Inactivation of the retinoblastoma tumor suppressor protein, Rb, is essential for the progression of cell cyclefrom G1 to the S phase. Rb gene or its gene product is inactivated in a wide variety of cancers, either bymutation of the gene or functional inactivation of the protein. During normal cell cycle progression, Rb isinactivated by a cascade of phosphorylation events, mediated by kinases associated with the D and E typecyclins. Studies from our lab have shown that growth factor stimulation of quiescent cells leads to the bindingof a cellular signaling kinase, Raf-1, to the Rb proteins. This binding of Raf-1 to Rb precedes the binding ofcyclins and cdks and appears to be essential for the subsequent phosphorylation of Rb. Disruption of thebinding of Raf-1 to Rb using a peptide inhibitor prevented Rb phosphorylation, cell cycle progression,angiogenesis as well as tumor growth in nude mice. Based on these findings, we propose to identify anddevelop small molecules that can disrupt the Rb-Raf-1 interaction. Such compounds can be expected tohave anti-proliferative as well as anti-neoplastic activities and might be of use as anti-cancer agents. Indeed,our preliminary screen of a drug library led tot he identification of two small molecules capable of disruptingthe binding of Raf-1 to Rb selectively and with reasonable potency. They were effective in arresting cellproliferation and one of the compounds could inhibit or retard tumor growth in nude mice. Based on theseresults, we propose to carry out the following studies: (1) To identify and synthesize small molecules todisrupt Rb-Raf-1 interaction (2) To assess the specificity and efficacy of the Rb-Raf-1 inhibitors in vitro (3) Toevaluate the effects of Rb-Raf-1 disrupters on cell proliferation and angiogenesis. (4) To evaluate the effectof Rb-Raf-1 disrupters on tumor growth and metastasis in vivo. Dr. Nick Lawrence's laboratory w illsynthesize combinatorial drug libraries based on initial hits from the drug screen. Additional screening oflarger libraries will also be done to identify more potent inhibitors than the ones already identified. Thesecompounds will be subjected to extensive in vitro and in vivo analysis for their efficacy as anti-tumor drugs.Further, these compounds will be used to study the biological events taking place in the cell during earlystages of cell cycle. These compounds will also be used extensively to study other signaling pathwaysexamined in the sister projects of this PO-1 application.
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