Department of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine , University of California, San Diego , La Jolla , California 92093-0601 , United States.
J Med Chem. 2019 Feb 28;62(4):1999-2007. doi: 10.1021/acs.jmedchem.8b01568. Epub 2019 Feb 4.
Assaying lipolytic enzymes is extremely challenging because they act on water-insoluble lipid substrates, which are normally components of micelles, vesicles, and cellular membranes. We extended a new lipidomics-based liquid chromatographic-mass spectrometric assay for phospholipases A to perform inhibition analysis using a variety of commercially available synthetic and natural phospholipids as substrates. Potent and selective inhibitors of three recombinant human enzymes, including cytosolic, calcium-independent, and secreted phospholipases A were used to establish and validate this assay. This is a novel use of dose-response curves with a mixture of phospholipid substrates, not previously feasible using traditional radioactive assays. The new application of lipidomics to developing assays for lipolytic enzymes revolutionizes in vitro testing for the discovery of potent and selective inhibitors using mixtures of membranelike substrates.
测定脂肪酶极具挑战性，因为它们作用于不溶于水的脂类底物，这些底物通常是胶束、囊泡和细胞膜的组成部分。我们扩展了一种基于脂质组学的新型液相色谱-质谱法磷脂酶 A 测定法，以使用各种市售的合成和天然磷脂作为底物进行抑制分析。三种重组人酶（包括细胞质、钙非依赖性和分泌型磷脂酶 A）的有效且选择性抑制剂用于建立和验证该测定法。这是首次使用脂质组学在具有混合磷脂底物的剂量反应曲线上的新应用，这在以前使用传统放射性测定法是不可行的。将脂质组学应用于开发脂肪酶测定法，为使用类似膜的底物混合物发现有效且选择性抑制剂的体外测试带来了革命性的变化。
