title: High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis.
abstract: Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, California, 94080, USA. halladay@gene.com
authors.forename: Jason S
authors.initials: JS
authors.lastname: Halladay
authors.forename: Erlie Marie
authors.initials: EM
authors.lastname: Delarosa
authors.forename: Daniel
authors.initials: D
authors.lastname: Tran
authors.forename: Leslie
authors.initials: L
authors.lastname: Wang
authors.forename: Susan
authors.initials: S
authors.lastname: Wong
authors.forename: S Cyrus
authors.initials: SC
authors.lastname: Khojasteh
mesh_terms: D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D007527:Isoenzymes; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes