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1.
ChemMedChem ; 17(7): e202100641, 2022 04 05.
Article in English | MEDLINE | ID: covidwho-1705258

ABSTRACT

The pentafluorosulfanyl (-SF5 ) functional group is of increasing interest as a bioisostere in medicinal chemistry. A library of SF5 -containing compounds, including amide, isoxazole, and oxindole derivatives, was synthesised using a range of solution-based and solventless methods, including microwave and ball-mill techniques. The library was tested against targets including human dihydroorotate dehydrogenase (HDHODH). A subsequent focused approach led to synthesis of analogues of the clinically used disease modifying anti-rheumatic drugs (DMARDs), Teriflunomide and Leflunomide, considered for potential COVID-19 use, where SF5 bioisostere deployment led to improved inhibition of HDHODH compared with the parent drugs. The results demonstrate the utility of the SF5 group in medicinal chemistry.


Subject(s)
Chemistry, Pharmaceutical , Dihydroorotate Dehydrogenase , Amides , Dihydroorotate Dehydrogenase/antagonists & inhibitors , Humans
2.
ChemMedChem ; 17(9): e202200016, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-1653198

ABSTRACT

The two SARS-CoV-2 proteases, i. e. the main protease (Mpro ) and the papain-like protease (PLpro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PLpro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing Mpro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro . Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro , highlighting the potential for dual PLpro /Mpro inhibition. MS-based PLpro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Coronavirus Papain-Like Proteases , Humans , Lactams , Leucine , Mass Spectrometry , Nitriles , Peptide Hydrolases , Proline , Protease Inhibitors/pharmacology
3.
Nucleic Acids Res ; 50(3): 1484-1500, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1624985

ABSTRACT

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/metabolism , Genome, Viral/genetics , Genomic Instability , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Exoribonucleases/antagonists & inhibitors , Genome, Viral/drug effects , Genomic Instability/drug effects , Genomic Instability/genetics , HIV Integrase Inhibitors/pharmacology , Isoindoles/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Organoselenium Compounds/pharmacology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Raltegravir Potassium/pharmacology , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Replication/drug effects , Virus Replication/genetics
4.
Chem Sci ; 12(41): 13686-13703, 2021 Oct 27.
Article in English | MEDLINE | ID: covidwho-1569290

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective 'peptibitors' inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.

5.
ChemMedChem ; 17(4): e202100582, 2022 02 16.
Article in English | MEDLINE | ID: covidwho-1540073

ABSTRACT

The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID-19) and other diseases. We report structure-activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro ), employing turnover and protein-observed mass spectrometry-based assays. The results reveal scope for optimisation of ebselen/ebselen derivative- mediated inhibition of Mpro , particularly with respect to improved selectivity.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Isoindoles/pharmacology , Organoselenium Compounds/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , COVID-19/virology , Humans , Isoindoles/chemistry , Organoselenium Compounds/chemistry , Protease Inhibitors/chemistry , Structure-Activity Relationship
7.
Chem Commun (Camb) ; 57(12): 1430-1433, 2021 Feb 15.
Article in English | MEDLINE | ID: covidwho-1387498

ABSTRACT

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Endopeptidases/drug effects , Mass Spectrometry/methods , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , beta-Lactams/pharmacology , Acylation , Antiviral Agents/chemistry , COVID-19/virology , Catalytic Domain , High-Throughput Screening Assays , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , beta-Lactams/chemistry
8.
Sci Rep ; 11(1): 13208, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1281733

ABSTRACT

Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.


Subject(s)
Antiviral Agents/chemistry , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Repositioning , Oligopeptides/chemistry , Cell Line , Humans , Serpins/chemistry , Viral Proteins/chemistry
9.
Chem Commun (Camb) ; 57(12): 1430-1433, 2021 Feb 15.
Article in English | MEDLINE | ID: covidwho-1035940

ABSTRACT

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Endopeptidases/drug effects , Mass Spectrometry/methods , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , beta-Lactams/pharmacology , Acylation , Antiviral Agents/chemistry , COVID-19/virology , Catalytic Domain , High-Throughput Screening Assays , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , beta-Lactams/chemistry
10.
Angewandte Chemie ; 132(52):23750-23754, 2020.
Article in English | Academic Search Complete | ID: covidwho-976961

ABSTRACT

The SARS‐CoV‐2 main protease (Mpro) cleaves along the two viral polypeptides to release non‐structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus‐2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro. Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd=0.14±0.03 μM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme‐substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x‐ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity. [ABSTRACT FROM AUTHOR] Copyright of Angewandte Chemie is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

11.
Angew Chem Int Ed Engl ; 59(52): 23544-23548, 2020 12 21.
Article in English | MEDLINE | ID: covidwho-728060

ABSTRACT

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.


Subject(s)
Coronavirus 3C Proteases/chemistry , Coronavirus Protease Inhibitors/chemistry , Models, Molecular , SARS-CoV-2/enzymology , Small Molecule Libraries/chemistry , Allosteric Regulation , Binding Sites , Biological Assay , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/pharmacology , Crystallography, X-Ray , Mass Spectrometry , Protein Binding , Protein Conformation , Protein Multimerization , SARS-CoV-2/physiology , Small Molecule Libraries/pharmacology , Substrate Specificity , Virus Replication
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