Abstract：
Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 μM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.
摘要：
白三烯A4水解酶（LTA4H）的氨肽酶（AP）活性的激活为解决慢性炎症提供了潜在的治疗策略。以前,在酶动力学测定中，使用丙氨酸-对硝基苯胺（Ala-pNA）作为报告基团，发现ARM1和衍生物可激活AP活性。作为先前工作的延伸,使用钯催化的Ullmann偶联反应合成了新型ARM1衍生物，并使用相同的测定法进行了筛选。发现类似物5是ARM1的氨基吡唑（AMP）类似物，是一种有效的AP激活剂，AC50为0.12μM。LTA4H与AMP配合物的X射线晶体结构在2.7进行了精制。尽管其与Ala-pNA底物的AP活性，AMP不影响先前提出的LTA4H的天然配体的水解，Pro-Gly-Pro（PGP）。该结果突出了通常用于测定的更方便监测的显色合成肽的水解与内源肽之间的差异。在体内测量AMP的环氧化物水解酶(EH)活性,并且该化合物在鼠细菌性肺炎模型中显著降低白三烯B4(LTB4)水平。然而,在14天的时间内，AMP并未增强鼠肺炎模型的存活率。肝微粒体稳定性分析显示AMP的代谢稳定性。结果表明，加速Ala-pNA裂解不足以预测治疗潜力。即使激活的完整机制是已知的。
