Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 27
Filter
1.
Commun Biol ; 5(1): 160, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1721596

ABSTRACT

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


Subject(s)
Coronavirus 3C Proteases/metabolism , Catalysis , Catalytic Domain , Circular Dichroism , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Models, Molecular , Mutation , Pyrrolidines/chemistry , Sulfonic Acids/chemistry , Thermodynamics
2.
Arch Virol ; 167(4): 1125-1130, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1694546

ABSTRACT

Given the structural similarities of the viral enzymes of different coronaviruses (CoVs), we investigated the potency of the anti-SARS-CoV-2 agents boceprevir and GC376 for counteracting seasonal coronavirus infections. In contrast to previous findings that both boceprevir and GC376 are potent inhibitors of the main protease (Mpro) of SARS-CoV-2, we found that GC376 is much more effective than boceprevir in inhibiting SARS-CoV-2 and three seasonal CoVs (NL63, 229E, and OC43) in cell culture models. However, these results are discordant with a molecular docking analysis that suggested comparable affinity of boceprevir and GC376 for the different Mpro enzymes of the four CoVs. Collectively, our results support future development of GC376 but not boceprevir (although it is an FDA-approved antiviral medication) as a pan-coronavirus antiviral agent. Furthermore, we caution against overinterpretation of in silico data when developing antiviral therapies.


Subject(s)
Antiviral Agents , COVID-19 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Molecular Docking Simulation , Proline/analogs & derivatives , Protease Inhibitors/pharmacology , Pyrrolidines , SARS-CoV-2 , Sulfonic Acids
3.
J Chem Inf Model ; 61(12): 6053-6065, 2021 12 27.
Article in English | MEDLINE | ID: covidwho-1541114

ABSTRACT

The main protease (Mpro) is a key enzyme responsible for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication that causes the spread of the global pandemic novel coronavirus (nCOVID-19) infection. In the present study, multiple computational approaches such as docking, long-range molecular dynamics (MD) simulations, and binding free-energy (BFE) estimation techniques were employed to investigate the mechanistic basis of the high-affinity inhibitors─GC-376, Calpain XII, and Calpain II (hereafter Calpain as Cal) from the literature─binding to Mpro. Redocking GC-376 and docking Cal XII and Cal II inhibitors to Mpro were able to reproduce all crucial interactions like the X-ray conformation. Subsequently, the apo (ligand-free) and three holo (ligand-bound) complexes were subjected to extensive MD simulations, which revealed that the ligand binding did not alter the overall Mpro structural features, whereas the heatmap analysis showed that the residues located in subsites S1 and S2, the catalytic dyad, and the 45TSEDMLN51 loop in Mpro exhibit a conformational deviation. Moreover, the BFE estimation method was used to elucidate the crucial thermodynamic properties, which revealed that Coulomb, solvation surface accessibility (Solv_SA), and lipophilic components contributed significant energies for complex formation. The decomposition of the total BFE to per-residue showed that H41, H163, M165, Q166, and Q189 residues contributed maximum energies. The overall results from the current investigation might be valuable for designing novel anti-Mpro inhibitors.


Subject(s)
COVID-19 , Protease Inhibitors , Carbonates , Coronavirus 3C Proteases , Humans , Leucine , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/pharmacology , SARS-CoV-2 , Sulfonic Acids
4.
Int J Mol Sci ; 22(20)2021 Oct 12.
Article in English | MEDLINE | ID: covidwho-1480792

ABSTRACT

The infection of mammalian cells by enveloped viruses is triggered by the interaction of viral envelope glycoproteins with the glycosaminoglycan, heparan sulfate. By mimicking this carbohydrate, some anionic polysaccharides can block this interaction and inhibit viral entry and infection. As heparan sulfate carries both carboxyl and sulfate groups, this work focused on the derivatization of a (1→3)(1→6)-ß-D-glucan, botryosphaeran, with these negatively-charged groups in an attempt to improve its antiviral activity. Carboxyl and sulfonate groups were introduced by carboxymethylation and sulfonylation reactions, respectively. Three derivatives with the same degree of carboxymethylation (0.9) and different degrees of sulfonation (0.1; 0.2; 0.4) were obtained. All derivatives were chemically characterized and evaluated for their antiviral activity against herpes (HSV-1, strains KOS and AR) and dengue (DENV-2) viruses. Carboxymethylated botryosphaeran did not inhibit the viruses, while all sulfonated-carboxymethylated derivatives were able to inhibit HSV-1. DENV-2 was inhibited only by one of these derivatives with an intermediate degree of sulfonation (0.2), demonstrating that the dengue virus is more resistant to anionic ß-D-glucans than the Herpes simplex virus. By comparison with a previous study on the antiviral activity of sulfonated botryosphaerans, we conclude that the presence of carboxymethyl groups might have a detrimental effect on antiviral activity.


Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/drug effects , Herpesviridae/drug effects , Sulfonic Acids/chemistry , beta-Glucans/chemistry , Animals , Antiviral Agents/chemistry , Cell Survival/drug effects , Chlorocebus aethiops , Dengue Virus/physiology , Glucans/chemistry , Glucans/pharmacology , Herpesviridae/physiology , Methylation , Vero Cells , Virus Internalization/drug effects , beta-Glucans/pharmacology
5.
Antiviral Res ; 195: 105183, 2021 11.
Article in English | MEDLINE | ID: covidwho-1458592

ABSTRACT

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system.


Subject(s)
Biosensing Techniques , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , Pyrrolidines/pharmacology , SARS-CoV-2/enzymology , Sulfonic Acids/pharmacology , Drug Evaluation, Preclinical , Fluorescence , HEK293 Cells , Humans
6.
Virology ; 564: 33-38, 2021 12.
Article in English | MEDLINE | ID: covidwho-1447220

ABSTRACT

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight that the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.


Subject(s)
Coronavirus 229E, Human/genetics , Coronavirus Infections/drug therapy , Coronavirus NL63, Human/genetics , Coronavirus OC43, Human/genetics , Cytidine/analogs & derivatives , Hydroxylamines/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Common Cold/drug therapy , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/physiology , Coronavirus NL63, Human/drug effects , Coronavirus NL63, Human/physiology , Coronavirus OC43, Human/drug effects , Coronavirus OC43, Human/physiology , Cytidine/pharmacology , Humans , Molecular Docking Simulation , Protein Binding/drug effects , Pyrrolidines/pharmacology , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Seasons , Sulfonic Acids/pharmacology , Virus Replication/drug effects , Virus Replication/genetics
7.
JCI Insight ; 6(20)2021 10 22.
Article in English | MEDLINE | ID: covidwho-1403154

ABSTRACT

Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2/angiopoietin axis. Primary HUVECs treated with plasma from patients with severe COVID-19 upregulated the expression of thromboinflammatory genes, inhibited the expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from patients with COVID-19 demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity, and the highest levels were associated with worse survival. These data highlight the disruption of Tie2/angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.


Subject(s)
COVID-19/drug therapy , Endothelial Cells/drug effects , Protective Agents/pharmacology , Receptor, TIE-2/metabolism , Adult , Aged , Aged, 80 and over , Angiopoietin-2/metabolism , Aniline Compounds , Female , Gene Expression , Humans , Lung , Male , Middle Aged , Receptor, TIE-2/genetics , SARS-CoV-2 , Signal Transduction , Sulfonic Acids , Vascular Diseases/metabolism , Young Adult
8.
ACS Appl Mater Interfaces ; 13(36): 43696-43707, 2021 Sep 15.
Article in English | MEDLINE | ID: covidwho-1392772

ABSTRACT

Graphene is a two-dimensional semiconducting material whose application for diagnostics has been a real game-changer in terms of sensitivity and response time, variables of paramount importance to stop the COVID-19 spreading. Nevertheless, strategies for the modification of docking recognition and antifouling elements to obtain covalent-like stability without the disruption of the graphene band structure are still needed. In this work, we conducted surface engineering of graphene through heterofunctional supramolecular-covalent scaffolds based on vinylsulfonated-polyamines (PA-VS). In these scaffolds, one side binds graphene through multivalent π-π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding. The construction of PA-VS scaffolds was demonstrated by spectroscopic ellipsometry, Raman spectroscopy, and contact angle measurements. The covalent binding of -SH, -NH2, or -OH groups was confirmed, and it evidenced great chemical versatility. After field-effect studies, we found that the PA-VS-based scaffolds do not disrupt the semiconducting properties of graphene. Moreover, the scaffolds were covalently modified with poly(ethylene glycol) (PEG), which improved the resistance to nonspecific proteins by almost 7-fold compared to the widely used PEG-monopyrene approach. The attachment of recognition elements to PA-VS was optimized for concanavalin A (ConA), a model lectin with a high affinity to glycans. Lastly, the platform was implemented for the rapid, sensitive, and regenerable recognition of SARS-CoV-2 spike protein and human ferritin in lab-made samples. Those two are the target molecules of major importance for the rapid detection and monitoring of COVID-19-positive patients. For that purpose, monoclonal antibodies (mAbs) were bound to the scaffolds, resulting in a surface coverage of 436 ± 30 ng/cm2. KD affinity constants of 48.4 and 2.54 nM were obtained by surface plasmon resonance (SPR) spectroscopy for SARS-CoV-2 spike protein and human ferritin binding on these supramolecular scaffolds, respectively.


