Structure Kinetics Relationships and Molecular Dynamics Show Crucial Role for Heterocycle Leaving Group in Irreversible Diacylglycerol Lipase Inhibitors

Abstract

Drug discovery programs of covalent irreversible, mechanism-based enzyme inhibitors often focus on optimization of potency as determined by IC50-values in biochemical assays. These assays do not allow the characterization of the binding activity (Ki) and reactivity (kinact) as individual kinetic parameters of the covalent inhibitors. Here, we report the development of a kinetic substrate assay to study the influence of the acidity (pKa) of heterocyclic leaving group of triazole urea derivatives as diacylglycerol lipase (DAGL)-α inhibitors. Surprisingly, we found that the reactivity of the inhibitors did not correlate with the pKa of the leaving group, whereas the position of the nitrogen atoms in the heterocyclic core determined to a large extent the binding activity of the inhibitor. This finding was confirmed and clarified by molecular dynamics simulations on the covalently bound Michaelis–Menten complex. A deeper understanding of the binding properties of covalent serine hydrolase inhibitors is expected to aid in the discovery and development of more selective covalent inhibitors.

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

“…A slightly higher substrate concentration ([S] ≥ 0.1K M ) resulted in underestimation of k inact and overestimation of K I , but a good estimation of overall second-order inactivation rate constant k inact /K I . Importantly, a calibration/titration (Dharadhar et al, 2019;Janssen et al, 2019) should be performed prior to data analysis to determine product coefficient r P (in AU/M) that transforms the detected signal F t (in AU) into product concentration [P] t (in M).…”

“…Therefore, substrate is added in a large excess over the enzyme ([S] 0 > 10[E] 0 ), and the uninhibited velocity of product formation (v ctrl ) is calculated over the linear part corresponding to less than 10% substrate conversion ([P] t < 0.1[S] 0 ) (Wu, Yuan, & Hodge, 2003). The signal corresponding to 10% substrate conversion can be estimated from a product calibration/titration curve (Dharadhar et al., 2019; Janssen et al., 2019) to avoid substrate depletion. The effect of substrate depletion can be investigated with the kinetic simulation script KinSubDpl .…”

Development and biological applications of sulfur–triazole exchange (SuTEx) chemistry

“…RSC Chemical Biology resulting compounds. 108 Combined with the synthetic accessibility of 1,2,3-and 1,2,4-triazoles 109,110 using diverse methodologies, [111][112][113][114][115][116] the triazole moiety offers multiple opportunities for tuning reactivity of electrophilic compounds including the type and position of functional group modifications and selection of regioisomers.…”

“… 20 Both 1,2,3- and 1,2,4-triazoles 105 – 107 can serve as an effective LG for SH inhibitor development, and recent evidence supports that regioisomers can affect the activity of resulting compounds. 108 Combined with the synthetic accessibility of 1,2,3- and 1,2,4-triazoles 109 , 110 using diverse methodologies, 111 – 116 the triazole moiety offers multiple opportunities for tuning reactivity of electrophilic compounds including the type and position of functional group modifications and selection of regioisomers.…”

Mechanistic Modeling of Monoglyceride Lipase Covalent Modification Elucidates the Role of Leaving Group Expulsion and Discriminates Inhibitors with High and Low Potency

“… 14 An interesting property of azole ureas is that their reactivity versus nucleophiles depends on the chemical nature of the azole ring, with tetrazoles being highly reactive, 22 followed by triazoles, imidazoles, and pyrazoles. 23 This trend, to some extent, can be explained by the ability of the azole ring to serve as the LG in carbamoylation reactions, with the p K a of the azole conjugate acid used as a coarse predictor of reactivity. Within this class, pyrazole derivatives are often neglected as they display low reactivity toward nucleophiles due to their poor ability to be expelled as anionic LG in acyl transfer reactions.…”