A high-throughput cell-based screening method for Zika virus protease inhibitor discovery

Abstract

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Keywords: Cell-based assay; HTS; Protease inhibitors; Zika virus.

Fig. 1.

Design and characterisation of cycLuc_ZIKV-NS2B reporters and ZIKV protease constructs. A. Schematic representation of the expression and activation of the cyclised circularly permuted firefly luciferase (cycLuc_ZIKV-NS2B) reporter. B. Schematic representation of NS2B-NS3 constructs generated in this study. C. Activation of cycLuc_ZIKV-NS2B reporters by recombinant ZIKV protease. Various cycLuc reporters were co-expressed with either wild-type or S135A alleles of NS2B_1–130—−NS3_Pro. Data are representative of two independent experiments performed in a technical singlicate. D. Dual_cycLuc_ZIKV-NS2B reporter assay for various ZIKV protease constructs. 293T cells were transfected with 10 - 40 ng plasmid DNA of four different ZIKV protease constructs. Normalised cycLuc activity is shown. Expression levels of various ZIKV protease constructs were probed using Western blot analysis using an anti-FLAG M2 antibody. Filled triangles represent the expected size of each overexpressed ZIKV protease variant. Data are representative of three independent experiments performed in a technical singlicate. E. Dual_cycLuc_ZIKV-NS2B reporter assay for various ZIKV protease constructs. 293T cells were co-transfected with the dual_cycLuc_ZIKV-NS2B reporter, 5 ng GFP-FLAG, and 20 ng of four different ZIKV protease constructs. The S135A alleles of respective ZIKV proteases were used as a negative control for protease activity. Normalised cycLuc activity is presented. Expression levels of various ZIKV protease constructs were probed through Western blot analysis using an anti-FLAG M2 antibody. Filled triangles represent the expected size of each overexpressed ZIKV protease variant. Co-transfected GFP-FLAG (open arrow) served as an internal loading control. Data are representative of three independent experiments performed in a technical singlicate.

Fig. 2.

Initial validation of dual cycLuc_ZIKV-NS2B reporter in 96-well plate format. A. Overall cycLuc and normalised cycLuc activities from low and high luminescence samples. Z’ and signal-to-background (S/B) ratios for 25 ng NS2B_1–130—NS3_FL (construct A). B. Similar results as in A are also shown for 10 ng NS2B_46–130—NS3_Pro (construct C). C. Dose-response inhibition curves for JNJ-40,418,677, MK-591, and MI-2110 compounds against recombinant ZIKV proteases bZiPro (left panel) and gZiPro (middle panel) as determined using biochemical protease assay. Data represent the mean ± SEM (n = 3). Summary of IC₅₀ and 95 % CI values for JNJ-40,418,677, MK-591, and MI-2110 against bZiPro and gZiPro proteins is shown in right panel. D. 293T cells co-transfected with dual_cycLuc_ZIKV-NS2B and NS2B₄₆–₁₃₀—NS3_Pro plasmids were treated with serially diluted JNJ-40,418,677 or MK-591. DMSO was used as a control. cycLuc was measured 24 h post-compound treatment using the Dual-Glo^® Luciferase assay. E. Cytotoxicity of JNJ-40,418,677 and MK-591 on 293T cells as measured using CCK-8 assay. Assays in (D-E) were conducted in technical quadruplicates and error bars represent SEM.

Fig. 3.

Optimisation of HTS assay in 1536-well plate format and LOPAC screening A. 1536-well HTS assay with cells from either cultured or frozen conditions, seeded at densities of 938, 1875, and 3750 cells per well. Data represent the mean ± SD of RLU (n = 40 wells). B. Assessment of 0.75 % v/v DMSO impact on the 1536-well HTS assay under varying cell densities. Data represent the mean ± SD of RLU (n = 24 wells). C. Summary of IC₅₀ and 95 % CI values for JNJ-40,418,677 and MK-591 against NS2B_46–130—NS3_Pro across various cell densities. D. Scatterplot representation of the inhibition efficiency values of the LOPAC compounds against NS2B_46–130—NS3_Pro. Black dots represent individual compound values and coloured dots depict the controls. The blue dashed line denotes the 38.86 % inhibition threshold for hits. The screening statistics are summarised in the adjacent box to the graph.