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1.
Viruses ; 13(8)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1524167

ABSTRACT

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , SARS-CoV-2/drug effects , Tretinoin/pharmacology , Animals , Cell Line, Tumor , Chlorocebus aethiops , Cysteine Proteinase Inhibitors/pharmacology , DEAD Box Protein 58/metabolism , High-Throughput Screening Assays , Humans , Receptors, Immunologic/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Vero Cells , Virus Replication/drug effects
2.
Angew Chem Int Ed Engl ; 60(48): 25428-25435, 2021 11 22.
Article in English | MEDLINE | ID: covidwho-1490696

ABSTRACT

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/chemistry , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Drug Evaluation, Preclinical , Microbial Sensitivity Tests , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Multimerization , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Vero Cells
4.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1387141

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
5.
Molecules ; 26(16)2021 Aug 05.
Article in English | MEDLINE | ID: covidwho-1362396

ABSTRACT

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.


Subject(s)
Alkaloids/pharmacology , Cysteine Proteases/metabolism , Alkaloids/chemistry , Animals , Antiviral Agents/pharmacology , Binding Sites , COVID-19/drug therapy , COVID-19/metabolism , Catalytic Domain , Cathepsins/pharmacology , Cell Line, Tumor , Cysteine Proteases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Humans , Mice , Molecular Docking Simulation/methods , Nuphar/chemistry , Papain/pharmacology , Plant Extracts/pharmacology , Protein Binding , SARS-CoV-2/drug effects
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.
Science ; 373(6557): 931-936, 2021 08 20.
Article in English | MEDLINE | ID: covidwho-1319369

ABSTRACT

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus OC43, Human/drug effects , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Thiazoles/pharmacology , A549 Cells , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Benzamides , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus OC43, Human/physiology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , HEK293 Cells , Humans , Inhibitory Concentration 50 , Mice , Mice, Transgenic , Microbial Sensitivity Tests , Piperidines , Pyridines , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/therapeutic use , Viral Load/drug effects , Virus Replication/drug effects
8.
Commun Biol ; 4(1): 93, 2021 01 20.
Article in English | MEDLINE | ID: covidwho-1301188

ABSTRACT

Emerging outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is a major threat to public health. The morbidity is increasing due to lack of SARS-CoV-2 specific drugs. Herein, we have identified potential drugs that target the 3-chymotrypsin like protease (3CLpro), the main protease that is pivotal for the replication of SARS-CoV-2. Computational molecular modeling was used to screen 3987 FDA approved drugs, and 47 drugs were selected to study their inhibitory effects on SARS-CoV-2 specific 3CLpro enzyme in vitro. Our results indicate that boceprevir, ombitasvir, paritaprevir, tipranavir, ivermectin, and micafungin exhibited inhibitory effect towards 3CLpro enzymatic activity. The 100 ns molecular dynamics simulation studies showed that ivermectin may require homodimeric form of 3CLpro enzyme for its inhibitory activity. In summary, these molecules could be useful to develop highly specific therapeutically viable drugs to inhibit the SARS-CoV-2 replication either alone or in combination with drugs specific for other SARS-CoV-2 viral targets.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Drug Discovery , SARS-CoV-2/drug effects , Virus Replication/drug effects , Antiviral Agents/chemistry , COVID-19/virology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , SARS-CoV-2/enzymology , Structure-Activity Relationship
9.
Bioorg Med Chem Lett ; 47: 128202, 2021 09 01.
Article in English | MEDLINE | ID: covidwho-1272320

ABSTRACT

Cathepsin C plays a key role in the activation of several degradative enzymes linked to tissue destruction in chronic inflammatory and autoimmune diseases. Therefore, Cathepsin C inhibitors could potentially be effective therapeutics for the treatment of diseases such as chronic obstructive pulmonary disease (COPD) or acute respiratory distress syndrome (ARDS). In our efforts towards the development of a novel series of Cathepsin C inhibitors, we started working around AZD5248 (1), an α-amino acid based scaffold having potential liability of aortic binding. A novel series of amidoacetonitrile based Cathepsin C inhibitors were developed by the application of a conformational restriction strategy on 1. In particular, this work led to the development of a potent and selective Cathepsin C inhibitor 3p, free of aortic binding liability.


