Identification and biochemical characterization of halisulfate 3 and suvanine as novel inhibitors of hepatitis C virus NS3 helicase from a marine sponge

Abstract

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

Figure 1

Structures of halisulfate 3 (hal3) and suvanine.

Figure 2

Inhibition of NS3 helicase-catalyzed RNA unwinding activity by hal3 and suvanine. (A,B) Inhibition curves of hal3 and suvanine generated using a fluorescence helicase assay. The NS3 helicase activities of samples containing inhibitor were calculated relative to control samples containing DMSO vehicle rather than inhibitor. The data are presented as mean ± standard deviation of three replicates; (C,D) Gel images representing the inhibitory effects of hal3 and suvanine in a gel-based helicase assay. Fluorescence-labeled ssRNA and dsRNA were applied to lanes 1 and 2, respectively. The dsRNA was incubated with NS3 in the presence of increasing concentrations of inhibitor (lanes 3–7, 0–100 µM). Lane 8 shows the control reaction in the absence of NS3.

Figure 3

Effects of hal3 and suvanine on NS3 ATPase activity demonstrated by autoradiography of an ATPase assay using [γ-³²P] ATP. Lane 1 contains the control reaction without NS3. Lanes 2–8 (A) and 2–9 (B) show the ATP hydrolysis reaction with poly(U) RNA at increasing concentrations (0–100 µM) of hal3 and suvanine, respectively.

Figure 4

Effects of hal3 and suvanine on NS3 RNA binding activity, assessed by autoradiography of a gel mobility shift assay using ³²P-labeled ssRNA. Lanes 1 and 2 contain control reactions consisting of heat-denatured ssRNA and 300 nM BSA instead of NS3, respectively. Lanes 3−9 (A) and 3−10 (B) show the RNA binding reaction with increasing concentrations (0−100 µM) of hal3 and suvanine, respectively.

Figure 5

Effects of hal3 (A) and suvanine (B) on NS3 serine protease activity. The NS3 serine protease activity of samples containing inhibitor was calculated relative to control samples containing DMSO vehicle rather than inhibitor. The data are presented as means ± standard deviation of three replicates.

Figure 6

Effects of hal3 (A) and suvanine (B) on DENV NS3 helicase activity, assessed using a gel-based helicase assay. Fluorescence-labeled ssRNA and dsRNA were applied to lanes 1 and 2, respectively. The dsRNA was incubated with NS3 in the presence of increasing concentrations of inhibitor (lanes 3–7, 0–100 µM). Lanes 8 and 9 contain the control reaction mixtures in the absence of NS3, and in the presence of 15 mM ATP-gamma-S as an inhibition control, respectively.