Objective

Over recent years there has been a resurgence in the focussed development of irreversible inhibitors that act via a covalent, time-dependent mechanism. Traditionally the potential for enzyme inhibition has been determined by steady state affinity measurements and quantified in terms of IC₅₀.  However this parameter in isolation may be misleading when applied to ranking different covalent compounds for structure-activity-relationship studies.

It has been proposed that in addition to IC₅₀, the rate of covalent modification should be taken into consideration (Strelow, 2017).  This is defined by the rate constant k_inact/K_i where K_i, the inhibition constant describes the potency of the first reversible binding event and k_inact the maximum rate in inactivation.

A commonly used technique is to derive k_inact/K_i by plotting the observed rate of inactivation (k_obs) as a function of inhibitor concentration.  Other methods employ an approach based on the effect of time on IC₅₀ which typically involves many pre-incubation steps.  In 2009 Krippendorff et al described a novel alternative to this whereby the pre-incubation step is omitted and data is fitted to a 3D model in IDBS Activitybase™.

At Charles River Discovery we have established a capability which utilises the Krippendorff time-dependent IC₅₀-based method in k_inact/K_i studies. Here, we introduce the technique and demonstrate how it can be applied in order to facilitate higher-throughput data analysis to drive projects forward.