RESUMEN
Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.
Asunto(s)
Proteasas 3C de Coronavirus/antagonistas & inhibidores , Inhibidores de Proteasas/química , SARS-CoV-2/enzimología , Sitios de Unión , COVID-19/patología , COVID-19/virología , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/metabolismo , Cristalografía por Rayos X , Humanos , Conformación Molecular , Simulación del Acoplamiento Molecular , Nitrilos/química , Nitrilos/metabolismo , Nitrilos/farmacología , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 Å resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Apoproteínas/química , Proteínas Bacterianas/química , Ácido Fólico/química , Lactobacillus delbrueckii/química , Vitamina B 12/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transporte Biológico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Expresión Génica , Prueba de Complementación Genética , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Lactobacillus delbrueckii/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Especificidad por Sustrato , Vitamina B 12/metabolismoRESUMEN
Archaeal glutamate transporter homologs catalyze the coupled uptake of aspartate and three sodium ions. After the delivery of the substrate and sodium ions to the cytoplasm, the empty binding site must reorient to the outward-facing conformation to reset the transporter. Here, we report a crystal structure of the substrate-free transporter GltTk from Thermococcus kodakarensis, which provides insight into the mechanism of this essential step in the translocation cycle.