A Cell-Based Renilla Luminescence Reporter Plasmid Assay for High-Throughput Screening to Identify Novel FDA-Approved Drug Inhibitors of HPV-16 Infection

Abstract

Like cervical cancer, anal cancer is caused by human papillomavirus (HPV). HPV is the most common sexually transmitted agent and is found in the anal canal of almost all HIV-positive men who have sex with men (MSM). Rates of HPV anal cancer are disproportionately higher in this population. Although the nanovalent HPV vaccine is efficacious in protecting against oncogenic HPV types, a substantial proportion of MSM remains unvaccinated and anal HPV infection continues to be an important public health burden. Therefore, it is important to identify strategies to prevent HPV infection. We report on two promising and interlinked strategies: (1) the development of a cell-based Renilla luminescence reporter assay using HPV-16 pseudovirions that encapsidate SV40-driven Renilla luminescence reporter expression plasmid and (2) use of this assay for high-throughput screening (HTS) of FDA- and internationally approved drugs to identify those that could be repurposed to prevent HPV infection. We conducted a screen of 1906 drugs. The assay was valid with a Z' of 0.67 ± 0.04, percent coefficient of variance of 10.0, and signal-to-background noise window of 424.0 ± 8.0. Five drugs were chosen for further analyses based on selection parameters of ≥77.0% infection of HPV-16 pseudovirion-driven Renilla expression with <20.0% cytotoxicity. Of these, the antifungal pentamidine and a gamma-amino butyric acid receptor agonist securinine exhibited ≥90.0% infection with <10.0% cytotoxicity. This luminescent cell-based reporter expression plasmid assay for HTS is a valid method to identify FDA- and internationally approved drugs with the potential to be repurposed into prevention modalities for HPV infection.

Keywords: HPV-16; high-throughput screening; papillomavirus; pseudovirus; repurposed.

Figure 1.

High-throughput screen for identifying inhibitors of HPV infection. Shown is a schematic illustrating the basic steps in the HTS. See Materials and Methods for additional details. Briefly, HPV16-PsVs containing the Renilla reporter genes are analyzed for their ability to infect as judged by the levels of luminescence. Cell viability was assessed in parallel 384-well plates. In the specific HTS carried out in this study, the compounds were added at a 8.3 μM/0.3% DMSO final concentration 2 h prior to infection.

Figure 2.

Assay validation. (A) Screen performance. Luminescence signals (gray diamonds) of positive and negative controls in six plates assayed during the screen (n = 32 for each control per plate, with mean and standard deviation bars). Panel A shows the signal-to-background window (S/B) and assay performance parameter Z′ of controls in each assay plate. The screen was robust with a wide assay window (S/B > 400) and excellent assay performance (control Z′ > 0.5). (B) Hit selection for inhibitors of HPV infection. Screening of 1906 clinical drugs (gray diamonds). The y axis represents percent inhibition of HPV infections and the x axis represents the fold change of cell toxicity. The black diamonds represent hits. WI-38 cells, normal human lung cells, were used as a positive control in a corresponding counterscreen to filter out compound toxicity.

Figure 3.

Two lead FDA drugs. (A) Pentamidine exhibited more than 100-fold selective inhibition of HPV16-PsV infection (IC₅₀ ≤ 0.002 μM) compared with cell viability (IC₅₀ = 0.08 μM). (B) Securinine exhibited 90-fold selective inhibition of HPV 16-PsV infection (IC₅₀ = 9 μM) compared with cell viability (IC₅₀ = 42.0 μM).

Figure 4.

Percent fold change of infection and cell viability. (A) Pentamidine and securinine were tested in a secondary stock threefold dose–response for their ability to prevent HPV infection compared with the positive control (PsV + cells) and negative control (cells only). (B) Pentamidine and securinine were tested in a secondary stock threefold dose–response for cell viability compared with the positive control (cell only).