Small molecular compounds and crude extracts derived from marine microorganisms were all obtained from Prof. Lixin Zhang’s lab, East China University of Science and Technology, Shanghai, China. Commercial compounds were purchased from National Compound Resource Center, China (Supplementary Data Sheets 1, 2).

Recombinant NS3h was expressed and purified as described below. cDNA genome of HCV 1b (Con1) was kindly provided by Prof. Charles M. Rice, the Rockefeller University, New York. NS3h (468 amino acids, corresponding sequence of pH77 genome GenBank No. AJ238799.1: 3918 to 5321) was expressed and purified in Escherichia coli BL21 (DE3) cells as recombinant proteins. The ns3h gene of HCV 1b (Con1) was cloned into pET21b(+) vector (Novagen). After sequencing, the right construct was transformed into E. coli BL21 (DE3) cells. Cells harboring plasmids were grown to an absorbance at 600 nm (A₆₀₀) of 0.6 and induced with 0.5 mM isopropyl-D-thiogalactopyranoside at 16°C overnight. Cells were harvested by centrifugation, washed in HEPES buffer (25 mM, pH 7.4) and resuspended in buffer A containing 25 mM HEPES (pH 7.4), 75 mM KCl, 10% glycerol, 1 mM DTT and 0.5 % protease inhibitor cocktail (Sigma–Aldrich). For purification, cells were lysed by sonication and centrifuged at 25,000 × g. The supernatant was then applied to a 5 mL HisTrap FF column (GE Healthcare) and equilibrated in buffer A. Then the column was eluted by increasing imidazole concentration to 300 mM using ÄKTA protein purification system (GE Healthcare). The fraction containing NS3h was confirmed by SDS-PAGE. Then the NS3h fraction was dialyzed against buffer A and loaded on a HiTrap Q FF column (GE Healthcare) to exclude nucleic acids contamination. The column was eluted by increasing KCl concentration to 500 mM. After SDS-PAGE confirmation, fractions containing NS3h were dialyzed again using buffer A, and then was concentrated and loaded onto HiLoad 16/600 Superdex 200 (GE healthcare) and eluted by buffer A for further purification. The purified NS3h was stored at -80°C. Protein concentration was quantified via spectrophotometry.

Plasmid pET11a::ns5b for expressing truncated NS5B (hydrophobic 21 amino acids were deleted at C terminal) of HCV 1b (Con1) was kindly provided by Prof. Helena Danielson, Uppsala University, Sweden (Abdurakhmanov, 2010). Recombinant protein NS5B was expressed, purified, stored and quantified as NS3h.

FP assays were performed in 96-well, flat bottom, black plates and the final volume of each reaction was 50 μL. The reaction system contained 5 nM Cy5-dT15 (synthesized by Sangon Biotech, China), 15 nM NS3h, 25 mM MOPS (pH 7.5), 1.25 mM MgCl₂, 0.0025 mg/mL BSA, 0.005 % (v/v) Tween 20 and 0.025 mM DTT. 0.5 μL compound dissolved in DMSO with a final concentration of 100 μM or 100 μg/mL was added. For each plate, blank wells (without NS3h and Cy5-dT15) and reference blank wells (without NS3h) were included. Positive control and negative control were primuline (Li et al., 2012) and DMSO, respectively. FP value was detected using a TECAN Infinite M1000 Pro multi-mode microplate reader, with detection wavelength of excitation 635 nm. Inhibition value was calculated as (mp_sample-mp_negative)/(mp_positive-mp_negative) × 100% (mp standards for polarization value).

Anti-NS5B assay was performed in 25 μL reaction system as reported (Eltahla et al., 2013). The reaction system contained 250 μM rGTP, 10 μg/mL poly(C) RNA (Sigma–Aldrich, p4903), 2.5 mM MnCl₂, 5 mM DTT, 0.01% BSA, and 0.005 % Tween-20 in 20 mM Tris-Cl (pH 7.5). 0.2 μL DMSO or compound dissolved in DMSO with a final concentration of 100 μM or 100 μg/mL was added to each well. Heat-inactivated NS5B was used as positive control. Plates were incubated at 30°C for 1 h. Then 175 μL PicoGreen dye (1:350 diluted in TE buffer) was added to a final volume of 200 μL. After covering from light for 5 min, FI was measured using a TECAN Infinite M1000 Pro multi-mode microplate reader at excitation wavelength of 485 nm and emission wavelength of 520 nm.

