Kukday Sayali S, Manandhar Surya P, Ludley Marissa C, Burriss Mary E, Alper Benjamin J, Schmidt Walter K
The University of Georgia, Athens, GA, USA.
J Biomol Screen. 2012 Dec;17(10):1348-61. doi: 10.1177/1087057112451921. Epub 2012 Jun 26.
The insulin-degrading enzyme (IDE) cleaves numerous small peptides, including biologically active hormones and disease-related peptides. The propensity of IDE to degrade neurotoxic Aβ peptides marks IDE as a potential therapeutic target for Alzheimer disease. Using a synthetic reporter based on the yeast a-factor mating pheromone precursor, which is cleaved by multiple IDE orthologs, we identified seven small molecules that stimulate rat IDE activity in vitro. Half-maximal activation of IDE by the compounds is observed in vitro in the range of 43 to 198 µM. All compounds decrease the K(m) of IDE. Four compounds activate IDE in the presence of the competing substrate insulin, which disproportionately inhibits IDE activity. Two compounds stimulate rat IDE activity in a cell-based assay, indicating that they are cell permeable. The compounds demonstrate specificity for rat IDE since they do not enhance the activities of IDE orthologs, including human IDE, and they appear specific for a-factor-based reporters since they do not enhance rat IDE-mediated cleavage of Aβ-based reporters. Our results suggest that IDE activators function in the context of specific enzyme-substrate pairs, indicating that the choice of substrate must be considered in addition to target validation in IDE activator screens.
胰岛素降解酶（IDE）可切割多种小肽，包括生物活性激素和疾病相关肽。IDE降解神经毒性Aβ肽的倾向使其成为阿尔茨海默病的潜在治疗靶点。我们使用一种基于酵母a因子交配信息素前体的合成报告分子，该前体可被多种IDE直系同源物切割，从而在体外鉴定出七种刺激大鼠IDE活性的小分子。这些化合物在体外43至198µM的范围内可观察到对IDE的半数最大激活作用。所有化合物均降低了IDE的米氏常数（K(m)）。四种化合物在存在竞争性底物胰岛素的情况下激活IDE，而胰岛素会不成比例地抑制IDE活性。两种化合物在基于细胞的测定中刺激大鼠IDE活性，表明它们具有细胞渗透性。这些化合物对大鼠IDE具有特异性，因为它们不会增强包括人IDE在内的IDE直系同源物的活性，并且它们似乎对基于a因子的报告分子具有特异性，因为它们不会增强大鼠IDE介导的基于Aβ的报告分子的切割。我们的结果表明，IDE激活剂在特定酶-底物对的背景下起作用，这表明在IDE激活剂筛选中，除了靶点验证外，还必须考虑底物的选择。