Subject(s)
Aorta/metabolism , COVID-19/drug therapy , Cathepsin C/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/pharmacology , Respiratory Distress Syndrome/drug therapy , Acetonitriles/chemistry , Acetonitriles/pharmacology , Amino Acids/chemistry , Amino Acids/pharmacology , Biphenyl Compounds/pharmacology , COVID-19/complications , Humans , Models, Molecular , Molecular Structure , Respiratory Distress Syndrome/etiology , Structure-Activity Relationship
10.
Eur J Med Chem ; 222: 113584, 2021 Oct 15.
Article in English | MEDLINE | ID: covidwho-1252810

ABSTRACT

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , Sulfonic Acids/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Humans , Micelles , Microbial Sensitivity Tests , Molecular Structure , Protein Binding , Pyrrolidines/chemical synthesis , Pyrrolidines/metabolism , SARS-CoV-2/enzymology , Solubility , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/metabolism , Vero Cells
11.
Chem Commun (Camb) ; 57(48): 5909-5912, 2021 Jun 15.
Article in English | MEDLINE | ID: covidwho-1233726

ABSTRACT

The SARS-CoV-2 main viral protease (Mpro) is an attractive target for antivirals given its distinctiveness from host proteases, essentiality in the viral life cycle and conservation across coronaviridae. We launched the COVID Moonshot initiative to rapidly develop patent-free antivirals with open science and open data. Here we report the use of machine learning for de novo design, coupled with synthesis route prediction, in our campaign. We discover novel chemical scaffolds active in biochemical and live virus assays, synthesized with model generated routes.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/enzymology , Antiviral Agents/chemical synthesis , Coronavirus OC43, Human/drug effects , Cysteine Proteinase Inhibitors/chemical synthesis , Drug Design , Drug Discovery/methods , Machine Learning , Microbial Sensitivity Tests
12.
J Virol ; 95(14): e0237420, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1207480

ABSTRACT

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.


Subject(s)
COVID-19/enzymology , Coronavirus 3C Proteases , Cysteine Proteinase Inhibitors , SARS-CoV-2/enzymology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , HEK293 Cells , Humans
13.
Curr Opin Virol ; 49: 36-40, 2021 08.
Article in English | MEDLINE | ID: covidwho-1201247

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. The coronavirus 3-chymotrypsin-like protease (3CLpro) controls virus replication and is therefore considered a major target and promising opportunity for rational-based antiviral discovery with direct acting agents. Here we review first-generation SARS-CoV-2 3CLpro inhibitors PF-07304814, GC-376, and CDI-45205 that are being delivered either by injection or inhalation due to their low intrinsic oral bioavailability. In addition, PF-07321332 is now emerging as a promising second-generation clinical candidate for oral delivery. A key challenge to the development of novel 3CLpro inhibitors is the poor understanding of the predictive value of in vitro potency toward clinical efficacy, an issue complicated by the involvement of host proteases in virus entry. Further preclinical and clinical validation will be key to establishing 3CLpro inhibitors as a bona fide class for future SARS-CoV-2 therapeutics for both hospitalized and outpatient populations.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/therapeutic use , Drug Administration Routes , Drug Development , Drug Discovery , Humans , SARS-CoV-2/enzymology
14.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1160225

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
15.
Biochem Biophys Res Commun ; 555: 134-139, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1157141

ABSTRACT

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.