The Z′ factor and coefficient of variation (CV) values were used to evaluate the quality of HTS systems and were calculated as follows:

Z′ = 1-(3SD of positive control + 3SD of negative control)/| mean of positive control - mean of negative control|, SD: Standard deviation. The theoretical value is between 0.5 and 1 (Sui and Wu, 2007). CV(%) = SD_max/Mean_max or CV(%) = SD_min/Mean_min. The acceptable value of CV for HTS assay is less than 10%.

Whether purified NS3 helicase can bind to DNA substrate in vitro was tested using EMSA. The binding assays containing 50 mM Tris-Cl (pH 7.4), 10% glycerol, 100 nM DNA substrate (5′-Cy5-CC TAC GCC ACC AGC TCC GTA GG–3′ annealed to 5′-GGA GCT GGT GGC GTA GG (T)20-3′) (Mukherjee et al., 2012) and 650 nM NS3h were incubated at 0°C for 20 min. Aurintricarboxylic acid was used as positive control (Shadrick et al., 2013). A non-denaturated PAGE gel was pre-run at 4 °C for 30 min at 120 V. Samples were loaded and ran 40 min at 120 V. Then the resulting gel was visualized by fluorescence imaging using Typhoon FLA 9500 with the wavelength of 635 nm.

ATPase assays were assembled in 96-well, flat bottom, transparent plates on a TECAN Infinite M1000 Pro multi-mode microplate reader, with an absorbance at 630 nm, and the total reaction volume of each well was 50 μL. The final reaction system contained 15 nM NS3h, 1 mM ATP, 25 mM MOPS (pH 6.5), 1.25 mM MgCl₂, 5% DMSO, 50 μg/mL BSA and 0.01 % Tween 20. Plates were incubated at 23°C for 15 min. Then 200 μL malachite green solution (0.034% malachite green, 1 M HCl, 1% ammonium molybdate, and 0.025% Tween 20 followed within 10 s by addition of 25 μL of 35% sodium citrate) was added to each well for 20 min at 23°C to terminate the reaction.

Expression plasmid pET21b(+)::ssb was constructed and SSB protein was obtained as NS3h. The procedure for this assay was the same as that for NS3h except that E. coli SSB was used at 20 nM instead of NS3h.

Establishing effective HTS systems and identifying potential new HCV replication inhibitors were the primary objectives of this study. A FP-based ssDNA binding assay was selected for NS3h inhibitors discovery. NS3h of HCV 1b (Con1) was successfully expressed in E. coli BL21(DE3) cells and purified by Ni-NTA column chromatography, Q column chromatography and gel filtration column chromatography. NS3h has a molecular mass of 51.0 kDa (Figure 1A) and the total yield was 0.75 mg/L LB broth. The in vitro binding capacity of NS3h with nucleic acids was verified by EMSA, which indicated that purified NS3h could tightly bind to the oligo-dT sequence of dsDNA and that the NS3h-dsDNA complex migrated much more slowly than free dsDNA through a non-denaturing polyacrylamide gel (Figure 1B).

After optimization, the reaction conditions for this assay were 10–20 nM NS3h, 5 nM Cy5-dT15, 25 mM MOPS (pH 7.5), 0–1.5 mM MgCl₂, 0.0025 mg/mL BSA, 0.005 % (v/v) Tween 20 and 0.025 mM DTT. There were significant differences in FP values for DMSO concentration ranges of 0–40% (v/v) and the best range for this assay was 0–5%. Decreased FP values were detected with increasing concentrations of the positive control (primuline) or competitive substrate non-labeled dT20. FP-based binding assays were then performed in a high-throughput format to evaluate necessary precision and reproducibility. To determine well-to-well variation by FP-based binding assays, 40 negative controls (1% v/v of DMSO only) and 40 positive controls (100 μM primuline) were performed, and resulted in a Z′ factor of 0.69 (>0.5) (Figures 1C,D), which is considered acceptable for HTS system. The CV values of HTS system were CV_max = 2.6% and CV_min = 9.5%. Both values were less than the threshold value of 10%, which is recognized as delineation of correct assays.

Sets of small molecular compounds and crude extracts were screened by the FP-based ssDNA binding assay in 96-well plates. Samples were added to a final concentration of 100 μM or 100 μg/mL to the reaction system (Figure 2A and Supplementary Data Sheet 1). In additional positive control assays, primuline was added to a final concentration of 100 μM (Figure 2A). Three parallel repeats were employed for the primary FP screening and positive hits were selected for activity confirmation. Natural compound No.3A5, isolated from marine fungi, exhibited significantly lower FP signals than other samples and inhibited the ability of NS3h to bind Cy5-dT15 in a concentration dependent manner. The IC₅₀ value of compound No.3A5 was determined to be 2.8 μM (Figure 2B). Thus, compound No.3A5 was proposed to disrupt the interaction between NS3h and ssDNA and was selected for further study.

