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1.
RSC Adv ; 11(33): 20457-20464, 2021 Jun 08.
Article in English | MEDLINE | ID: covidwho-1387504

ABSTRACT

An improved and expedient technique for the synthesis of peptidyl-fluoromethyl ketones is described. The methodology is based on prior coupling of an aspartate fluoromethyl ketone to a linker and mounting it onto resin-bound methylbenzhydrylamine hydrochloride. Subsequently, by utilising standard Fmoc peptide procedures, a number of short Z-protected peptides were synthesised and assessed as possible inhibitors of the main protease from SARS-CoV-2 (3CLpro).

2.
Biochem J ; 478(13): 2405-2423, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1292181

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , RNA Helicases/antagonists & inhibitors , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Chlorocebus aethiops , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , RNA Helicases/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Suramin/pharmacology , Vero Cells , Viral Nonstructural Proteins/metabolism
3.
Biochem J ; 478(13): 2499-2515, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1291175

ABSTRACT

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


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Azoles/pharmacology , Chlorocebus aethiops , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Isoindoles , Leupeptins/pharmacology , Organoselenium Compounds/pharmacology , Peptidomimetics , RNA-Binding Proteins/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism
4.
Biochem J ; 478(13): 2517-2531, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1290988

ABSTRACT

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Aniline Compounds/pharmacology , Animals , Benzamides/pharmacology , Chlorocebus aethiops , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/isolation & purification , Coronavirus Papain-Like Proteases/metabolism , Drug Synergism , Enzyme Assays , Flavins/pharmacology , Fluorescence Resonance Energy Transfer , Furans/pharmacology , High-Throughput Screening Assays , Inhibitory Concentration 50 , Naphthalenes/pharmacology , Phenanthrenes/pharmacology , Quinones/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/growth & development , Small Molecule Libraries/chemistry , Vero Cells , Virus Replication/drug effects
5.
Biochem J ; 478(13): 2533-2535, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1290318

ABSTRACT

In response to the COVID-19 pandemic, we began a project in March 2020 to identify small molecule inhibitors of SARS-CoV-2 enzymes from a library of chemical compounds containing many established pharmaceuticals. Our hope was that inhibitors we found might slow the replication of the SARS-CoV-2 virus in cells and ultimately be useful in the treatment of COVID-19. The seven accompanying manuscripts describe the results of these chemical screens. This overview summarises the main highlights from these screens and discusses the implications of our results and how our results might be exploited in future.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Enzyme Assays , Humans , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Viral Nonstructural Proteins/metabolism
6.
Biochem J ; 478(13): 2445-2464, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1290093

ABSTRACT

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/antagonists & inhibitors , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Animals , Aurintricarboxylic Acid/pharmacology , Chlorocebus aethiops , Enzyme Assays , Exoribonucleases/metabolism , Fluorescence , High-Throughput Screening Assays , Patulin/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism
7.
Biochem J ; 478(13): 2465-2479, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1290092

ABSTRACT

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Endoribonucleases/antagonists & inhibitors , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Allosteric Regulation , Animals , Chlorocebus aethiops , Endoribonucleases/isolation & purification , Endoribonucleases/metabolism , Enzyme Assays , Fluorescence , High-Throughput Screening Assays , In Vitro Techniques , Kinetics , Naphthoquinones/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/growth & development , Small Molecule Libraries/chemistry , Solutions , Vero Cells , Viral Nonstructural Proteins/isolation & purification , Viral Nonstructural Proteins/metabolism
8.
Biochem J ; 478(13): 2425-2443, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1289982

ABSTRACT

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Animals , Benzoates/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Holoenzymes/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Suramin/pharmacology , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
9.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1289949

ABSTRACT

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/antagonists & inhibitors , Methyltransferases/antagonists & inhibitors , RNA Caps/metabolism , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/chemistry , Chlorobenzenes/pharmacology , Chlorocebus aethiops , Enzyme Assays , Exoribonucleases/genetics , Exoribonucleases/isolation & purification , Exoribonucleases/metabolism , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Indazoles/pharmacology , Indenes/pharmacology , Indoles/pharmacology , Methyltransferases/genetics , Methyltransferases/isolation & purification , Methyltransferases/metabolism , Nitriles/pharmacology , Phenothiazines/pharmacology , Purines/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Substrate Specificity , Trifluperidol/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/isolation & purification , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/isolation & purification , Viral Regulatory and Accessory Proteins/metabolism
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