Subject(s)
Biomarkers/analysis , COVID-19/diagnosis , Graphite/chemistry , Immunoassay/methods , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Ethylenes/chemistry , Ferritins/immunology , Ferritins/metabolism , Humans , Point-of-Care Systems , Polyamines/chemistry , Polyethylene Glycols/chemistry , Pyrenes/chemistry , Quantum Theory , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Semiconductors , Spike Glycoprotein, Coronavirus/immunology , Sulfonic Acids/chemistry , Surface Plasmon Resonance
10.
Molecules ; 26(17)2021 Aug 28.
Article in English | MEDLINE | ID: covidwho-1374472

ABSTRACT

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/pharmacology , Ginger/chemistry , Plant Extracts/pharmacology , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/ultrastructure , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/isolation & purification , Coronavirus Protease Inhibitors/therapeutic use , Crystallography, X-Ray , Enzyme Assays , Gas Chromatography-Mass Spectrometry , Humans , Magnetic Resonance Spectroscopy , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/therapeutic use , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Structure-Activity Relationship , Sulfonic Acids/pharmacology
11.
Brief Bioinform ; 22(2): 1476-1498, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1352121

ABSTRACT

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic by the World Health Organization, and the situation worsens daily, associated with acute increases in case fatality rates. The main protease (Mpro) enzyme produced by SARS-CoV-2 was recently demonstrated to be responsible for not only viral reproduction but also impeding host immune responses. The element selenium (Se) plays a vital role in immune functions, both directly and indirectly. Thus, we hypothesised that Se-containing heterocyclic compounds might curb the activity of SARS-CoV-2 Mpro. We performed a molecular docking analysis and found that several of the selected selenocompounds showed potential binding affinities for SARS-CoV-2 Mpro, especially ethaselen (49), which exhibited a docking score of -6.7 kcal/mol compared with the -6.5 kcal/mol score for GC376 (positive control). Drug-likeness calculations suggested that these compounds are biologically active and possess the characteristics of ideal drug candidates. Based on the binding affinity and drug-likeness results, we selected the 16 most effective selenocompounds as potential anti-COVID-19 drug candidates. We also validated the structural integrity and stability of the drug candidate through molecular dynamics simulation. Using further in vitro and in vivo experiments, we believe that the targeted compound identified in this study (ethaselen) could pave the way for the development of prospective drugs to combat SARS-CoV-2 infections and trigger specific host immune responses.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Heterocyclic Compounds/pharmacology , Protease Inhibitors/pharmacology , Selenium/analysis , Antiviral Agents/chemistry , Computational Biology , Computer Simulation , Coronavirus 3C Proteases/chemistry , Heterocyclic Compounds/chemistry , Humans , Ligands , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Protein Structure, Tertiary , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Reproducibility of Results , Sulfonic Acids
12.
World J Microbiol Biotechnol ; 36(11): 164, 2020 Sep 30.
Article in English | MEDLINE | ID: covidwho-1343003

ABSTRACT

Laccases (EC 1.10.3.2) are multi-copper oxidases that can degrade several xenobiotics, including textile dyes. Present study investigated the nature of laccase isoforms induced by 2,6-dimethylaniline in Cyathus bulleri cultivated on basal salt medium. Two isoforms, LacI and LacII were identified and purified by a combination of ultrafiltration and ion-exchange chromatography. The MS spectrum of the two proteins displayed a number of non-identical and identical molecular peaks (m/z), and, the latter were mapped to protein originating from the previously reported Laccase (Lcc) 1 gene. The LacI isoform exhibited higher catalytic efficiency (Kcat/Km) towards 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol and pyrogallol and was tolerant to high levels of chloride ions and resistant to EDTA. Higher decolorization of several dyes such as Direct Scarlet B (67%), Reactive Brilliant blue-R (96%), Direct Orange 34 (50%) and Reactive Red198 (95%) by the LacI isoform makes it a good candidate for degradation of synthetic dyes. The decolorization of Direct Orange 34 by laccases is being reported for the first time. Many of the properties exhibited by this isoform make it a good candidate for large scale production and applications for use in the dyeing industry.


