دانلود رایگان مقاله غربالگری برای شناسایی مانع ریز مولکول پروتئاز ویروسی

Abstract

The mosquito-borne dengue virus serotypes 1e4 (DENV1-4) and West Nile virus (WNV) cause serious illnesses worldwide associated with considerable morbidity and mortality. According to the World Health Organization (WHO) estimates, there are about 390 million infections every year leading to ~500,000 dengue haemorrhagic fever (DHF) cases and ~25,000 deaths, mostly among children. Antiviral therapies could reduce the morbidity and mortality associated with flaviviral infections, but currently there are no drugs available for treatment. In this study, a high-throughput screening assay for the Dengue protease was employed to screen ~120,000 small molecule compounds for identification of inhibitors. Eight of these inhibitors have been extensively analyzed for inhibition of the viral protease in vitro and cell-based viral replication using Renilla luciferase reporter replicon, infectivity (plaque) and cytotoxicity assays. Three of these compounds were identified as potent inhibitors of DENV and WNV proteases, and viral replication of DENV2 replicon and infectious RNA. Fluorescence quenching, kinetic analysis and molecular modeling of these inhibitors into the structure of NS2B-NS3 protease suggest a mode of inhibition for three compounds that they bind to the substrate binding pocket.

4. Discussion

In this study, we performed in vitro HTS for small molecule inhibitors of the DENV2 protease. We initially performed our screen with 24,846 compounds without 0.1% CHAPS detergent in the reaction buffer. From this first round of screening, we obtained 436 (1.75%) hits which were mostly false positives (89%). We then modified our two succeeding rounds of HTS by adding 0.1% CHAPS in the reaction buffer. This yielded lower hit rates of 0.1% and 0.04%. Lower hit rates are likely due to several possible reasons. First, the presence of CHAPS reduces aggregation of compounds in solution, which decreases false positives (Ezgimen et al., 2009; Feng and Shoichet, 2006). Second, the in vitro protease assay requires a high pH (9.5) for optimal activity (Leung et al., 2001; Nall et al., 2004), which leads to protonation of some compounds resulting in false positives/negatives. Third, the assay buffers contain 20e30% glycerol which might have resulted in lower hit rates leading to pipetting errors in the automated liquid handling facilities (Noble et al., 2010). Fourth, the crystal structures of NS2B hydrophilic peptide-NS3 protease domain shows the active site to be relatively flat and negatively charged thus potentially contributing to lower hit rates observed (Erbel et al., 2006).