Development of Abiraterone-based Photoaffinity Probe and Evaluation of their Labeling Efficiency for CYP17A1

Abiraterone, a drug approved by US Food and Drug Administration at the end of 2012 for Castration-Resistant Prostate Cancer, is an inhibitor of CYP17A1, which catalyzes the steroid biosynthesis to produce the androgen in adrenal cortex. Up to date, most in vitro tumor cell lines research or in vivo clinic trials focused on the evaluation of the abiraterone effects on relative protein up/down regulation or the level of specific sterols in the hormonal signal pathways. Research works involving the drug mechanism of action as well as ligand-based protein screening are relatively rare. In this study, I applied the Activity-Based Protein Profiling (ABPP) concept to develop abiraterone-based photoaffinity probes to profile abiraterone’s possible working targets. The probes are composed of abiraterone (ligand) as a recognition unit, daizirine moiety as a photoreactive group and a biotin tag as an output. The working hypothesis is that once the ligand recognized by the target protein, the daizirine could be brought to the vicinity of the target protein and subsequently forms a covalent bond with the target protein upon irradiation at 365 nm. After washing off the nonspecific binding proteins, the covalent-linked proteins can be further purified and characterized either by the tag purification or tag immunohistochemistry via biotin. Based on the molecular docking result and the reported X-ray structure of abiraterone-CYP17A1 ternary complex, abiraterone was derivatized at the C7 position with α configuration to minimize the structural perturbation to bind CYP17A1. The probes, denoted by Ab-C7α-PB and Ab-C7α-P, were successfully synthesized by assembling different functional modules via the designed connectivities. The configuration at C7 was confirmed to be α by several different NMR techniques. To exploit the utility of the probes, H295 cell line was used since it contains abiraterone binding protein-CYP17A1. In vitro photolabeling results showed that Ab-C7α-PB failed to label CYP17A1 by using many kinds of lysis buffers. On the other hand, if Ab-C7α-PB was incubated and photoactivated under native cellular environments in vivo, subsequent Western Blot showed that Ab-C7α-PB could successfully label CYP17A implicating that Ab-C7α-PB could function as an effective photoaffinity probe for labeling CYP17A1 and profiling the abiraterone regulating proteins.