Drug Target Identification and Elucidation of Mechanism of Action of ARKILs

Prostate cancer cells require the transcription factor Androgen Receptor (AR) for their growth and survival. The current standard of care for patients diagnosed with prostate cancer is anti-androgen therapy, which is not curative and leads to relapse and more advanced Castration Resistant Prostate Cancer (CRPC). ARKILs (Androgen Receptor Kil-ling) are small molecules that were developed to inhibit the AR in CRPC. The purpose of this study was to find a protein target for ARKILs and further explore its mechanism of action. To find protein targets two methods were chosen; DARTs (Drug affinity Responsive Target Stability) and affinity pull down of biotinylated c52. DARTs uses the principle that a small molecule-protein complex is less susceptible to protease degradation. Affinity pull down involves fishing out a biotin small molecule probe and target using streptavidin beads. Both of these methods were performed in parallel and several candidate protein targets were found. ATP-dependent helicase DDX3X and exportin 7 are two main targets that are now undergoing validation. Our studies also revealed a broader profile of toxicity that included several AR negative breast cancer cell lines. This indicated that AR inhibition might not be the only way ARKILs cause cell death. Another very interesting characteristic observed is that ARKILs cause apoptosis (cellular death) in both wild type p53 and mutant p53 sensitive cell lines, which suggested a dual role of ARKILs action. The tissue specificity and p53 dependent/independent mechanism of ARKILS is very important and finding a target for ARKILs could help reveal an unknown common feature. In the future this target could be used as potential biomarker for patient selection and help develop better, more specific therapies for breast and prostate cancer.