Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation

Abstract

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2μM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

Figure 1

Ability of primuline derivatives to inhibit NS3 catalyzed RNA-unwinding, DNA unwinding, ATP hydrolysis and peptide cleavage. (A) An RNA-based NS3 helicase assay where NS3 helicase must separate an RNA duplex (blue) to enhance Cy5 fluorescence. Unwinding only occurs upon addition of ATP, which fuels helicase action. (B), (C), (D) Effects of various concentrations of primuline derivatives on the kinetics of NS3 catalyzed RNA unwinding. Data obtained after ATP addition were fitted to a first order rate equation (solid curves) (E), (F), (G) Comparison of the ability of various primuline derivatives (for structures see Table 1) to inhibit NS3 catalyzed DNA unwinding (squares), peptide cleavage (circles), and ATP hydrolysis (triangles). Points are averages of three independent assays and error bars show standard deviations. Data were fitted to a normalized dose response equation with IC₅₀ values listed in Table 1.

Figure 2

Interaction of primuline derivatives with NS3h and scNS4A-NS3. (A) Effect of various compounds on NS3h intrinsic protein fluorescence. Corrected fluorescence values for titrations of 200 nM NS3h_2a(JFH1) (F_c, Equation 1). Data were fitted to Equation 2. (B) Average K_d values from three titrations with each compound performed at 50 nM, 100 nM and 200 nM NS3h_2a(JFH1). Error bars show standard deviations. (C) Corrected fluorescence values for titrations of either 100 nM NS3h_2a(JFH1) or scNS4-NS3. Data were fitted to Equation 2.

Figure 3

Use of fluorescence microscopy to visualize helicase inhibitors in cells. (A) Absorbance (solid lines, left y-axis) spectra, and fluorescence emission spectra when excited at peak absorbance ± 5 nm (see Table 1 for peak absorbance, dotted lines, right y-axis) of 10 µM aliquot of compound 1, the most active helicase inhibitor isolated from primuline (Li et al., 2012). (B) Optical properties of compound 2, which was used to synthesize compounds 3–13 (Table 1) (Li et al., 2012). (C), (D), (E) Optical properties of the most potent helicase inhibitors derived from primuline. (F), (G), (H), (I), (J) Fluorescence of cells exposed to the most potent derivatives (excitation 340 ± 40 nm, emission 435 ± 50). Scale bar - 10 µm.

Figure 4

Effect of Compound 3 on Huh7.5 hepatoma cells harboring a stably transfected subgenomic rLuc HCV replicon. (A) Schematic genome map of the HCV replicon. (B) Percent Renilla luciferase remaining (circles) after 72 hours exposure of replicon containing cells to 3 when grown in media containing 0.5% DMSO. Cell viability was measured with the Cell Titer-Glo luminescent cell viability kit (Promega) and is expressed compared to DMSO controls. (C) Amount of HCV RNA remaining in cells exposed to the indicated amounts of 3. RNA was measured using 1 µg of total cellular RNA with specific Taqman probes and quantitative reverse transcriptase PCR (qRT-PCR). (D) Time-dependent clearance of HCV RNA in cells exposed to 10 µM of 3 (triangles) or 100 units of interferon (circles). RNA levels are expressed relative to RNA levels observed in cells grown in the presence of media and 0.5% DMSO. All assays were performed in triplicate, and error bars mark standard deviations. (E) Western blot of cell extracts treated with 3 or standard HCV drugs for indicated times.

Figure 5

Effect of Compound 3 on the cellular location of HCV Replication complexes observed in replicon-containing Huh7.5/Con1sg-Rluc cells. Cells were fixed, permeabilized, and stained with 9E10 α-NS5A antibody (obtained from Charles Rice, Rockefeller University) and Alexa 546 secondary antibody 72 hours after treatment with (A) 0.5% DMSO, (B) 100 units of interferon, (C) 10 µM primuline, or (D) 10 µM of Compound 3. Scale bar - 10 µm. (E) Compound 3 fluorescence. Note: scale is the same as the graphs for the more potent inhibitors shown in Fig. 2. To examine Compound 3 location in cells, Huh7.5/Con1sg-Rluc cells were treated with compound 3 for 72 hours, permeabilized and stained with 9E10 α-NS5A antibody and Alexa 546 secondary antibody. (F) Localization of compound 3 fluorescence observed when the cells are excited at 350 nm. (G) NS5A localization observed when the same cells were excited to observe the NS5A replication complexes. (H) Overlay of the images with compound 3 colored green and NS5A complexes colored red. Scale bar – 5 µm.

Figure 6

Effects of helicase and protease inhibitors on cells harboring HCV replicons. (A) HCV replicon content in cells following treatment with various concentrations of telaprevir and 3 for 72 hrs. (B) Effect of 10 µM of various compounds (when administered in DMSO for 72 hrs.) on the location of a recombinant NS3 protease substrate: red fluorescent- nuclear localization signal- interferon (IFN)-β promoter stimulator protein 1 (RFP-NLS-IPS) (Jones et al., 2010). Note that when NS3 is present, the reporter is in the nucleus, but in the presence of protease inhibitors, the reporter remains tethered to mitochondria. Scale bars – 10 µm.