Subject(s)
Amides/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Disease Models, Animal , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Amides/pharmacokinetics , Animals , COVID-19/virology , Cathepsin L/antagonists & inhibitors , Cell Line , Cricetinae , Cysteine Proteinase Inhibitors/pharmacokinetics , Female , Humans , Inhibitory Concentration 50 , Male , Mesocricetus/virology , Reproducibility of Results , SARS-CoV-2/growth & development , Serine Endopeptidases , Substrate Specificity , Virus Replication/drug effects
16.
Signal Transduct Target Ther ; 6(1): 134, 2021 03 27.
Article in English | MEDLINE | ID: covidwho-1152831

ABSTRACT

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/metabolism , Cathepsin L , Cysteine Proteinase Inhibitors/pharmacology , Drug Development , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Adolescent , Adult , Aged , Animals , COVID-19/drug therapy , COVID-19/genetics , Cathepsin L/antagonists & inhibitors , Cathepsin L/genetics , Cathepsin L/metabolism , Female , Humans , Male , Mice , Mice, Transgenic , Middle Aged , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
17.
Biochem Biophys Res Commun ; 538: 72-79, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1139451

ABSTRACT

SARS-CoV-2 papain-like protease is considered as an important potential target for anti-SARS-CoV-2 drug discovery due to its crucial roles in viral spread and innate immunity. Here, we have utilized an in silico molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 papain-like protease, by screening 21 antiviral, antifungal and anticancer compounds. Among them, Neobavaisoflavone has the highest binding energy for SARS-CoV-2 papain-like protease. These molecules could bind near the SARS-CoV-2 papain-like protease crucial catalytic triad, ubiquitination and ISGylation residues: Trp106, Asn109, Cys111, Met208, Lys232, Pro247, Tyr268, Gln269, His272, Asp286 and Thr301. Because blocking the papain-like protease is an important strategy in fighting against viruses, these compounds might be promising candidates for therapeutic intervention against COVID-19.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Coronavirus Protease Inhibitors/chemistry , Cysteine Proteinase Inhibitors/chemistry , Drug Discovery/methods , Isoflavones/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Humans , Isoflavones/pharmacology , Ligands , Molecular Docking Simulation , Protein Binding
18.
ChemMedChem ; 16(2): 340-354, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-1044678

ABSTRACT

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PLpro ) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PLpro . Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PLpro are valuable starting points for the development of new pan-coronaviral inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Isoindoles/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Benzamides/chemical synthesis , Benzamides/metabolism , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Isoindoles/chemical synthesis , Isoindoles/metabolism , Molecular Docking Simulation , Molecular Structure , Protein Binding , Structure-Activity Relationship , Vero Cells , Virus Replication/drug effects
19.
J Enzyme Inhib Med Chem ; 36(1): 147-153, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1024061

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19). Since its emergence, the COVID-19 pandemic has not only distressed medical services but also caused economic upheavals, marking urgent the need for effective therapeutics. The experience of combating SARS-CoV and MERS-CoV has shown that inhibiting the 3-chymotrypsin-like protease (3CLpro) blocks the replication of the virus. Given the well-studied properties of FDA-approved drugs, identification of SARS-CoV-2 3CLpro inhibitors in an FDA-approved drug library would be of great therapeutic value. Here, we screened a library consisting of 774 FDA-approved drugs for potent SARS-CoV-2 3CLpro inhibitors, using an intramolecularly quenched fluorescence (IQF) peptide substrate. Ethacrynic acid, naproxen, allopurinol, butenafine hydrochloride, raloxifene hydrochloride, tranylcypromine hydrochloride, and saquinavir mesylate have been found to block the proteolytic activity of SARS-CoV-2 3CLpro. The inhibitory activity of these repurposing drugs against SARS-CoV-2 3CLpro highlights their therapeutic potential for treating COVID-19 and other Betacoronavirus infections.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Drug Repositioning , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Drug Evaluation, Preclinical , Fluorescent Dyes , Humans , Molecular Docking Simulation , Substrate Specificity
20.
ChemMedChem ; 16(6): 942-948, 2021 03 18.
Article in English | MEDLINE | ID: covidwho-959133

ABSTRACT

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Alanine/analogs & derivatives , Alanine/metabolism , Alanine/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Cysteine/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Humans , Microbial Sensitivity Tests , Protein Binding , Pyrrolidinones/chemical synthesis , Pyrrolidinones/metabolism , Pyrrolidinones/pharmacology , SARS-CoV-2/enzymology , Vero Cells
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