title: High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic vivid substrates.
abstract: CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.
authors.affiliation: PanVera, Madison, WI 53719, USA.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Marks
authors.forename: Tony A
authors.initials: TA
authors.lastname: Goossens
authors.forename: Heidi A
authors.initials: HA
authors.lastname: Braun
authors.forename: Mary S
authors.initials: MS
authors.lastname: Ozers
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Smith
authors.forename: Connie
authors.initials: C
authors.lastname: Lebakken
authors.forename: Olga V
authors.initials: OV
authors.lastname: Trubetskoy
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical; D065702:Cytochrome P-450 CYP2B6; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012997:Solvents; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D012997:Solvents; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating