Targeting Calpain for Novel Anticancer Agents

描述(申请人提供)：钙蛋白酶是一类细胞内的半胱氨酸蛋白酶，受第二信使钙2调节。已有几种钙蛋白酶亚型的报道，其中钙蛋白酶I和II是哺乳动物细胞中最普遍存在的。钙蛋白酶被认为是细胞中许多生理和病理事件的重要调节器。该酶具有广泛的底物特异性，许多钙蛋白酶底物，如转录因子c-Fos和c-jun，肿瘤抑制蛋白P53，多种信号酶(如蛋白激酶C，pp60 src)，以及黏附分子整合素和E-钙粘素参与了不同人类肿瘤的发病机制。在细胞培养研究中也证明了抑制Calain会在癌细胞系中触发细胞凋亡。这些研究表明，Calain是一种潜在的新的癌症靶点，有望发现一类新的抗肿瘤药物。然而，很少有体内研究(动物研究)证实Calain是抗癌药物发现的一个可行的癌症靶点。在体外钙蛋白酶抑制和体内抗癌活性之间的联系还没有得到充分的研究，部分原因是大多数钙蛋白酶抑制剂的代谢不稳定。这构成了一个重要的缺口，必须加以解决，才能验证CalPain作为新的癌症靶点。这项建议的直接目标是通过开发一系列独特的钙蛋白酶抑制剂来填补这一空白，这些抑制剂是有效的、选择性的和代谢稳定的，可以用作工具来验证钙蛋白酶作为新的癌症靶点。长期目标是开发钙蛋白酶抑制剂作为一类新的抗癌药物。其具体目的在于：(1)利用基于结构的分子设计方法设计和合成代谢稳定的钙蛋白酶抑制剂；(2)确定化合物相对于其他蛋白水解酶的抑制活性和选择性；(3)测定化合物的代谢稳定性；(4)测定化合物的体外细胞毒性和体内药效；(5)确定抑制剂的抑制效力(KI)是否与其体外细胞毒作用和体内抗肿瘤作用有关；(6)确定Calain抑制剂诱导细胞毒性的机制(S)。拟议的研究结果将验证Calain作为抗肿瘤药物发现的新癌症靶点的有效性。此外，由于它们的代谢稳定性，这些化合物是研究钙蛋白酶在细胞内的作用的有用的生物医学工具。这些化合物也是潜在的抗癌药物。

DESCRIPTION (provided by applicant): Calpains are a class of intracellular cysteine proteases regulated by the second messenger, Ca 2. Several Calpain isoforms have been reported of which Calpains I and II are the most ubiquitous in mammalian cells. Calpains are believed to be important modulators of a number of physiologic and pathologic events in cells. The enzyme has a broad substrate specificity and many Calpain substrates such as the transcription factors c-Fos and c-Jun, the tumor suppressor protein p53, multiple signaling enzymes (e.g., protein kinase C, pp60 src) and the adhesion molecules integrin and E-cadherin have been implicated in the pathogenesis of different human tumors. It has also been demonstrated in cell culture studies that inhibition of Calpain triggers apoptosis in cancer cell lines. These studies suggest that Calpain is a potential new cancer target for the discovery of a new class of antitumor agents. There is however, a paucity of in vivo studies (animal studies) that validate Calpain as a viable cancer target for anticancer drug discovery. The link between in vitro Calpain inhibition and in vivo anticancer activity is under-explored due in part to the metabolic instability of most Calpain inhibitors. This constitutes a significant gap that must be addressed in order to validate Calpain as a new cancer target. The immediate goal of this proposal is to fill this gap by developing a unique series of Calpain inhibitors that are potent, selective, and metabolically stable for use as tools to validate Calpain as a new cancer target. The long-term goal is to develop Calpain inhibitors as a new class of anticancer agents. The specific aims are to: (1) use structure-based molecular design approaches to design and synthesize metabolically stable Calpain inhibitors; (2) determine the potency and selectivity of the compounds towards Calpain inhibition versus other proteases; (3) determine the metabolic stability of the compounds; (4) determine the in vitro cytotoxicity and in vivo efficacy of the compounds; (5) determine if the inhibitory potency (Ki) of the inhibitors correlate with their in vitro cytotoxicity profiles as well as in vivo antitumor efficacy; (6) determine the mechanism(s) of Calpain inhibitor-induced cytotoxicity. The results of the proposed studies will validate Calpain as a new cancer target for anti-tumor drug discovery. Additionally, the compounds are useful biomedical tools for investigating the intracellular roles of Calpain due to their metabolic stability. The compounds are also potential anticancer agents.