RESUMEN
Despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. Persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. A combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. Initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. We provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Fenómenos Fisiológicos de los Virus/efectos de los fármacos , Animales , Cápside/efectos de los fármacos , Cápside/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Due to the high number of drug-resistant HIV-1 mutants generated by highly active antiretroviral therapy (HAART), there is continuing demand for new types of inhibitors. Both the assembly of the Gag polyprotein into immature and mature HIV-1 particles are attractive candidates for the blocking of the retroviral life cycle. Currently, no therapeutically-used assembly inhibitor is available. One possible explanation is the lack of a reliable and simple assembly inhibitor screening method. To identify compounds potentially inhibiting the formation of both types of HIV-1 particles, we developed a new fluorescent high-throughput screening assay. This assay is based on the quantification of the assembly efficiency in vitro in a 96-well plate format. The key components of the assay are HIV-1 Gag-derived proteins and a dual-labelled oligonucleotide, which emits fluorescence only when the assembly of retroviral particles is inhibited. The method was validated using three (CAI, BM2, PF74) reported assembly inhibitors.
Asunto(s)
Fármacos Anti-VIH/farmacología , Evaluación Preclínica de Medicamentos/métodos , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Ensamble de Virus/efectos de los fármacos , Productos del Gen gag/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , HumanosRESUMEN
We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.