Use of stable isotope labeled probes to facilitate liquid chromatography/mass spectrometry based high-throughput screening of time-dependent CYP inhibitors.

Inhibition curve shift is a commonly used approach for screening of time-dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co-incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time-dependent incubation (TDI), the test compound is pre-incubated with NADPH-fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP-specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre-incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP-specific probes are used for the TDI, and non-labeled substrates are included in the control incubation. Because CYP-specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96-well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource.