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

• Published in: Rapid communications in mass spectrometry Vol. 24; no. 15; pp. 2177 - 2185
• Main Authors: Dasgupta, Malini, Tang, Weimin, Caldwell, Gary W., Yan, Zhengyin
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15-08-2010
• Subjects: Chromatography, Liquid - methods; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors - analysis; Fortified; High-Throughput Screening Assays - methods; Humans; Inhibition; Inhibitors; Isotope Labeling; Isotopes; Liquid chromatography; Mass spectrometry; Metabolites; Microsomes, Liver - chemistry; Microsomes, Liver - enzymology; Screening; Tandem Mass Spectrometry - methods
• ISSN: 0951-4198; 1097-0231; 1097-0231
• DOI: 10.1002/rcm.4610

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. Copyright © 2010 John Wiley & Sons, Ltd.