High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

Abstract

The mosquito-borne dengue virus serotypes 1-4 (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.

Keywords: Fluorescence quenching; High-throughput screening; Protease inhibitors; Renilla luciferase reporter replicon; Therapeutic index; Virus infectivity assays.

Figure 1

Active-Site titration of DENV2 protease with BPTI. X-intercept indicates the active concentration of DENV2 protease (25 nM protein was used based on protein concentration).

Figure 2

Pie-chart of primary and secondary assays for selection of inhibitors.

Figure 3

Structures of 8 selected compounds identified by HTS

Figure 4

EC50 values for inhibition of DENV2-infected BHK-21 cells determined by plaque assays. Compound C (A), Compound D (B), Compound F (C), Compound G (D), and Compound H (E).

Figure 5

Inhibition of DENV2 replication analyzed by qPCR. BHK-21 cells were infected with DENV2 (MOI 1) and the viral RNA were isolated after 24h and the RNA copy number was determined as described under Materials and Methods. DMSO treated control is taken as 100% and percent reduction of viral RNA by treatment of DENV-2 infected BHK-21 cells with the compounds at the indicated concentrations is plotted. Compounds C, D and H at 10 μM; Compounds F and G at 2 μM concentrations.

Figure 6

(A) Absorbance spectra of compounds C, F, G and H. (B) Fluorescence spectra of DENV2 protease (2μM) with various concentrations of compounds, C, F, G and H. (C) Competition of BPTI on the fluorescence quenching in the presence of 50 μM compound and 10 μM BPTI. (D) Kinetic analysis of compounds C, F and G. Lineweaver-Burk (LB) plots, of compounds, C, F, and G.

Figure 7

Molecular modeling using the DENV3 NS2B-NS3 protease structure (3U1I) (Noble et al., 2012). (A): Compound C (magenta); (B): Compound F (cyan); (C): Compound G (blue). Active site triad (H51, D75 and S135) is highlighted in yellow.