2型血小板活化因子乙酰水解酶（PAFAH2）三唑脲抑制剂的优化与表征

Optimization and characterization of a triazole urea inhibitor for platelet-activating factor acetylhydrolase type 2 (PAFAH2)

作者信息

Adibekian Alexander, Hsu Ku-Lung, Speers Anna E, Monillas Elizabeth S, Brown Steven J, Spicer Timothy, Fernandez-Vega Virneliz, Ferguson Jill, Bahnson Brian J, Cravatt Benjamin F, Hodder Peter, Rosen Hugh

机构信息

The Scripps Research Institute, La Jolla CA

University of Delaware, Newark, DE

PMID:23658960

Abstract

Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. In support of this theory, over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death [1]. However, no selective inhibitors of PAFAH2 are known for investigation of PAFAH2 biology. We initiated a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) HTS campaign to identify potential inhibitors of PAFAH2 (AIDs 492956 and 493030). The assay also served as a counterscreen for inhibitor discovery for the related enzyme, plasma PAFAH (pPAFAH; AIDs 463082, 463230). Interestingly, the triazole urea SID 7974398—a top lead in the lysophospholipase (LYPLA1) inhibitor screen from which we derived a dual inhibitor of LYPLA1/LYPLA2 (ML211) and inhibitor of ABHD11 (ML226)—was also a top hit in the PAFAH2 HTS assay. Given that triazole ureas were previously found to have tunable potency and selectivity, low cytotoxicity, and good activity , we endeavored to derive a PAFAH2-selective probe from the triazole urea scaffold. The medchem optimized probe (ML225, SID 103913572) is highly potent against its target enzyme (IC50 = 3 nM), and is active at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling, and is selective for other PAFAH enzymes. ML225 inhibits PAFAH2 by carbamoylating the active site serine. The complete properties, characterization, and synthesis of ML225 are detailed in this Probe Report.

摘要

氧化应激被认为是包括癌症、动脉粥样硬化、衰老和各种神经退行性疾病在内的多种疾病病理过程中的潜在炎症因子。磷脂尤其容易受到氧化损伤，据认为，胞质酶II型血小板活化因子乙酰水解酶（PAFAH2）可能通过水解氧化磷脂的氧化截断酰基链来促进其周转。支持这一理论的是，PAFAH2的过表达已被证明可减少氧化应激诱导的细胞死亡[1]。然而，目前尚不知道有用于研究PAFAH2生物学的PAFAH2选择性抑制剂。我们启动了一项基于荧光偏振活性的蛋白质谱分析（FluoPol-ABPP）高通量筛选活动，以鉴定PAFAH2的潜在抑制剂（AIDs 492956和493030）。该测定还用作相关酶血浆PAFAH（pPAFAH；AIDs 463082、463230）抑制剂发现的反筛选。有趣的是，三唑脲SID 7974398——我们从中获得了LYPLA1/LYPLA2双重抑制剂（ML211）和ABHD11抑制剂（ML226）的溶血磷脂酶（LYPLA1）抑制剂筛选中的顶级先导化合物——在PAFAH2高通量筛选测定中也是顶级命中物。鉴于先前发现三唑脲具有可调节的效力和选择性、低细胞毒性和良好的活性，我们努力从三唑脲支架衍生出一种PAFAH2选择性探针。经药物化学优化的探针（ML225，SID 103913572）对其靶酶具有高效力（IC50 = 3 nM），并且在亚纳摩尔浓度下具有活性。通过基于凝胶的竞争性活性蛋白质谱分析评估，ML225对所有其他丝氨酸水解酶（约20种）的选择性至少为333倍，并且对其他PAFAH酶具有选择性。ML225通过使活性位点丝氨酸氨甲酰化来抑制PAFAH2。本探针报告详细介绍了ML225的完整特性、表征和合成。