Exploitation of the cancer biomarker alpha methylacyl coenzyme A racemase for cancer imaging and therapeutics

Alpha-methylacyl coenzyme A racemase (AMACR) is a metabolic enzyme whose over-expression has been shown to be a diagnostic indicator of prostatic adenocarcinoma as well as other solid tumors. The central purpose of this dissertation is to discover inhibitors of this enzyme that may be useful either as chemotherapeutic agents or potentially as radiolabeled imaging agents for cancers that over-express this enzyme. First we confirm that attenuation of AMACR expression diminishes the growth of prostate cancer cell lines using stably expressed shRNA constructs. This observation strongly suggests that the AMACR enzyme may be a target for therapeutic inhibition in prostate cancer. To this end, we report here a novel assay capable of screening libraries of diverse small molecules for inhibitors of AMACR activity. This assay facilitated the screening of approximately 5,000 unique compounds and the discovery of seven distinct chemical entities capable of inhibiting AMACR at low micromolar concentrations. The most potent inhibitor discovered is the seleno-organic compound ebselen oxide (IC50:0.80 microM). The parent compound, ebselen (IC50:2.79 microM), is a covalent inactivator of AMACR (KI(inact):24 microM). Two of the AMACR inhibitors appear selectively toxic to prostate cancer cell lines (LAPC4/LNCaP/PC3) that express AMACR compared to a normal prostate fibroblast cell line (WPMY1) that does not express the protein. This selective AMACR specific toxicity indicates that not only does chemical inhibition of AMACR offer some promise of the chemotherapeutic efficacy of AMACR inhibition, but also that there is a therapeutic window in which cancer cells that over-express AMACR can be specifically targeted for cytotoxicity while sparing those normal cells that do not express high levels of the AMACR protein. This dissertation demonstrates the first high-throughput screen for the discovery of novel AMACR inhibitors, characterizes the first non-substrate based inhibitors, and validates that AMACR is a viable chemotherapeutic target in-vitro.