Tuesday, August 18, 2009

Automation of Luminescence-based CYP Inhibition Assays in Drug Discovery

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 affect the metabolism of another, leading to possible toxic effects. The ability to determine the effects that lead compounds have on these enzymes is an important part of today’s drug discovery routine. One desired component of this process is the ability to profile compounds against multiple CYP450 enzymes using the same basic procedure. The second is easy to use, yet dependable instrumentation that can be used to dilute compounds, as well as dispense assay reagents, that will create accurate titration curves.
To demonstrate automated CYP profiling, we used BioTek’s Precision™ XS Automated Pipetting System and Synergy™ MX Monochromator-Based Multi-Mode Microplate Reader, along with Promega’s P450-Glo™ 2C9, 3A4, and 2D6 Screening Systems. The Precision was used to perform compound titrations, as well as dispense all assay components to the wells of the assay plates. The Synergy was used to detect the luminescent signal from each well. Titrations were carried out in 96-well format, with a 20 µL transfer volume. 5 µL of compound was transferred in quadruplicate to the assay plate, followed by 5 µL of enzyme/substrate mix. 10 µL of NADPH Regeneration System was then dispensed to start the reaction. After the appropriate incubation time, 20 µL of Luciferin Detection Reagent was added to the plate.
Z’-Factor assays were run to validate the automated methods. Compounds were then profiled against all three CYP450 isoforms on the same 384-well assay plate to demonstrate the ease of the combined procedure. Agreement of IC50 values for control compounds with established literature values confirms that the combination of robotics, detection, and chemistry creates an ideal solution for automated cytochrome P450 profiling of lead compounds in drug discovery campaigns.
Where do you see cytochrome P450 profiling demands going in the future? Will there be an increased need for cell-based CYP450 assays? What type of throughput do you feel is essential for automated CYP450 profiling?
Table: Compound IC50 values using P450-Glo™ and Control Assays
Table: Compound IC50 values using P450-Glo™ and Control Assays
 
Graph: Compounds Inhibition Curves for CYP3A4 Isoform
Graph: Compounds Inhibition Curves for CYP3A4 Isoform
 
 
 
By: BioTek Instruments
 
 

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