ABROGATION OF CELL CYCLE ARREST BY STAUROSPORINE ANALOGS

多年来，咖啡因被认为可以增强DNA损伤剂（如顺铂）引起的细胞毒性。这种增强的机制是通过其废除S和G2检查点的能力，这些检查点通常阻止受损细胞进行致命的有丝分裂。 不幸的是，这种治疗组合从未实现，因为在患者体内无法获得足够浓度的咖啡因。 一种新的化合物，7-hydroxystaurosporine（UCN-01），在消除顺铂诱导的S期和G2期阻滞的能力方面比咖啡因强至少100，000倍，并显著增强顺铂介导的细胞杀伤。此外，所需浓度似乎在动物中耐受良好。 在人类和啮齿类动物细胞系中的实验已经确定，这种废除只发生在p53肿瘤抑制因子有缺陷的时候。 因此，UCN-01可以选择性地增强肿瘤中的化疗，同时保留正常组织。不幸的是，正在进行的UCN-01的I期试验显示，与人血浆蛋白的结合限制或阻止其进入肿瘤。 该提议基于观察到新的类似物K252 a不与人血浆蛋白结合但仍消除顺铂诱导的S期阻滞，解决了该问题。 然而，它未能废除G2检查站，因为单独使用时会导致G2逮捕。 提出了两种方法。 首先，K252 a将用于选择性地驱动p53突变细胞通过S期，此时将添加S期特异性药物如伊立替康;这可能增强细胞毒性，而不需要通过有丝分裂。 第二，基于结构/活性分析的结果，提出合成新的类似物，预测其将消除S和G2停滞并增强细胞毒性，同时不能结合人血浆蛋白。 这些药物组合和类似物将在细胞培养模型和可移植鼠肿瘤模型中进行测试。同时机制研究将致力于了解这些类似物废除顺铂或伊立替康后的S期阻滞的机制，并确定野生型p53如何防止废除细胞周期阻滞。 这些实验应该确定一种药物组合，可以在未来的临床试验中测试其对p53缺陷肿瘤的选择性靶向。

Caffeine has been known for many years to enhance the cytotoxicity elicited by DNA damaging agents such as cisplatin. The mechanism of this enhancement is through its ability to abrogate the S and G2 checkpoints that normally prevent a damaged cell from undergoing a lethal mitosis. Unfortunately, this therapeutic combination has never been realized because adequate concentrations of caffeine can not be obtained in a patient. A new compound, 7-hydroxystaurosporine (UCN-01), is at least 100,000 times more potent than caffeine in its ability to abrogate the cisplatin-induced S and G2 arrest, and causes a marked enhancement in cisplatin-mediated cell killing. Furthermore, the concentrations required appear to be well tolerated in animals. Experiments in human and rodent cell lines have established that this abrogation occurs only when the p53 tumor suppressor is defective. Thus, UCN-01 may selectively enhance chemotherapy in the tumor while sparing normal tissue. Unfortunately, the ongoing Phase I trial of UCN-01 has shown avid binding to human plasma proteins limiting or preventing its access to the tumor. This proposal addresses a solution to this problem based on the observation that a novel analog, K252a, does not bind to human plasma proteins yet still abrogates S phase arrest induced by cisplatin. However, it fails to abrogate the G2 checkpoint because when used alone it causes a G2 arrest. Two approaches are proposed. First, K252a will be used to selectively drive p53-mutant cells through S phase, at which time an S phase-specific drug such as irinotecan will be added; this may enhance cytotoxicity without the requirement for passage through mitosis. Second, based on results of a structure/activity analysis, it is proposed to synthesize novel analogs that it is predicted will abrogate both S and G2 arrest and enhance cytotoxicity while failing to bind to human plasma proteins. These drug combinations and analogs will be tested in cell culture models and in a transplantable murine tumor model. Concurrent mechanistic studies will be directed toward understanding the mechanism by which these analogs abrogate S phase arrest following cisplatin or irinotecan, and determine how wildtype p53 prevents abrogation of cell cycle arrest. These experiments should identify a drug combination that can be tested for its selective targeting to p53-defective tumors in future clinical trials.