Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and primuline

Abstract

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.

Figure 1

Discovery of thioflavine S as a HCV helicase inhibitor. (A) Schematic drawing of the MBHA mechanism. (B) The NCI Mechanistic Set of 827 compounds was screened with an MBHA, each at 20 μM. Fluorescence was read before and 30 minutes after ATP addition and compound inhibition was calculated from F₃₀/F₀ ratios. Hits were defined as compounds inhibiting more than 50% and interfering less than 20%. (C) Hits from the MBHA primary screen were tested for their DNA-binding capacity with an FID counterscreen at 1.5 μM compound concentration, and percent binding was calculated. Numbers refer to NSC numbers. (D) Concentration response curves for thioflavine S when assayed in MBHAs using either a DNA or RNA substrate.

Figure 2

Structures of isolated, pure compounds from thioflavine S (T) and primuline (P) dyes.

Figure 3

Effects of primuline and its components on HCV helicase-catalyzed DNA-unwinding and ATP hydrolysis. (A) MBHAs performed at various concentrations of primuline. Reactions were initiated by adding ATP at the indicated time. (B) Initial rates of DNA unwinding in MBHAs containing indicated concentrations of primuline (*), compound P1a (circles), P3 (triangles), or P4 (diamonds). Data are fit to Equation 3 (methods). Individual reaction time-courses and curve-fits used to calculate initial rates are shown in Figure S1 (supplemental data). IC₅₀'s listed in Table 1 are the averages from three separate titrations with each compound. (C) ATPase assays were performed in the absence of RNA (squares), 15 μM (circles) or 1 mM (triangles) poly(U) RNA (measured as μM UMP). ATP was present at 1 mM, the reactions were initiated by rapidly mixing in NS3h, and the amount of phosphate released was measured after 15 minutes at 26 °C. Various concentrations of P4 were present as indicated.

Figure 4

Effect of various compounds on Huh7.5 hepatoma cells harboring a stably transfected subgenomic rLuc HCV replicon. All compounds were tested at 10 μM such that cell media contained 0.5% (v/v) DMSO. Percent renilla luciferase, which is proportional to HCV RNA content is expressed with regard to cells grown in media and 0.5% DMSO. Cell viability was measured with the Titer-Glo luminescent cell viability kit (Promega) and is also expressed compared to DMSO controls.

Figure 5

Comparative effects of benzothiazoles in HCV helicase assays. (A) Potency of each compound in MBHAs is plotted against the interference observed in an MBHA performed in the presence of 10 μM of each compound. Points are labeled with compound number, although some numbers are omitted for clarity. Compounds that bind the helicase DNA substrate and quench its fluorescence lie on the left side of the plot while those that enhance substrate fluorescence are on the right. (B) Effects of various concentrations of the most potent compound that does not interfere with the MBHA (compound 17) in standard MBHAs where Cy5 fluorescence was monitored.

Scheme 1

^a Reagents: (a) Substituted benzoyl chloride, pyridine, 80 °; (b) arylisocyanate, DMF, 80 °; (c) arylsulfonyl chloride, pyridine, 80 °.