A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.
Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each.
AbbVie, Drug Metabolism, North Chicago, Illinois.
Xiaofeng
X
Li
AbbVie, Drug Metabolism, North Chicago, Illinois.
Tom
T
Suhar
AbbVie, Drug Metabolism, North Chicago, Illinois.
Lateca
L
Glass
AbbVie, Drug Metabolism, North Chicago, Illinois.
Ganesh
G
Rajaraman
D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D010641:Phenotype; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System
cytochrome P450; enzyme reaction phenotyping; recombinant human CYP