Primuline derivatives that mimic RNA to stimulate hepatitis C virus NS3 helicase-catalyzed ATP hydrolysis

Abstract

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μM. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.

Keywords: ATPases; Chemical Biology; Drug Discovery; High Throughput Screening (HTS); Molecular Motors; RNA Helicase.

FIGURE 1.

Primuline derivatives that stimulate HCV helicase-catalyzed ATP hydrolysis. A, ATP hydrolysis catalyzed by 100 nm NS3h_1b(con1) was monitored using assay 1 (“Experimental Procedures”) in the presence of 88 different primuline derivatives. All reactions contained 5% DMSO and 100 μm compound (circles). Percent activity remaining (y axis) was calculated by normalizing observed phosphate concentrations to average phosphate observed in reactions containing DMSO only (x). The reactions were incubated for 30 min at 23 °C. B, structures of two hits (1 and 2), a compound with the terminal pyridine replaced with benzene (compound 3) and the related soluble dye titan yellow. C, NS3h-catalyzed phosphate released from ATP in the presence of indicated concentrations of compounds 1–3 and titan yellow. D, kinetics of NS3h catalyzed ATP hydrolysis in the presence of indicated concentrations of compound 1. Data are fit to a first order rate equation. E, NS3h-catalyzed ADP release from ATP in the presence of various concentrations of compound 1, as measured by either the ADP-Glo assay (Promega, Assay 2 methods) or the ADP transcreener assay (BellBrook labs, Assay 3 methods).

FIGURE 2.

Effect of primuline derivatives on NS3h-catalyzed ATP hydrolysis in the presence of various concentrations of ATP and RNA. A, specific activities (nmol PO₄/s/nmol NS3h) observed in the presence of various concentrations of compound 1 at the indicated initial concentrations of ATP. B, specific activities observed in the presence of various concentrations of compound 3 at the indicated initial concentrations of ATP. C, specific activities observed in the presence of compound 1 and various concentrations of PolyU RNA. Data are fitted to Equation 1 (“Experimental Procedures”), with k_slow of 3 ± 2 s⁻¹, k_fast of 142 ± 7 s⁻¹, K_RNA of 26 ± 7 nm, K_i of 4 ± 1.2 μm, and K_ii of 920 ± 400 μm. Uncertainties represent 95% confidence intervals for the global non-linear regression. D, specific activities observed in the presence of compound 1 and various concentrations of the oligonucleotide dT20 (5′-TTT TTT TTT TTT TTT TTT TT). In panels A, B, and D, data are fitted to a standard 4-parameter concentration-response equation using GraphPad Prism (v. 6).

FIGURE 3.

Compound 1 only stimulates NS3h encoded by HCV genotype 1b strains. A, relative rates of ATP hydrolysis catalyzed in the presence of various concentrations of compound 1 by NS3h proteins derived from various HCV genotypes and dengue virus (DENV). B, relative rates of ATP hydrolysis catalyzed by various NS3h enzymes in the presence of 100 μm of compound 1. Data are expressed relative to control reactions containing the same proteins that were performed in the absence of compound 1. All reactions were performed in triplicate, points are average values, and error bars depict standard deviations.

FIGURE 4.

Interaction of primuline derivatives and titan yellow with NS3 helicase in the absence of RNA and ATP. Intrinsic protein fluorescence was monitored in the presence of various concentrations of compounds 1–3 and titan yellow. A, shown are two representative titrations of either 200 nm NS3h_1b(con1) or 200 nm NS3h_2a(JFH1) with compound 1. The inset shows average (± S.D.) K_d and ΔF_max values (Equation 3, methods) obtained from 3 separate titrations. B, shown are three representative titrations of 200 nm NS3h_2a(JFH1) with each compound. Three data sets were obtained with each compound and either 50 nm, 100 nm or 200 nm NS3h. Titrations were fitted to Equation 3 (“Experimental Procedures”), and the resulting dissociation constants were averaged to yield the dissociation constants shown on the inset. Error bars are standard deviations from the three repeats.

FIGURE 5.

Possible molecular interactions between compound 1 and HCV helicase. A, molecular model of compound 1 docked in PDB file 3KQN (genotype 1b(con1)). NS3 helicase is depicted as ribbons colored by conservation with a gradient from cyan (identical) to maroon (most variable). Compound 1 is shown as a space-filled model, which is bound between the motor domains perpendicular to the nucleic acid binding cleft. The inset shows the surface of NS3 that contacts compound 1 (sticks) in a model of the binding surface colored based on electrostatic potential using UCSF Chimera. Residues targeted for site directed mutagenesis are shown as sticks and labeled. B, response of NS3 harboring amino acid substitution in the putative compound 1 binding cleft to various concentrations of compound 1.