Assay Method Information

Assay Name:  Inhibition Assay
Description:  A dimethyl sulfoxide (DMSO) solution of a test compound with a concentration 1000 times higher than the evaluation concentration was prepared, and a reaction solution was prepared by diluting the solution. Enzyme reaction was performed by incubating in a potassium phosphate buffer solution (pH 7.4) containing 1 nM to 20 μM test compound, 3.2 mM magnesium chloride, 0.2 pmol human CYP3A4 (BD Biosciences), 0.5 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 3 μM Luciferin-IPA (Promega) at 37° C. for 10 minutes. The volume of the reaction solution was 50 μL/well. The 30-minute pre-incubation group was incubated at 37° C. for 30 minutes before adding tire substrate, the Luciferin-IPA solution (12.5 μL/well). At the end of the enzyme reaction, 50 μL/well of a Luciferin detection reagent (Promega) was added to the wells, and the plate was left at room temperature for 20 minutes. Then, the emission intensities were measured with Infinite M1000 (TECAN). The enzyme activities (%) relative to the value of the group to which the test compound was not added were calculated. A dose-response curve was drawn using analysis software, GraphPad Prism (GraphPad Software), and the concentration of each compound that exhibited 50% inhibition, IC50, was calculated. As a comparative example, aprepitant, which is an NK1 receptor antagonist, was tested in the same manner.
Affinity data for this assay
 

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