Abstract
Cytochrome P450 assays employ probe substrates to interrogate the influence of NCE's towards CYP enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates which are oxidized by CYP enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified CYP enzymes. In particular, one pro-luciferin acetal has demonstrated sensitive and selective CYP3A4 catalyzed oxidation to a luciferin ester - Km and kcat are 2.88 uM and 5.87 pmol metabolite/min/pmol enzyme, respectively. The pro-luciferin acetal was used as a probe substrate to measure IC50. values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC50 values for the known inhibitors correlate strongly with IC50 values calculated from the traditional HPLC based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.
- cytochrome P450
- cytochrome P450 catalyzed oxidations
- cytochrome P450 function
- drug toxicity
- drug-drug interactions
- drug-induced hepatotoxicity
- hepatocytes
- human CYP enzymes
- liver microsomes
- Received July 14, 2011.
- Accepted September 2, 2011.
- The American Society for Pharmacology and Experimental Therapeutics