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.

Probing the activity of cytochrome P450 3A4 (CYP3A4) is critical for monitoring the metabolism of pharmaceuticals and identifying drug–drug interactions. A library of Ir(III) probes that detect occupancy of the CYP3A4 active site were synthesized and characterized. These probes show selectivity for CYP3A4 inhibition, low cellular toxicity, K d values as low as 9 nM, and are highly emissive with lifetimes up to 3.8 μs in cell growth media under aerobic conditions. These long emission lifetimes allow for time-resolved gating to distinguish probe from background autofluorescence from growth media and live cells. X-ray crystallographic analysis revealed structure–activity relationships and the preference or indifference of CYP3A4 toward resolved stereoisomers. Ir(III)-based probes show emission quenching upon CYP3A4 binding, then emission increases following displacement with CYP3A4 inhibitors or substrates. Importantly, the lead probes inhibit the activity of CYP3A4 at concentrations as low as 300 nM in CYP3A4-overexpressing HepG2 cells that accurately mimic human hepatic drug metabolism. Thus, the Ir(III)-based agents show promise as novel chemical tools for monitoring CYP3A4 active site occupancy in a high-throughput manner to gain insight into drug metabolism and drug–drug interactions.

The research being presented herein is focused on making a drug that competitively and selectively inhibits cyclooxygenase‐2 (COX‐2) in response to a given wavelength of light, thereby reducing side effects. Cyclooxygenase‐2 is an enzyme primarily responsible for synthesizing prostaglandin, which generates inflammation in response to an infection or injury. Overproduction of prostaglandin can lead to painful inflammation like rheumatoid arthritis and osteoarthritis. Therefore, a COX‐2 inhibitor can solve this issue of inflammation by inhibiting the synthesis of prostaglandin.Before the advent of selective COX‐2 inhibitors, drugs targeting prostaglandin production were not as selective and also inhibited Cyclooxygenase‐1 (COX‐1), which could lead to unwanted side effects, like ulcers and gastrointestinal toxicity. To address this problem, “coxib” drugs were created to selectively inhibit COX‐2. While these drugs worked well in reducing inflammation, long‐term use was found to increase the risk of adverse vascular side effects. These side effects are thought to arise, in part, as a result of the role of COX‐2 in the maintenance of the vascular system. The research being presented aims to eliminate this side effect by making a drug that can target COX‐2 at the site of inflammation, without affecting the vascular system.The drug being synthesized is an azologue of a validated COX‐2 selective inhibitor. Azologues are modified to contain a nitrogen‐nitrogen double bond between two aromatic rings. Molecules containing this moiety can change conformation trans to cis or cis to trans in response to near UV or visible light, respectively. It is this change in conformation that creates a difference in target binding in the presence or absence of the appropriate wavelength of light. The target compound was designed to exist in the trans conformation in the absence of light. In silico binding analysis suggests that this is the active conformation of the inhibitor. The synthesis of the target molecule will be disclosed and its photophysical characterization by UV/Vis spectrophotometry will be outlined.Support or Funding InformationAlbion College Foundation for Undergraduate Research, Scholarship and Creativity ActivityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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