Quintara Discovery

TDI/MBI – kinact/KI

When a drug candidate exhibits TDI and/or MBI, it is important to determine the kinetic parameters of inhibition constant, KI, and inactivation rate constant, kinact. KI and kinact, with the knowledge of the degradation rate of CYP450 proteins, are important in predicting the in vivo DDI effects.

Assay Methodology

To determine KI and kinact, the inhibitor is preincubated with HLM in the presence of NADPH for various time intervals at multiple concentrations. Upon adding the substrate, the remaining enzymatic activity is monitored by LC-MS/MS, and plotted as a function of preincubation time. The observed inactivation rate (kobs), determined from the slope of the plot, is analyzed over a range of inhibitor concentrations. Values of KI and kinact can be determined by nonlinear fit to the classic Michaelis-Menten enzyme kinetics.

Time-Dependent Inhibition – kinact/Ki: Standard Assay Conditions (Customizable)

CYP Isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (other isoforms are available)
Matrix Pooled human liver microsomes
Testing Compound Concentration 0, 0.25xIC50, 0.5xIC50, 0.75xIC50, 1xIC50, 2.5xIC50, 5xIC50, 10xIC50
Preincubation 0, 5, 10, 20, 30, 60 min with 1 mM NADPH
Controls Known time-dependent inhibitors
Detection Method Specific metabolites by LC-MS/MS
Deliverables KI, kinact, detailed Report
Turnaround Time 2-3 days
Compound requirements 2 mg solid