title: Evaluation of time-dependent cytochrome p450 inhibition in a high-throughput, automated assay: introducing a novel area under the curve shift approach.
abstract: Early in the drug discovery process, the identification of cytochrome P450 (CYP) time-dependent inhibition (TDI) is an important step for compound optimization. Here we describe a high-throughput, automated method for the evaluation of TDI utilizing human liver microsomes and conventional CYP-specific mass spectrometer-based probes in a 384-well format. One of the key differences from other published TDI assays is the use of a shift in area the under curve of the percent activity remaining versus inhibitor concentration plot (AUC shift) rather than the traditional fold-shift in IC50, to determine the magnitude of TDI. An AUC shift of < 15% suggests negative TDI and > 15% suggests potential TDI. This AUC shift was used to achieve quantitative data reporting, even in the case of weak inhibitors for which IC50 values cannot be quantified. An Agilent Technologies BioCel 1200 System was programmed such that the TDI liability of up to 77 test compounds, incubated at four test concentrations, with and without NADPH in the pre-incubation, can be analyzed in a single run. The detailed automated methodology, assay validation, data reporting and the novel TDI AUC shift approach to describe magnitude of TDI are presented.
authors.affiliation: Genentech Inc., 1 DNA Way, MS412A, South San Francisco, CA 94080, USA.
authors.forename: Sophie
authors.initials: S
authors.lastname: Mukadam
authors.forename: Suzanne
authors.initials: S
authors.lastname: Tay
authors.forename: Daniel
authors.initials: D
authors.lastname: Tran
authors.forename: Leslie
authors.initials: L
authors.lastname: Wang
authors.forename: Erlie M
authors.initials: EM
authors.lastname: Delarosa
authors.forename: S Cyrus
authors.initials: SC
authors.lastname: Khojasteh
authors.forename: Jason S
authors.initials: JS
authors.lastname: Halladay
authors.forename: Jane R
authors.initials: JR
authors.lastname: Kenny
mesh_terms: D019540:Area Under Curve; D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D009249:NADP