RESUMEN
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
Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading- and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.
Asunto(s)
Replicación del ADN , ADN Bacteriano/química , Escherichia coli/genética , Secuencias Invertidas Repetidas , ADN/metabolismo , ADN Bacteriano/metabolismo , Desoxirribonucleasas/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Exonucleasas/metabolismo , Modelos Genéticos , Plásmidos/genéticaRESUMEN
The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at â¼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3'-5' exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end.
Asunto(s)
División del ADN , ADN Cruciforme/metabolismo , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Exonucleasas/metabolismo , ADN/química , ADN/metabolismoRESUMEN
The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/química , Dimerización , Zinc/metabolismo , Ácido Anhídrido Hidrolasas/metabolismo , Adenosina Trifosfatasas , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Recombinación Homóloga , Humanos , Proteína Homóloga de MRE11/química , Proteína Homóloga de MRE11/metabolismo , Microscopía de Fuerza Atómica , Complejos Multiproteicos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , CohesinasRESUMEN
Phenelfamycins G and H are new members of the family of elfamycin antibiotics with the basic structure of phenelfamycins E and F, respectively, which are also well known as ganefromycins α and ß. Phenelfamycins G and H differ from phenelfamycins E and F by an additional hydroxy group at position C-30, which is not described so far for any of the elfamycin-type antibiotics. The actinomycete strain that produced phenelfamycins G and H was identified to be Streptomyces albospinus based on its 16S rRNA gene sequence. Phenelfamycins G and H exhibit a narrow antibacterial spectrum with a pronounced inhibitory activity against Propionibacterium acnes.