RESUMEN
INTRODUCTION: This study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. METHODS: Recombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood-brain barrier permeability. RESULTS: Five compounds showed non-competitive inhibition of ATPase activity with an EC50 ⩽ 15 µM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52 µM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100 µM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34 µM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. CONCLUSION: LTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antígenos Virales de Tumores/metabolismo , Antivirales/aislamiento & purificación , Antivirales/farmacología , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Virus JC/enzimología , Adenosina Trifosfatasas/genética , Animales , Antígenos Virales de Tumores/genética , Línea Celular , Chlorocebus aethiops , Colorimetría/métodos , Evaluación Preclínica de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Virus JC/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
BK virus large T antigen (LTA) is a hexameric protein with a helicase activity that is powered by ATP hydrolysis. A mutant virus with Lys420Ala, Arg421Ala, and Asp504Ala mutations at the ATP binding sites showed marked reduction in viral fitness. This observation indicates that high throughput screening for ATPase inhibitors will be valid strategy to discover anti-BKV drugs. Pilot screening of 300 compounds from the Tim Tec ActiTarg K library identified a compound, STO18584, with selectivity index of 19.2.