Quintara Discovery

TDI – IC50 Shift

CYP450 enzymes have been shown to be subjective to time-dependent inhibition (TDI), a kinetic phenomenon where inhibition increases with incubation time between the inhibitor and the enzyme (or HLM). A major subset of TDI by a chemical compound is mechanism-based inhibition (MBI) in which the compound is a CYP450 “substrate“and subsequently inactivates the CYP450 enzyme during the catalytic cycle by an irreversible or quasi-irreversible mechanism to the active site.

In early drug discovery, knowing the mechanism of TDI will inform medicinal chemists on how to remove this property through drug design and optimization. TDI information is required for accurate DDI prediction, as the amount of CYP450 enzyme available becomes time-dependent in the presence of TDI.

Assay Methodology

Quintara Discovery can conduct time-dependent inhibition assays for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (two probe substrates) using human hepatic microsomes. In the standard assay format, the test compound is preincubated with HLM in the presence and absence of NADPH for 30 min. Subsequently an excess of CYP450 substrate is added to initiate the CYP450-catalyzed reaction, and the IC50 values of the compounds are obtained. The ratio of the two IC50 values (+/- NADPH) is an indicator of potential time-dependent inhibition.

Time-Dependent Inhibition – IC50 Shift: 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 and 30 min +/- NADPH
Controls Known time-dependent inhibitors
Detection Method Specific metabolites by LC-MS/MS or RapidFire-MS/MS
Deliverables IC50, Standard error of IC50, IC50 Shift, Report
Turnaround Time 2-3 days
Compound requirements 2 mg solid