Subject(s)
Coloring Agents/metabolism , Cyathus/metabolism , Laccase/metabolism , Textiles , Amino Acid Sequence , Aniline Compounds/metabolism , Culture Media/chemistry , Hydrogen-Ion Concentration , Oxidoreductases/metabolism , Protein Isoforms/metabolism , Substrate Specificity , Sulfonic Acids/metabolism
13.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Article in English | MEDLINE | ID: covidwho-1294550

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Protease Inhibitors/therapeutic use , Pyrrolidines/therapeutic use , SARS-CoV-2/drug effects , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/pathology , Coronavirus 3C Proteases/chemistry , Coronavirus Papain-Like Proteases/chemistry , Crystallography, X-Ray , Deuterium , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Lung/pathology , Mice , Mice, Transgenic , Models, Molecular , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation , Pyrrolidines/chemistry , SARS-CoV-2/enzymology , Sulfonic Acids , Transgenes
14.
J Mol Biol ; 433(13): 167003, 2021 06 25.
Article in English | MEDLINE | ID: covidwho-1272546

ABSTRACT

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug's properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug's P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cats , Coronavirus 3C Proteases/metabolism , Molecular Docking Simulation , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Sulfonic Acids , Thermodynamics , Viral Nonstructural Proteins/chemistry
15.
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
16.
Sci Rep ; 11(1): 9609, 2021 05 05.
Article in English | MEDLINE | ID: covidwho-1217711

ABSTRACT

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice. Overall outcome of clinical symptoms and survival upon SARS-CoV-2 challenge were not improved in mice treated with GC-376 compared to controls. The treatment with GC-376 slightly improved survival from 0 to 20% in mice challenged with a high virus dose at 105 TCID50/mouse. Most notably, GC-376 treatment led to milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls in mice challenged with a low virus dose at 103 TCID50/mouse. This was particularly the case in the brain where a 5-log reduction in viral titers was observed in GC-376 treated mice compared to vehicle controls. This study supports the notion that GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection and that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/pharmacology , COVID-19/drug therapy , Carbonates/pharmacology , Leucine/pharmacology , Sulfonic Acids/pharmacology , Animals , Brain/drug effects , Brain/pathology , COVID-19/pathology , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Female , Keratin-18/genetics , Lung/drug effects , Lung/pathology , Lung/virology , Mice, Transgenic , Vero Cells , Viral Load
17.
J Med Chem ; 65(4): 2848-2865, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1199254

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Chlorocebus aethiops , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Proline/analogs & derivatives , Proline/chemical synthesis , Proline/chemistry , Proline/pharmacology , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS-CoV-2/enzymology , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistry , Sulfonic Acids/pharmacology , Vero Cells
18.
Nat Commun ; 12(1): 2016, 2021 04 01.
Article in English | MEDLINE | ID: covidwho-1164851

ABSTRACT

We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , SARS-CoV-2/drug effects , Virus Replication/drug effects , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Pyrrolidines/pharmacology , Sulfonic Acids
19.
ACS Infect Dis ; 7(3): 586-597, 2021 03 12.
Article in English | MEDLINE | ID: covidwho-1108883

ABSTRACT

As the COVID-19 pandemic continues to unfold, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII, and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. In this study, we further characterized the mechanism of action of these four compounds using the SARS-CoV-2 pseudovirus neutralization assay. It was found that GC-376 and calpain inhibitors II and XII have a dual mechanism of action by inhibiting both viral Mpro and host cathepsin L in Vero cells. To rule out the cell-type dependent effect, the antiviral activity of these four compounds against SARS-CoV-2 was also confirmed in type 2 transmembrane serine protease-expressing Caco-2 cells using the viral yield reduction assay. In addition, we found that these four compounds have broad-spectrum antiviral activity in inhibiting not only SARS-CoV-2 but also SARS-CoV, and MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift-binding assay and enzymatic fluorescence resonance energy transfer assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 might be promising starting points for further development against existing human coronaviruses as well as future emerging CoVs.


Subject(s)
Antiviral Agents/pharmacology , Carbonates/pharmacology , Glycoproteins/pharmacology , Leucine/pharmacology , Oligopeptides/pharmacology , Proline/analogs & derivatives , SARS-CoV-2/drug effects , Sulfonic Acids/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , COVID-19/drug therapy , Caco-2 Cells , Cathepsin L/antagonists & inhibitors , Cell Line , Chlorocebus aethiops , Coronavirus 229E, Human/drug effects , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus NL63, Human/drug effects , Coronavirus OC43, Human/drug effects , Drug Combinations , HEK293 Cells , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Proline/pharmacology , Serine Endopeptidases/metabolism , Vero Cells
20.
Antiviral Res ; 187: 105020, 2021 03.
Article in English | MEDLINE | ID: covidwho-1051448

ABSTRACT

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , Rhinovirus/drug effects , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Binding Sites , Cathepsin L/metabolism , Drug Discovery , Glycoproteins/pharmacology , Humans , Kinetics , Models, Molecular , Protease Inhibitors/chemistry , Pyrrolidines/pharmacology , Sulfonic Acids
SELECTION OF CITATIONS
SEARCH DETAIL