Detection of endogenous and drug-modulated CYP3A activity in cells using LC-MS/MS

Abstract Most currently-prescribed drugs are metabolized by the Cytochrome P450 3A (CYP3A) family. It is therefore critical to know whether drugs or compounds of interest inhibit these enzymes (primarily CYP3A4 and CYP3A5). Here, we describe an LC-MS/MS based method to measure CYP3A catalytic activity in cells. Our method allows for highly sensitive detection of drug-induced modulations in CYP3A activity, as well as endogenous (non-treated) activity. This is accomplished by exploiting the CYP3A-catalyzed hydroxylation of midazolam. We include DMSO and inhibitor controls to obtain a signal window capable of producing normalized dose-response curves. We also obtain tight error bars suitable for statistical interpretation, which highlights the reproducibility of the assay between replicates. While our method is performed in 384-well plates and is amenable to high-throughput screening – an optional plate freezing step allows for storage of samples before metabolite detection, making our protocol suitable for lower throughputs as well. We present a stepwise protocol starting from treatment of cells with a compound of interest to obtaining its IC50 value and determining CYP3A inhibition.