Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047 and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Nucleic Acids Research (Impact Factor: 9.11). 06/2012; 40(17):8607-21. DOI: 10.1093/nar/gks623
Source: PubMed


Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC(50) = 1.4 μM), suramin sodium salt (IC(50) = 3.6 μM), NF 023 hydrate (IC(50) = 6.2 μM) and tyrphostin AG 538 (IC(50) = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

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    • "Understanding the mechanism of action of this molecular motor at the atomistic level is fundamental since the NS3 helicase domain has been proposed as a target for the development of a antiviral agents [6]. NS3 helicase has been characterized by single-molecule experiments [7] [8] [9] [10] [11] and biochemical essays [12] [13] [14]. Although sometime acting as a dimer or as an oligomer [15] [16], NS3 also functions as a monomer, similarly to other SF1 and SF2 helicases [17] [18]. "
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    ABSTRACT: Non structural protein 3 (NS3) helicase from hepatitis C virus is an enzyme that unwinds and translocates along nucleic acids with an ATP-dependent mechanism and has a key role in the replication of the viral RNA. An inchworm-like mechanism for translocation has been proposed based on crystal structures and single molecule experiments. We here perform atomistic molecular dynamics in explicit solvent on the microsecond time scale of the available experimental structures. We also construct and simulate putative intermediates for the translocation process, and we perform non-equilibrium targeted simulations to estimate their relative stability. For each of the simulated structures we carefully characterize the available conformational space, the ligand binding pocket, and the RNA binding cleft. The analysis of the hydrogen bond network and of the non-equilibrium trajectories indicates an ATP-dependent stabilization of one of the protein conformers. Additionally, enthalpy calculations suggest that entropic effects might be crucial for the stabilization of the experimentally observed structures.
    Nucleic Acids Research 08/2015; DOI:10.1093/nar/gkv872 · 9.11 Impact Factor
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    • "Suramin appears to inhibit (re)initiation of CHIKV RNA synthesis, maybe by interfering with binding of the template RNA. This would be in line with earlier in vitro studies, reporting that suramin inhibits various RNA-binding enzymes like viral polymerases (De Clercq, 1979; Ono et al., 1988; Tarantino et al., 2014) and helicases (Basavannacharya and Vasudevan, 2014; Mukherjee et al., 2012). Several suramin-related compounds were analyzed, and though these compounds were not more effective, they provided insight into the (different) structural elements that are Table 2 Antiviral and cytotoxic effects of suramin-containing and empty control liposomes, determined by CPE protection assay with Vero E6 cells. "
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    ABSTRACT: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC50 of ∼5 μM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC50 of ∼80 μM (CC50 >5 mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections. Copyright © 2015. Published by Elsevier B.V.
    Antiviral research 06/2015; 121. DOI:10.1016/j.antiviral.2015.06.013 · 3.94 Impact Factor
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    • "All but NF 023 inhibited replication of subgenomic HCV replicons (EC50 = 18–98 μM). Unfortunately, none of these inhibitors were specific to NS3 helicase [67]. "
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    ABSTRACT: Currently, hepatitis C virus (HCV) infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs) against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir) have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin). The new therapy has significantly improved sustained virologic response (SVR); however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.
    10/2013; 2013(15):467869. DOI:10.1155/2013/467869
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