Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl2 (tpy = 2,2:6,2″-terpyridine) with L = 6,6-dimethyl-2,2-bipyridine (Me2bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2,3-c]phenazine (Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2,3-c]phenazine (Me2Ph2dppn; 11), [Ru(tpy)(Me2bpy)(4)]Cl2 (7) and [Ru(tpy)(Me2dppn)(4)]Cl2 (9). Photochemical release of 4 or 6 from 7-11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9-11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.