Mechanism-Based Inhibition: Deriving KI and kinact Directly from Time-Dependent IC50 Values

Abstract

The potential of enzyme inhibition of a drug is frequently quantified in terms of IC50 values. While this is a suitable quantity for reversible inhibitors, concerns arise when dealing with irreversible, or mechanism-based inhibitors (MBI). IC50 values of MBI are time-dependent, causing serious problems when aiming at ranking different compounds with respect to their inhibitory potential. As a consequence, most studies and ranking schemes related to MBI rely on the inhibition constant (K I ) and the rate of enzyme inactivation (k inact ) rather than on IC50 values. In this article we derive a novel relation between potentially time-dependent IC50 values and K I , k inact parameters for different types of inhibition. This allows for direct estimation of K I and k inact values from time-dependent IC50 values, even without the need of additional pre-incubation experiments. The application of this approach is illustrated using a fluorimetric assay to access the drug-drug interaction potential associated with new chemical entities. The approach can easily be implemented using standard software tools (e.g., XLfit) and may also be suitable for applications where mechanism-based inhibition is a desired mode of actions, e.g., at particular pharmacological drug targets.

Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis

“…Covalent inhibitors display time-dependent changes in IC 50 values, making it difficult to derive enzyme kinetic parameters (21). Thus, we adapted a liquid chromatography (LC)-MS-based GPX4 assay (2) to measure GPX4 inhibition by (1S, 3R)-RSL3 in a concentration series at a specific time point to determine an effective IC 50 for (1S, 3R)-RSL3.…”

An Evaluation of the Dilution Method for Identifying Metabolism-Dependent Inhibitors of Cytochrome P450 Enzymes

“…The ability to estimate k inact and K I from shifted IC 50 values [based on a modified Cheng and Prusoff (1973) equation] was also validated by Krippendorff et al (2009) both theoretically (in silico) and experimentally using a nondilution approach in which the rate of formation of fluorescent metabolites (one for CYP1A2 and one for CYP3A4) was measured in situ at 2-min intervals (the "progress curve" method). Obach et al (2007) reported a strong empirical correlation between shifted IC 50 values and k inact /K I (as would be expected from the equations above).…”

Defining Human Pathways of Drug Metabolism In Vivo through the Development of a Multiple Humanized Mouse Model

“…The published method of time-dependent inhibition data analysis using an IC 50 -shift experimental setup (Maurer et al, 2000;Berry and Zhao, 2008;Krippendorff et al, 2009) was modified to allow the simultaneous nonlinear regression of the IC 50 curves. It was assumed that substrate metabolism follows Michaelis-Menten kinetics with the rapid equilibrium approximation, the inhibitor is competing with a substrate for the enzyme active site, and the enzyme-inhibitor complex can produce an inactive enzyme (Scheme 3).…”