RESUMEN
Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Antivirales/síntesis química , Antivirales/química , Sitios de Unión/efectos de los fármacos , COVID-19/metabolismo , Proteasas Similares a la Papaína de Coronavirus/aislamiento & purificación , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Humanos , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Pandemias , Resonancia por Plasmón de Superficie , Células Tumorales CultivadasRESUMEN
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.