Inhibition potential against CYP isoforms (IC50) measured in the presence of different concentrations of test compound after pre-incubation with NADPH (multiple time points) and without NADPH by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.
Constants KI (equilibrium binding) and kinact (irreversible CYP inactivation) for test compounds are determined from kinetic data using a non-linear regression analysis to the four-parameter logistic model fit with XLFit 5.3 from IDBS software.
Test Compound Concentration
Eight concentrations ranging around values of IC50 shift
Percent Organic Solvent in Incubation
CYP Isoforms Available for Testing
CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
up to 30 minutes (depending on CYP isoform)
Number of Repeats
Deliverable: Values of KI (µM), kinact(min-1), and kinact/KI (mL/min/µmol) for test compounds and positive control inhibitors
Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.