The tumor suppressor gene p53, which is among the most commonly mutated gene in human cancers, is a highly attractive target for the design of novel, mechanism-based anti-cancer agents that might have broadly utility. About half of all human cancers have been estimated to carry p53 mutations. In other cases p53 can be rendered functionally inactive by overexpression of proteins such as MDM2. p53 can induce the expression of WAF1/CIP1, which mediates the tumor suppressor activity of p53 by inhibiting cyclin- dependent kinase (CDK) activity. The overall goal of this research is to identify from high-throughput screening of natural product extracts of plant or microbial origin active compounds that restore lost p53 function or substitute for WAF1/CIP1 activity and to convert these leads by synthetic chemistry into pharmacologically active analogs. Xenova Limited (Laboratory Program 1) will prepare these extracts from their natural product resource and will carry out ELISA screens, designed and validated by PharmaGenics, Inc. (Laboratory Program 2), to identify lead compounds that can: (A) restore functional DNA binding activity to p53 mutants; (B) block the p53/MDM2 interaction; (C) inhibit the activity of a cyclin CDK complex. Xenova will purify milligram quantities of lead compound from a scaled-up fermentation or plant extract, as appropriate, and will conduct a complete structural analysis. Secondary cell-based assays will be employed at both Xenova and PharmaGenics, Inc. to confirm the activity of the purified leads and to probe their mechanism of action. These assays will be designed and validated by PharmaGenics, Inc. X-ray crystallography of the lead compound/protein target complex will be performed at the Memorial Sloan-Kettering Cancer Center in the laboratory of Nikola P. Pavletich (Laboratory Program 3) in order to generate information on structure-activity relationships and mechanism of action that will provide a basis for the rational synthesis of analogs. These analogs and the original leads will be tested for in vivo efficacy in relevant human tumor models designed and conducted by PharmaGenics, Inc. It is anticipated that these data will provide a basis for the initiation of clinical trials.