The leukotriene A4 hydrolase (LTA₄H) protein is a zinc metalloenzyme with both pro- and anti-inflammatory properties. The prevailing understanding is that as an epoxide hydrolase (EH), LTA₄H participates in pro-inflammatory pathways, but as an aminopeptidase (AP), LTA₄H participates in anti-inflammatory pathways. Herein, we designed a series of modulators that activate the AP activity of the LTA₄H enzyme. To better understand the kinetic mechanisms of AP activity, we solved the X-ray crystal structure of LTA₄H bound to small molecule modulators. We also carried out enzyme kinetic assays using different amino acids containing p-nitroanilide as a reporter group. We demonstrated both a ligand and substrate-dependent mechanism of LTA₄H AP activity. The small molecule 4MDM was shown to have anti-inflammatory properties in several murine models of pulmonary inflammation but not in LTA₄H knockout mice. Our aminopyrazole (AMP) analogue followed the same activation mechanism as 4MDM in modulating LTA₄H AP activity with improved potency but did not translate to anti-inflammatory effects in our model for pulmonary inflammation. In other words, the reporter group used in our enzyme assay did not track with therapeutic outcome. This raises an important consideration in the choice of reporter group when optimizing ligands in designing activators for the AP activity of the LTA₄H enzyme. We also realized that a modulator bound to LTA₄H determines the water network in the LTA₄H binding pocket, which we anticipate being a key component for LTA₄H AP activation. More careful assessment and mechanistic characterization of LTA₄H enzymatic activities are being pursued to further inform strategies for developing therapeutics for this target.

Schroeder Noble

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