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Combining Luminescence-based CYP Inhibition Assays and Simple, Robust Instrumentation for Use in Automated Cytochrome P450 Profiling下載
September 10, 2010
Authors: Brad Larson, Peter Banks, BioTek Instruments, Inc., Winooski, Vermont; Mary Sobol, James J Cali, Promega Corporation, Madison, Wisconsin
Most small molecule drugs are metabolized predominantly in the liver by cytochrome P450 (CYP) enzymes, particularly CYP isoforms 3A4, 2C9 and 2D6. It is important to assess metabolism for appropriate dosing, but also for establishing metabolism-related drug-drug interactions where one drug may inhibit the metabolism of another leading to possible toxic effects. While the gold standard method for in vitro determination of lead compound inhibition of CYP isoforms involves monitoring the metabolism of drug substrates by human liver microsomes or primary hepatocytes using LC-MS/MS, the use of recombinant CYP isoforms with optical readouts based on labeled drug substrates specific to the isoform is gaining favor as a low cost, higher throughput alternative that can assess metabolic profiles of leads earlier in the drug discovery process. One desired component of this change is the ability to profile compounds against multiple CYP enzymes using the same basic procedure. The second is an easy, yet dependable way to dilute compounds that will create accurate titration curves.
Here we demonstrate the automation of the profiling process, from compound titration through assay component transfer, using simple, yet robust instrumentation. IC50’s of small molecule drugs were determined using recombinant CYP isoforms 3A4, 2C9 and 2D6 as well as luminogenic substrates specific to each. Compounds were profiled against all three isoforms on the same 384-well assay plate to demonstrate the ease of this combined procedure.The combination of chemistry and instrumentation creates an ideal solution for high-throughput cytochrome P450 profiling of lead compounds in drug discovery campaigns.