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

Abstract

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC 50 ¼ 1.7 AE 0.2 mM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

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].…”

“…The FRET-based assay can be performed with various fluorophore-quencher pairs at multiple wavelengths. Furthermore, the FRET-based assay can be replaced with a similar fluorescence helicase assay based on photoinduced electron transfer between fluorescent dyes and guanine bases by preparing a double-stranded substrate with a 5′-fluorescent dye (such as BODIPY FL)-labeled strand hybridized to a complementary strand consisting of guanine bases at the 3′-end [19]. Because this assay requires only one type of fluorescent dye, it is more cost-effective than the FRET-based assay.…”

“…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].…”

Synthesis and characterization of physicochemical and immunological properties of recombinant NS3-G2 dendrimer conjugate

“…The non-structural protein 3 (NS3) is bi-functional containing serine protease and RNA helicase activity and is directly involved in the virus replication [5,6]. The NS3 has been utilized as an important immune target in various vaccine and targeted antiviral therapy studies [7][8][9][10][11]. An effective vaccine for HCV infection should elicit strong cellular immune responses; however, subunit vaccines without a Th1-specific adjuvant formulation induce humoral responses, generally [12].…”