Compound No.3A5 has a molecular weight of 182.1 Da and has no previously reported biological activity. Therefore, EMSA assay was performed to further validate the effects of compound No.3A5 on the interaction of NS3h and nucleic acids. The reaction system included 50 mM Tris-Cl (pH 7.4), 10% glycerol, 100 nM DNA substrate and 650 nM NS3h. Notably, in the presence of compound No.3A5, no NS3h-dsDNA complex band was detected, which showed the same result with aurintricarboxylic acid, a known NS3h inhibitor (Figure 3A). Protein NS3h catalyzes ATP hydrolysis and dsDNA/RNA unwinding and the unwinding ability of NS3h is dependent on ATP binding and ATP hydrolysis (Frick, 2007). We explored the effects of compound No.3A5 on the ATPase activity of NS3h. Firstly, the ATP hydrolysis ability of purified NS3h was investigated and we found that increasing dT20 concentration enhanced ATP hydrolysis (Figure 3B). This phenomenon verified that binding capacity of NS3h with nucleic acids needs energy produced by ATP hydrolyzation. Then, compound No.3A5 was added to the ATPase reaction solution. As shown in Figure 3C, with increasing concentrations of compound No.3A5 from 0 to 64 μM, decreased concentrations of Pi were detected, suggesting that compound No.3A5 could affect the ATPase activity of NS3h. Next, the ability of compound No.3A5 to function as a reversible inhibitor was tested. We incubated 1 μM NS3h with 3 μM compound No.3A5 for 60 min and then diluted the protein 60-fold while adding it to reaction system to determine if the protein retained the ability to bind ssDNA with a similar affinity as the protein when it was treated with DMSO only. Interestingly, when NS3h was treated with 3 μM compound No.3A5, the protein retained the ability to bind ssDNA after dilution. This suggests that there was no formation of covalent bond between compound No.3A5 and NS3h and the inhibition was reversible. Finally, to test if compound No.3A5 inhibited NS3h specifically, the same FP-based assay was repeated, substituting NS3h with the E. coli single-stranded DNA binding (SSB) protein. Gene ssb from E. coli DH5α genome was cloned and SSB protein (MW: 19.0 kDa) was expressed and purified as NS3h. Control primuline inhibited the ability of SSB to bind Cy5-dT15. Similarly, compound No.3A5 also inhibited the ability of E. coli SSB to bind Cy5-dT15, consistent with the result that there was no covalent bond formation between compound No.3A5 and NS3h. In contrast, competitive substrate non-labeled dT20 showed high selectivity on NS3h (>60% inhibition) and SSB protein (<10% inhibition) (Figures 3D,E). So non-specific interaction of compound No.3A5 with ssDNA binding proteins was detected.

In this study, the FI-based dsRNA formation assay was selected for discovery of NS5B inhibitors. Truncated NS5B of strain 1b (Con1) (21 hydrophobic amino acids deletion at C terminal, MW: 63.2 kDa) was expressed and purified as described (Figure 4A). As reported by Yamashita et al. (1998), NS5B catalyzes the formation of dsRNA from ssRNA template in vitro. Combined with PicoGreen, a reagent that preferentially binds dsRNA compared with ssRNA, can be used to quantify dsRNA by detecting FI excited at wavelength 485 nm (Mastrangelo et al., 2012). The catalytic ability of purified truncated NS5B was confirmed, with a significant increase of dsRNA (Figure 4B). Meanwhile, with increasing concentrations of truncated NS5B, the FI was greatly enhanced (Figure 4C). Therefore, we sought to establish another HTS system based on the truncated NS5B [HCV 1b (Con1)] and ssRNA, and FI was used for dsRNA amount detection. Tolerance to BSA, DMSO and Tween-20 was tested to optimize concentrations for this HTS system. The final reaction solution in 96-well plate was considered as 230 μM rGTP, 10 μg/mL poly(C) RNA, 2.5 mM MnCl₂, 5 mM DTT, 0–0.01% BSA, and 0–0.005 % Tween-20 in 20 mM Tris-Cl (pH 7.5), 0–4% DMSO. The final value of Z’ factor was 0.67.

Of all the tested samples (Figure 4D and Supplementary Data Sheet 2), compounds No.39 and No.94 with low FI values were confirmed after primary and secondary screening. They both reacted in a dose-dependent manner and the IC₅₀ values were determined to be 62.9 and 18.7 μM, respectively (Figure 5). These two compounds are both known as tyrosine kinase inhibitors. In this study, disrupting dsRNA formation is the novel function of them, which is worth further study.