Abstract

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester—K_m and k_cat are 2.88 μM and 5.87 pmol metabolite · min⁻¹ · pmol enzyme⁻¹, respectively. The proluciferin 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 high-performance liquid chromatography-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.