Ir(III)-Based Agents for Monitoring the Cytochrome P450 3A4 Active Site Occupancy

Abstract

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 K d and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

Dynamic Ir(III) Photosensors for the Major Human Drug-Metabolizing Enzyme Cytochrome P450 3A4

“…Consistent with our previous results, 42 pq-containing complexes 5−7, 14, and 15 were the most emissive overall, with emission quantum yields as high as 0.18 (2) in deaerated H 2 O (Table 2). Similar Φ em values were measured for the dfpp-based complexes, 8−10, whereas those containing btq cyclometallating ligands, 11 and 12, exhibit lower quantum yields.…”

“…Both the IC 50 and K d values were in the nanomolar range with no strong correlation (Table ). The IC 50 data indicate that the larger pq-containing complexes were more potent inhibitors than the smaller dfpp analogues, which is consistent with our previous observations on pq vs 2-phenylpyridine (ppy) analogues . The btq-containing complexes followed the trend and, on average, were more potent than the dfpp probes.…”

“…We recently reported a potent Ir(III)-based CYP3A4 inhibitor that detects active site occupancy by emission assays (Figure 2). 42 This work indicated that CYP3A4 bound more strongly to monocationic Ir(III) complexes compared to related dicationic Ru(II) systems. 42 This finding led us to conserve the Ir(III) center in our new series of CYP3A4 sensors.…”

“…42 This work indicated that CYP3A4 bound more strongly to monocationic Ir(III) complexes compared to related dicationic Ru(II) systems. 42 This finding led us to conserve the Ir(III) center in our new series of CYP3A4 sensors. Moreover, we found that the complex containing the large, hydrophobic ligand 2phenylquinoline (pq) showed a higher affinity for CYP3A4 than those derived from smaller phenylpyridine (ppy) ligands.…”

“…Our previous pq-based compound exhibited the largest emission yield (Φ = 0.54 ± 0.06, deoxygenated CH 3 CN and 0.086 ± 0.009, deoxygenated H 2 O) and emission lifetime (τ = 1.6 μs, deoxygenated H 2 O). 42 Ir(III) complexes containing dfpp ligands have been reported to have Φ = 0.96 ± 0.10 and long phosphorescence lifetimes, τ = 0.66 μs, in deoxygenated CH 3 CN. 44 In addition, the larger extended π-electronic system of the btq ligand results in emission wavelengths that are redshifted compared to those with ligands with smaller π-systems.…”