Fluorescent Probe Based Cyp Inhibition Assay: a High Throughput Tool for Early Drug Discovery Screening

Inhibition of cytochrome P450 (CYP450) enzymes can result in potential clinical drug-drug interaction liabilities. Because of the safety and economic concern leading to clinical failure or product withdrawal, screening of lead molecules for CYP450 inhibition potential is nowadays conducted much earlier in the drug development process. At this early phase, a large number of lead molecules are in need of evaluation. Thus, safety, economic, and throughput pressures have forced the development of assays that are rapid throughput but have high predictive value. Fluorometric assays using recombinant human enzymes are ideally suited for this purpose. Objective: This study was mainly designed with an objective to address the reliability of fluorogenic probes for CYP450 inhibition studies. Methods: Individual cDNA-expressed CYP isoforms (1A2, 2D6, 3A4, 2C9 and 2C19) were applied to microtitre plate assays. Standard inhibitors were co-incubated with fluorogenic substrates and their inhibitory potential (IC50) was determined. Assay conditions in terms of CYP450 protein concentration and time of incubation were standardized, enzyme kinetics parameters of each fluorescent probe substrate were estimated and IC50 values of inhibitors were determined and validated on different days to check reproducibility. Results: The fluorescent probe based CYP Inhibition assay methods were found to be robust, efficient and highly reproducible. Conclusion: These assays can be quite useful in preliminary high throughput screening of inhibitory potential of new drugs on individual P450 enzymes and predicting clinical drug-drug interactions.