title: Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor.
abstract: Leukotriene (LT) A4 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 LTA4 hydrolase. Here, we determined the crystal structure of LTA4 hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. 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 LTA4 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 ∼0.5 μM) and conversion of LTA4 into LTB4 by purified LTA4H with a Ki of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA4 hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA4, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA4 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 LTA4 hydrolase inhibitor that holds promise for improved anti-inflammatory properties.
authors.affiliation: Divisions of Physiological Chemistry II and Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Alena
authors.initials: A
authors.lastname: Stsiapanava
authors.forename: Ulrika
authors.initials: U
authors.lastname: Olsson
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.forename: Thea
authors.initials: T
authors.lastname: Kleinschmidt
authors.forename: Dorothea
authors.initials: D
authors.lastname: Rutishauser
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
authors.forename: Bengt
authors.initials: B
authors.lastname: Samuelsson
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000201:enzymology*; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism*; D011392:Proline / Q000031:analogs & derivatives* / Q000378:metabolism; D011487:Protein Conformation; D053719:Tandem Mass Spectrometry; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D014961:X-Ray Diffraction
chemical_list: C000588605:4-(4-benzylphenyl)thiazol-2-amine; D009842:Oligopeptides; D013844:Thiazoles; C113164:prolyl-glycyl-proline; D011392:Proline; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase
keywords: X-ray crystallography; drug development; enzyme mechanism; inflammation; leukotriene B4