2024 AIChE Annual Meeting

Screening Small Molecule Inhibitors of Prostate Cancer with an Adaptable Reporter Gene Assay

The androgen receptor (AR) is the predominant therapeutic target for men with prostate cancer. Patients with androgen-dependent prostate cancer are often treated with surgical castration or androgen ablation therapy. Patients with castration-resistant prostate cancer (CRPC) are treated with small-molecule AR inhibitors or chemotherapy. However, patients treated with FDA-approved AR small molecule inhibitors (e.g. enzalutamide) often develop resistance (1). In order to leverage a model system that would allow the screening of novel drug candidates for enzalutamide resistance, we developed an AR-based cell line that facilitates high-throughput in vitro drug screening.

The PC3 prostate cancer cell line does not have endogenous AR. We ectopically expressed AR with a modified lentiviral luciferin linked to an androgen response element (2). To benchmark these cells, we screened a 12-point dilution of small molecule AR antagonists with R1881 (20 nM, synthetic testosterone) and a vehicle control. Our results indicate that this cell line can provide a quantitative readout of androgen receptor activity with R1881 within physiological levels of androgen-dependent cell lines (ENZ IC50: 358.28 nM ± 14.74 nM). This system can also be genetically modified with AR mutations that have been found in men with enzalutamide resistance. This would facilitate a readout of the continuum of prostate cancer, starting from androgen-dependent to CRPC-resistant disease in an isogenic model.

This cell line and screening can also be applied beyond small molecule inhibitors. Small interfering RNAs (siRNA) are able to silence gene expression by degrading mRNA. This model can detect a knockdown of AR activity by testing siRNAs that target AR against R1881, a scramble and transfection reagent alone. We have screened two different siRNAs that target a different binding site of AR and received 82% and 86% knockdown when compared against scramble with R1881. We are currently optimizing this model to test multiple dilutions of siRNA and produce a dose response curve.

This assay successfully characterized an AR-based prostate cancer cell line while leaving room for novel studies on enzalutamide resistance. This system is also not limited to drug screenings, but can also be used to detect knockdown of AR activity. Our preliminary data with siRNAs targeting AR achieved detectable levels of knockdown, and ongoing optimization of this model will enable more insightful dose-response studies. This assay is a versatile and robust tool for drug screening and the development of new therapeutics.





References:

1. P. A. Watson, V. K. Arora, and C. L. Sawyers, “Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer,” vol. 15, no. 12. Springer Science and Business Media LLC, p. 701, Feb. 29, 2016, doi: 10.1038/nrc4016.

2. K. Patsch et al., “Paradoxical androgen receptor regulation by small molecule enantiomers,” vol. 118, no. 12, p. e2100918118, 2021, doi: 10.1073/pnas.2100918118.