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.
