RESUMEN
There are currently no treatments for life-threatening infections caused by human polyomaviruses JCV and BKV. We therefore report herein the first crystal structure of the hexameric helicase of JCV large T antigen (apo) and its use to drive the structure-based design of dual JCV and BKV ATP-competitive inhibitors. The crystal structures obtained by soaking our early inhibitors into the JCV helicase allowed us to rapidly improve the biochemical activity of our inhibitors from 18 µM for the early 6-(2-methoxyphenyl)- and the 6-(2-ethoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole hits 1a and 1b to 0.6 µM for triazolopyridine 12i. In addition, we were able to demonstrate measurable antiviral activity in Vero cells for our thiazolopyridine series in the absence of marked cytotoxicity, thus confirming the usefulness of this approach.
Asunto(s)
Virus BK/enzimología , ADN Helicasas/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Virus JC/enzimología , ADN Helicasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antígenos Virales de Tumores/metabolismo , Antivirales/farmacología , Virus BK/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Virus 40 de los Simios/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/aislamiento & purificación , Virus BK/enzimología , Virus BK/fisiología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus 40 de los Simios/enzimología , Virus 40 de los Simios/fisiología , Ensayo de Placa ViralRESUMEN
Our objective was to determine whether quantitative polymerase chain reaction (PCR) can be used to measure the effect of tyrosine kinase (TK) inhibition on polyomavirus BK (BKV) replication. The BKV was grown in a cell culture system. The rate of viral replication in the presence or absence of the drug being tested was assessed by amplifying the viral genome using primers directed against the viral capsid 1 protein. Dasatinib, erlotinib, gefitinib, imatinib, sunitinib, and sorafenib all showed antiviral activity at micromolar concentrations. The 50% effective concentration for erlotinib and sorafenib was within blood concentrations readily achieved in human subjects. Quantitative PCR is a convenient method for viral drug sensitivity testing for slow-growing viruses that do not readily produce cytopathic effect. TK inhibitors deserve further consideration as a potential therapeutic option for BKV-associated nephropathy and hemorrhagic cystitis.
Asunto(s)
Antivirales/farmacología , Virus BK/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/efectos de los fármacos , Virus BK/enzimología , Virus BK/genética , Proteínas de la Cápside/genética , ADN Viral/análisis , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la PolimerasaRESUMEN
An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested.