CYP Time-Dependent Inhibition (TDI) Using an IC50 Shift Assay with Stable Isotopic Labeled Substrate Probes to Facilitate Liquid Chromatography/Mass S

In this chapter we have provided a step-by-step protocol for a time-dependent inhibition (TDI) IC₅₀ shift assay using stable isotopic labeled probe substrates. The assay is performed in a 96-well format and can be fully automated and extended to a 384-well format if desired. Since the IC₅₀ shift assay requires parallel paired incubations to obtain two inhibition curves for comparison, the use of stable isotopic labeled probe substrates and non-labeled probe substrates allows the two sets of incubation samples to be combined and then simultaneously analyzed by LC/MS/MS in the same batch run. Compared to the traditional method, this sample pooling approach in combination with a short LC/MS/MS sample analysis time significantly enhances the throughput of the TDI screening assay by reducing sample analysis time and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resources.