High-throughput screening identifies a bisphenol inhibitor of SV40 large T antigen ATPase activity

Abstract

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC(50), cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC(50) of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.

Figure 1

Comparison of assay sensitivity using the malachite green and adenosine diphosphate (ADP) Hunter endpoint methods. (A) The increase in signal during substrate titration (phosphate or ADP, respectively) is shown. The shaded area is expanded for clarity. (B) The dependence of the T antigen (TAg)–mediated ATPase activity on enzyme concentration and time. In total, 1 μM adenosine triphosphate (ATP) was incubated with increasing amounts of TAg, and the amount of ATP hydrolysis (fluorescence determined using ADP Hunter) was measured at 60, 120, or 240 min. Each point represents the mean of three assays. Linear regression was used to derive equations for each line (60, 120, and 240 min) of y = 524072x +10⁶ (R² = 0.9748), y = 473438x +10⁶ (R² = 0.9978), and y = 310381x +2×10⁶ (R² = 0.9885), respectively. To simplify the figure, error bars are not shown; standard deviation for each point was <2%.

Figure 2

Compounds confirmed as active against SV40 T antigen (TAg) ATPase activity in a biochemical assay. Structures of confirmed hits and control compound MAL2-11B are depicted. The corresponding biochemical inhibitory concentrations (IC₅₀), the cell-based effective concentrations (EC₅₀), and the cytotoxic concentrations (TC₅₀) are tabulated for each compound. ND, values were not determined because of high compound cytotoxicity.

Figure 3

Specificity for T antigen (TAg) is demonstrated by DNA replication assays. The figure compares the effects of increasing concentrations of MAL2-11B (2-11B) and SIDs 85281215 and 92277586 on TAg-dependent DNA replication. Products were analyzed by urea–polyacrylamide (PA) gel electrophoresis. Polymerization of high molecular weight DNA (HMW DNA) increases in uninhibited lanes and decreases where compound-mediated enzymatic inhibition occurs. The concentration of compound in μM is indicated at the top. Increasing reaction time is indicated by a black wedge.

Figure 4

(A) Analog design based on hit SID 85281215 leading to bisphenol A, SID 92277586, and (B) regions of optimization and structure–activity relationship (SAR) investigation of the bisphenol scaffold, indicated by shading.

Figure 5

In vitro inhibition of SV40 virus replication. Compounds were tested (200–1.26 μM) in cytoprotection assays using BSC-40 cells, and % cell viability was measured 5 days post–drug or post–viral infection. The dashed line represents cells + compound only (cytotoxicity), and the solid line represents cells + compound + virus (cytoprotection). The curves are plotted but not fit to a specific mathematical model. The EC₅₀ and TC₅₀ values given in Figure 2 were derived using the equation described in Materials and Methods.