Biology subjects

Kuzmic, P.

Publications and source records attributed to Kuzmic, P..

3 recordsLinked to original sources

bioRxiv2020

A two-point IC50 method for evaluating the biochemical potency of irreversible enzyme inhibitors

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 {rightleftharpoons} E{middle dot}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.

biochemistry↗

bioRxiv2020

A steady-state algebraic model for the time course of covalent enzyme inhibition

This report describes a double-exponential algebraic equation for the time course of irreversible enzyme inhibition following the two-step mechanism E + I {rightleftharpoons} E{middle dot}I [->] EI, under the steady-state approximation. Under the previously invoked rapid-equilibrium approximation [Kitz & Wilson (1962) J. Biol. Chem. 237, 3245] it was assumed that the rate constant for the reversible dissociation of the initial noncovalent complex is very much faster than the rate constant for the irreversible inactivation step. The steady-state algebraic equation reported here removes any restrictions on the relative magnitude of microscopic rate constants. The resulting formula was used in heuristic simulations designed to test the performance of the standard rapid-equilibrium kinetic model. The results show that if the inactivation rate constant is significantly higher than the dissociation rate constant, the conventional "kobs" method is incapable of correctly distinguishing between the two-step inhibition mechanism and a simpler one-step variant, E + I [->] EI, even for inhibitors that have very high binding affinity in the reversible noncovalent step.

biochemistry↗

bioRxiv2020

Deciding between one-step and two-step irreversible inhibition mechanisms on the basis of "kobs" data: A statistical approach

This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition from kinetic experiments based on the standard "kobs" method. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. Recently published experimental data [Hopper et al. (2020) J. Pharm. Exp. Therap. 372, 331-338] on the irreversible inhibition of Bruton tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) by ibrutinib (PCI-32765) and acalabrutinib are used as an illustrative example. The results show that the kinetic mechanism of inhibition was misdiagnosed in the original publication for at least one of the four enzyme/inhibitor combinations. In particular, based on the available kobs data, ibrutinib behaves effectively as a one-step inhibitor of the TEC enzyme, which means that it is not possible to reliably determine either the inhibition constant Ki or the inactivation rate constant kinact, but only the covalent efficiency constant keff = kinact/Ki. Thus, the published values of Ki and kinact for this system are not statistically valid.

biochemistry↗