A two-point IC₅₀method for evaluating the biochemical potency of irreversible enzyme inhibitors

Abstract

Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50 values determined at a single point in time, because the same IC50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC50 values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanism E + I ⇔ E·I → EI, the two IC50 values alone can be used to estimate both the inhibition constant (Ki) as a measure of binding affinity and the inactivation rate constant (kinact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanism E + I → EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (kinact/Ki) from a single experimental value of IC50. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.

A Comprehensive Guide for Assessing Covalent Inhibition in Enzymatic Assays Illustrated with Kinetic Simulations

“…An implicit algebraic model based on multipoint IC 50 ( t ) values has been derived (Krippendorff, Neuhaus, Lienau, Reichel, & Huisinga, 2009) for two‐step irreversible covalent inhibitors ( Data Analysis 2 ). Additionally, a two‐point IC 50 ( t ) method for two‐step irreversible covalent inhibitors as well as a one‐point IC 50 ( t ) method for one‐step irreversible covalent inhibitors have been reported in a preprint (Kuzmič, 2020b). To our knowledge, algebraic methods to calculate K i *app (two‐step reversible covalent inhibitors) from (endpoint) IC 50 ( t ) values have not been reported.…”

A Blueprint for High Affinity SARS-CoV-2 Mpro Inhibitors from Activity-Based Compound Library Screening Guided by Analysis of Protein Dynamics

“…Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC 50 values, because the same IC 50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. 104 In other words, the important quantity to be considered would be the rate of covalent modification, (kinact/K i ), that describes the efficiency of covalent bond formation resulting from the potency (K i ) of the first reversible binding event and the maximum potential rate (kinact) of inactivation. 105 This information is unfortunately not available for most of the ligands here considered.…”

Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-CoV-2 activation

“…For the 10s timepoint, a kinetic read was performed after manual addition of enzyme, followed by substrate, using a multichannel pipette. Kinetic parameters Kiapp and kinact were determined with the simplified Equations 2 and 3, respectively, assuming a one-step kinetic inhibition mechanism, A 8 .…”