Levels of the enzyme α-methylacyl-CoA racemase (AMACR) are increased ca. 9-fold in prostate cancer cells. AMACR is a very promising novel drug target as reducing AMACR levels converts castrate-resistant prostate cancer cells to androgen-dependent cells which will respond to androgen-deprivation. Despite the importance of AMACR in prostate and other cancers, there are very limited numbers of AMACR inhibitors described to-date. This is mainly due to the absence of a high-throughput assay for the screening of inhibitors against AMACR. The active-site residues and catalytic mechanism of human AMACR are still unknown, which make the rational design of drugs targeting AMACR very difficult. A range of novel potential inhibitors were synthesised using a rational drug design approach to explore the structure-activity relationship (SAR) on the side-chains of AMACR inhibitors. Their potencies were assessed using the fluoride elimination assay based on 1H and 19F NMR. Potency, mode of binding and kinetic parameters of these inhibitors were assessed using the multi-well colorimetric assay, which is the first AMACR high-throughput continuous assay reported to-date. A site-directed mutagenesis study was carried out to identify the active-site residues and catalytic mechanism of human AMACR. His-122, Asp-152, Met-184 and Glu-237 were identified as potential active-site residues, so the cDNA was mutated and expressed. The activity of wild-type and mutant AMACR enzymes were assessed using the deuterium wash-in, fluoride elimination and multi-well colorimetric assays. Results from these assays showed that human AMACR does not operate using a ‘two-base’ mechanism. Instead, it operates using a ‘one-base’ mechanism, most likely via water molecules acting as intermediaries within the hydrogen-bondings network in the active site. The knowledge obtained from this research informs rational drug design for this castrate-resistant prostate cancer target.

The androgen receptor’s (AR) resurgence following treatment leads to castration resistant prostate cancer (CRPC). Studies show that the aryl hydrocarbon receptor (AhR) regulates AR signaling, is constitutively active, and enhances AR signaling in CRPC. AhR has ligands with carcinogenic properties and interacts with phytochemicals with anti-tumorigenic properties. Curcumin inhibits AhR activity and multidrug transporter ABCG2 activity, which mediates substrates out of the cell. Elevated ABCG2 expression causes resistance to anticancer drugs. AhR transcriptionally activates ABCG2 and our hypothesis is that inhibition of AhR activity by curcumin will decrease ABCG2 expression and activity in CRPC cells. C4-2 cells were treated with increasing concentrations of curcumin (0, 10, 25, 50µM) and CH223191 (50µM). Results show that curcumin decreases AhR, CYP1B1 and ABCG2 gene expression. Higher concentrations of curcumin diminish AhR and ABCG2 protein expression, ABCG2 activity, and cell proliferation. These results will help reveal a role for AhR in drug resistance.