Enzyme-substrate-inactivator complex formation can impact time-dependent inhibition kinetics

In order to predict drug-drug interactions (DDIs), screening and evaluating time-dependent inhibition (TDI) of cytochrome P450 is an important part of the drug discovery process. In vitro experiments are performed for obtaining TDI parameters (inhibitor binding constant, K_I, and inactivation rate constant, k_inact), which are further used for the prediction of clinical DDI. This work used numerical methods for analysis of in vitro TDI datasets to estimate K_I and k_inact. Specifically, the impact of enzyme-substrate-inhibitor (ESI) complex formation on the estimation of K_I and k_inact was evaluated. A total of 13 datasets were evaluated, all of which exhibited non-Michaelis-Menten kinetics, including activation, biphasic inactivation, partial inactivation, and multiple binding kinetics. Datasets were modeled with and without ESI formation. The difference in inactivation efficiency between models with and without ESI ranged from 0.67 to 2.94-fold across all compounds. This resulted in different DDI predictions ranging from moderate to strong DDI predictions for the same compound. These results suggest that in vitro TDI models should incorporate the observed in vitro kinetic complexities to improve confidence in in vivo DDI prediction. SIGNIFICANCE STATEMENT: In vitro time-dependent inhibition (TDI) analysis is often performed assuming that substrate kinetics do not impact the estimates of K_I and k_inact. By incorporating enzyme-substrate-inhibitor formation in the enzyme kinetic models, activation- and substrate-dependent kinetics can be modeled simultaneously with TDI, thereby providing a more accurate prediction of drug-drug interactions.