Control of Executioner Caspases with an Allosteric Switch

描述（由申请人提供）：蛋白质功能的正交控制允许来自相关蛋白质家族的一种蛋白质失活，以便评估其独特的生物学功能。我们建议在caspase家族中开发一种新的正交控制，称为变构开关。半胱天冬酶是一种半胱氨酸蛋白酶，它通过切割分离的靶蛋白来导致细胞死亡，最终导致细胞凋亡。半胱天冬酶有一个大的中心空腔，可以被小分子变构抑制。半胱天冬酶被认为是治疗癌症（半胱天冬酶激活剂）或中风、心脏病发作和阿尔茨海默病（半胱天冬酶抑制剂）的良好靶点。不幸的是，目前可用的结合在caspase活性位点的小抑制剂的相似性使12种不同的caspase的精确作用无法明确识别。我们将结合定向进化和计算蛋白设计来引入caspase -3、-6和-7的变构开关。当我们选择或设计的小分子效应物与进化的变构位点结合时，caspase活性将被抑制。这个开关将允许我们一次关闭一种caspase，以确定哪些蛋白质被caspase-3或-6或-7特异性切割。被切割的蛋白质本身就很有趣，因为它们的切割是促凋亡的，这意味着它们会导致细胞死亡。诱导细胞凋亡的能力是一种有用的抗癌药物的标志。我们的研究可能提示哪种半胱天冬酶应该靶向治疗哪种疾病。这些半胱天冬酶的变构开关的成功实现将为其他9种半胱天冬酶的变构开关铺平道路，并最终进入缺乏生物信息的其他蛋白质家族。

DESCRIPTION (provided by applicant): Orthogonal control of protein function allows one protein from a family of related proteins to be inactivated so that its unique biological function can be assessed. We propose development of a new kind of orthogonal control, called allosteric switches, in the caspase family. Caspases are cysteine proteases that execute cell death by cleaving a discrete selection of target proteins, which ultimately leads to apoptosis. Caspases have a large central cavity that can be allosterically inhibited by small molecules. Caspases are believed to be good targets for treatment of cancer (caspase activators) or stroke, heart attack and Alzheimer's Disease (caspase inhibitors). Unfortunately the similarities in currently available small inhibitors that bind at the caspase active sites have not allowed unambiguous identification of the precise roles of the twelve different caspases. We will use a combination of directed evolution and computational protein design to introduce an allosteric switch in caspases-3, -6, and -7. When the small molecule effector we have selected or designed against binds to the evolved allosteric site, caspase activity will be inhibited. This switch will allow us to turn off one type of caspase at a time to determine what proteins are specifically cleaved by caspase-3 or -6 or -7. The cleaved proteins themselves are inherently interesting, because their cleavage is proapoptotic, meaning they lead to cell death. The ability to induce apoptosis is the hallmark of a useful cancer drug. Our studies may suggest which caspase should be targeted for treating which disease. Successful implementation of allosteric switches in these caspases will pave the way for allosteric switches in the other nine caspases and ultimately into other families of proteins where biological information is lacking. NON-TECHNICAL SUMMARY: The central problem in disease treatment is knowing which protein should be targeted to treat which disease. The allosteric switch technology we will develop in caspases allows just one type of protein to be turned off by a drug, to determine which disease could be effectively treated by targeting that particular protein. We focus on caspase proteins, which can cause cancer cells to die.