Comparing these structures revealed residue interaction network changes associated with N3 inhibition. Comparing network clustering with and without inhibitor N3 identified the formation of a cluster of residues 17, 18, 30-33, 70, 95, 98, 103, 117, 122, and 177 as a network change in both viral proteases when bound to inhibitor N3. Betweenness and stress centrality differences as well as differences in bond energies and relative B-factors when comparing free Mpro to inhibitor-bound Mpro identified residues 131, 175, 182, and 185 as possibly conformationally relevant when bound to the inhibitor N3. Taken together, these results provide insight into conformational changes of betacoronavirus Mpros when bound to an inhibitor.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/enzymology , Enzyme Inhibitors/pharmacology , SARS Virus/enzymology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , COVID-19 , Catalytic Domain , Cluster Analysis , Coronavirus 3C Proteases , Coronavirus Infections/drug therapy , Cysteine Endopeptidases , Drug Design , Humans , Pandemics , Pneumonia, Viral/drug therapy , Polyproteins , Protein Binding , SARS-CoV-2
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