Inhibition of Hepatitis C Virus NS3 Helicase by Manoalide

Abstract

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world, hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose-dependent manner, with IC(50) values of 15 and 70 μM, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K(m) value (0.31 mM) of NS3 ATPase activity, suggesting that 1 acts as a noncompetitive inhibitor. The binding of NS3 to single-stranded RNA was inhibited by 1. Manoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.

A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

“…Here, we describe an evaluation of the effects of test compounds on fluorescence intensity that can also be adapted to identify false-positive and false-negative hits interfering with the FRET-based assay. An optional gel-based HCV NS3 helicase assay with RI-labeled dsRNA can also be conducted to directly visualize the RNA strands unwound by helicase [18][19][20][21] (see Note 13).…”

“…For characterization of the inhibitory mechanism, radioactive assays that evaluate the ATPase and RNA-binding activities of NS3 are employed [18][19][20][21][22][23], because these activities are linked to helicase activity and are well-known targets of HCV NS3 helicase inhibitors [24].…”

Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase

“…NS3 RNA helicase assay was performed as described previously 7,19 with some modifications. In brief, the substrate for annealing two complementary RNA oligonucleotides, 5 0 -AGAGAGAGAGGUUGAGAGAGAGAGAGUUUGAGAGA-GAGAG-3 0 (40-mer, template strand) and 5 0 -CAAACUCUCUC UCUCUCAACAAAAAA-3 0 (26-mer, release strand), was purchased from Shanghai GenePharma (Shanghai, China).…”

“…ATPase assay NS3 ATPase activity was directly determined by monitoring [g-32 P] ATP hydrolysis by thin-layer chromatography as described in previous reports 7,19 with slight modifications. The reaction mixture contained 25 mM MOPS-NaOH (pH 7.0), 1 mM dithiothreitol, 5 mM MgCl 2 , 5 mM CaCl 2 , 1 mM [g-32 P] ATP (Muromachi Yakuhin, Tokyo, Japan), 300 nM NS3, 0.1 mg/mL poly (U) single-stranded RNA (ssRNA) (Sigma-Aldrich, MO) and increasing concentrations of CS (as indicated) in a volume of 10 mL.…”

“…NS3-RNA binding activity was determined by gel mobility shift assay as previously described 19,22 . The ssRNA (5 0 -UGAGGUAGUAGGUUGUAUAGU-3 0 ) synthesized by Gene Design (Osaka, Japan) was labeled at the 5 0 -end with [g-32 P] ATP (Muromachi Yakuhin, Tokyo, Japan) using T4 polynucleotide kinase (Toyobo, Osaka, Japan) at 37 C for 60 min and purified by the phenol-chloroform extraction method.…”