title: Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition.
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 IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) 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.
authors: R&D, Promega Biosciences, San Luis Obispo, CA 93401, USA. pmeisenh@promega.com
authors: Poncho L
authors: PL
authors: Meisenheimer
authors: H Tetsuo
authors: HT
authors: Uyeda
authors: Dongping
authors: D
authors: Ma
authors: Mary
authors: M
authors: Sobol
authors: Mark G
authors: MG
authors: McDougall
authors: Cesear
authors: C
authors: Corona
authors: Dan
authors: D
authors: Simpson
authors: Dieter H
authors: DH
authors: Klaubert
authors: James J
authors: JJ
authors: Cali
mesh_terms: D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D049454:Firefly Luciferin / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D008862:Microsomes, Liver / Q000201:enzymology; D015335:Molecular Probes; D013379:Substrate Specificity
chemical_list: D065692:Cytochrome P-450 CYP3A Inhibitors; D015335:Molecular Probes; D049454:Firefly Luciferin; D051544:Cytochrome P-450 CYP3A