Rational Optimization of Mechanism-Based Inhibitors through Determination of the Microscopic Rate Constants of Inactivation

Abstract

Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine due to their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated due to time-dependent inhibition resulting from multi-step inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min−1).

Synthesis of a 3-Amino-2,3-dihydropyrid-4-one and Related Heterocyclic Analogues as Mechanism-Based Inhibitors of BioA, a Pyridoxal Phosphate-Dependent Enzyme

“…Compound 3 had a slightly improved k inact as compared to 1 that was not consistent with our expectations that lowering the p K a of the α-proton would lead to more rapid deprotonation by BioA. However, further computational studies on 3 suggest our initially calculated p K a ’s significantly overestimated the acidity of 2 and 3 . , A cocrystal structure of the final inactivated BioA-PLP adduct of 3 confirms it behaves as a covalent mechanism-based inhibitor analogous to 1 , lending support to our expectation that 3 operates via a similar four-step kinetic mechanism wherein deprotonation of the external aldimine to form the quinonoid is rate-limiting . The increased k inact value of 3 relative to 1 indicates that the rate-limiting step is faster, but the modest 2.7-fold rate increase could be caused by other factors such as a more favorable reaction trajectory of the key general base (Lys283) or perturbation of the p K a in the active site.…”

“…Compound 3 had a significantly increased k inact as compared to 1, validating our hypothesis that lowering the p K a of the α-proton would lead to more rapid deprotonation by BioA, though this improvement was less than anticipated based on the predicted difference in p K a between 3 and 1. 15,51 The increased k inact value means that the dihydro-4-pyridone scaffold has greater potential for future development when compared to the dihydro-2-pyridone scaffold of 1. The concomitant increase in K I makes it quite likely that binding affinity was lost, but we believe this can be overcome through the replacement of the 3-hydroxypropyl chain.…”

“…14 Unfortunately, both of these parameters are complex conglomerates of the individual microscopic rate constants of inactivation, and as such, neither of the parameters definitively assess binding affinity nor the identity of the rate limiting step. 15 In order to rationally optimize 1 , we undertook the first detailed kinetic characterization of an MBI, using pre-steady-state stopped-flow kinetic experiments to determine both the mechanism and the rate of each individual step. 15 This investigation found the kinetic bottleneck is the removal of the α-proton of the initial external aldimine to form the quinonoid intermediate.…”

“…15 In order to rationally optimize 1 , we undertook the first detailed kinetic characterization of an MBI, using pre-steady-state stopped-flow kinetic experiments to determine both the mechanism and the rate of each individual step. 15 This investigation found the kinetic bottleneck is the removal of the α-proton of the initial external aldimine to form the quinonoid intermediate. We hypothesized a simple way to facilitate this specific step would be to lower the p K a of this key proton.…”

“…k inact and K I are semianalogous to Michaelis–Menten parameters and describe, respectively, the maximum possible value of k obs at infinite inhibitor concentration and the concentration of inhibitor that produces a k obs that is equal to one-half of k inact . Unfortunately, both of these parameters are complex conglomerates of the individual microscopic rate constants of inactivation, and as such, neither of the parameters definitively assess binding affinity nor the identity of the rate-limiting step . To rationally optimize 1 , we undertook the first detailed kinetic characterization of an MBI, using presteady-state stopped-flow kinetic experiments to determine both the mechanism and the rate of each individual step .…”