Enhancing the cardioprotective effect of diadenosine tetraphosphate: designing inhibitors against Ap4A hydrolase

缺血描述了心脏血管中的血流减少或阻塞，阻止向心脏输送氧气和营养物质的情况。缺血预处理是一种短暂的缺血，随后再灌注，实际上保护心脏免受随后更长时间的缺血的现象。预处理中涉及的生化信号事件是复杂的且不完全定义的，但最可能涉及多个途径，尽管线粒体ATP依赖性钾通道可能与大多数途径相同。用化合物二腺苷四磷酸（Ap4A）预处理模拟缺血预处理，组织坏死（细胞死亡）明显减少。这种治疗已在实验工作中显示出在压力期间保护心脏，例如在心脏手术或从缺血事件中恢复。Ap4A的生物学作用位点可能是线粒体ATP依赖性钾通道或相关蛋白。Ap4A可以被位于心脏细胞内部和外部的酶降解，特别是被两种形式的Ap4A水解酶降解。我们将使用抗体检测来了解Ap4A水解酶在缺血前后和缺血预处理后在人心肌和血管中的特异性定位和量。我们建议确定酶的结构，并使用新的计算机方法来筛选潜在的抑制剂的数据库。这些Ap4A水解酶活性的抑制剂可以帮助设计一种有效的抑制剂，该抑制剂可以防止Ap4A水解酶降解Ap4A，从而增强Ap4A的心脏保护特性，并使Ap4A分解的副作用最小化。我们还将在生物测定中使用这些抑制剂和其他已知的不可降解的Ap4A类似物，以测试不同细胞位置中存在的Ap4A水解酶的相对意义。

Ischemia describes the condition where blood flow in the blood vessels of the heart is decreased or blocked, preventing delivery of oxygen and nutrients to the heart. Ischemic preconditioning is a phenomenon where short bursts of ischemia, followed by reperfusion, actually protect the heart from a subsequent longer period of ischemia. The biochemical signalling events involved in preconditioning are complex and incompletely defined, but most likely involve multiple pathways, although the mitochondrial ATP-dependent potassium channel may be in common with most pathways. Pretreatment with the compound diadenosine tetraphosphate (Ap4A) mimics ischemic preconditioning with noticeable reductions in tissue necrosis (cell death). This treatment has been shown in experimental work to protect the heart during periods of stress such as in heart surgery or recovery from an ischemic event. The biological site of action by Ap4A may be the mitochondria ATP-dependent potassium channel or an associated protein. Ap4A can be degraded by enzymes located inside and on the outside of heart cells, notably by two forms of Ap4A hydrolase. We will use antibody assays to understand the specific localization and amount of Ap4A hydrolase before and after ischemia and after ischemic preconditioning in human heart muscle and blood vessels. We propose to determine the structure of the enzyme and use novel computer methods to screen databases for potential inhibitors. These inhibitors of Ap4A hydrolase activity could aid the design of a potent inhibitor that would prevent Ap4A hydrolase from degrading Ap4A and therefore enhance the cardioprotective properties of Ap4A as well as minimizing side effects from the break down of Ap4A. We will also use these inhibitors and other known non-degradable Ap4A analogues in bioassays to test the relative significance of Ap4A hydrolase present in different cellular locations.