Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB4. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA(4) hydrolase. Here, we determined the crystal structure of LTA(4) hydrolase in complex with a Pro-Gly-Pro analog at 1.72 angstrom. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA(4) hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl) thiazol-2-amine, denoted ARM1, that inhibits LTB4 synthesis in human neutrophils (IC50 of similar to 0.5 mu M) and conversion of LTA(4) into LTB4 by purified LTA(4)H with a K-i of 2.3 mu M. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-angstrom crystal structure of LTA(4) hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the.-end of LTA(4), distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA(4) into LTB4, although sparing the enzyme's anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA(4) hydrolase inhibitor that holds promise for improved anti-inflammatory properties.