RESUMEN
A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.
Asunto(s)
Genes cdc/genética , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Daño del ADN/genética , ADN de Hongos/química , ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Mutación , Fase S , Saccharomyces cerevisiae/crecimiento & desarrollo , Estrés Fisiológico/genéticaRESUMEN
The eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases (CDKs). CDK activity rises over 50-fold during the cell cycle, from a low level in G1 to a high level in mitosis. However, it is not known whether the entire range of CDK activity is necessary for cell cycle progression, or whether cells can tolerate a reduction in CDK activity level. Here, in fission yeast, we show that sublethal CDK inhibition lengthens the time cells spend in mitosis but does not cause misordering of mitotic events. Maximum attainable CDK activity exceeds the amount necessary for mitosis, and thus forms a CDK activity buffer between sufficient and maximal possible CDK activities. This CDK activity buffer is needed for mitotic completion when CDK activity is compromised, and CDK inhibition only becomes lethal to cells when this buffer is exhausted. Finally, we explore what factors influence this CDK activity buffer, and find that it is influenced by CDK-counteracting phosphatases. Therefore, maximum attainable CDK activity is not necessary for mitosis but provides robustness to CDK activity reduction to ensure mitotic completion.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Mitosis , Fosforilación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
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.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Compuestos de Anilina/farmacología , Animales , Benzamidas/farmacología , Chlorocebus aethiops , Proteasas Similares a la Papaína de Coronavirus/genética , Proteasas Similares a la Papaína de Coronavirus/aislamiento & purificación , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Sinergismo Farmacológico , Pruebas de Enzimas , Flavinas/farmacología , Transferencia Resonante de Energía de Fluorescencia , Furanos/farmacología , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Naftalenos/farmacología , Fenantrenos/farmacología , Quinonas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Exorribonucleasas/antagonistas & inhibidores , Metiltransferasas/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Antivirales/química , Clorobencenos/farmacología , Chlorocebus aethiops , Pruebas de Enzimas , Exorribonucleasas/genética , Exorribonucleasas/aislamiento & purificación , Exorribonucleasas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Indazoles/farmacología , Indenos/farmacología , Indoles/farmacología , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Nitrilos/farmacología , Fenotiazinas/farmacología , Purinas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Especificidad por Sustrato , Trifluperidol/farmacología , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/aislamiento & purificación , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
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.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Azoles/farmacología , Chlorocebus aethiops , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/aislamiento & purificación , Proteasas 3C de Coronavirus/metabolismo , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Isoindoles , Leupeptinas/farmacología , Compuestos de Organoselenio/farmacología , Peptidomiméticos , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Proteínas no Estructurales Virales/metabolismoRESUMEN
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).