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.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

“…A fluorescence helicase assay based on FRET [ 26 , 27 ], with modifications in the fluorescent dyes, was used to examine NS3 helicase inhibition by different compounds. Since hydroxyanthraquinone is known to exhibit a wide range of absorption wavelengths in aqueous solution, ranging from shorter to longer wavelengths (e.g., ~200 up to 700 nm) [ 28 ], we used a dsRNA substrate prepared by annealing the 5′ Alexa Fluor 700 (maximum excitation/emission = 702/723 nm)-labeled fluorescence strand to the 3′ Black Hole Quencher (BHQ)-3-labeled quencher strand with the same RNA sequences, as described in previous reports [ 27 , 29 ], to avoid interference due to hydroxyanthraquinone absorption. The concentration of the capture strand was optimized to 400 nM based on the Z ′ value [ 30 ]; the Z ′ value validates assay conditions as having reliability and reproducibility when its value is close to 1.…”

Suvanine analogs from a Coscinoderma sp. marine sponge and their cytotoxicities against human cancer cell lines

“…In addition to the biological activities of the sulfated sestertepenoids mentioned above, two individual studies on the biological activity of the crude extracts of marine organisms revealed that suvanine is responsible for the high activity of some sponge extracts against hepatitis C virus NS3 helicase (Furuta et al 2014) and adenosine A1 receptor (Ali et al 2013), respectively. It was also found that suvanine effectively antagonize the mammalian bile acid sensor farnesoid X receptor, which resulted in the discovery of molecular bases of the antagonism and more potent semi-synthetic derivatives (Di Leva et al 2013).…”

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

“…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].…”

“…Alternatively, the gel-based assay can be performed with a double-stranded substrate labeled with a single fluorophore (such as Alexa Fluor 488) [23]. The fluorescent signal from the fluorophore-labeled dsRNA can be visualized using a fluorescent gel scanner, such as a Typhoon 9210 imager (GE Healthcare).…”

“…Remove gel plates, transfer gel on chromatography paper, and expose it to a phosphor storage screen. Image screen with a phosphorimager and quantify gel band intensities with associated software [18][19][20][21][22][23].…”