SYNTHESIS OF ACTIVE SITE-DIRECTED SRC INHIBITORS

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pp60c-src (Src) is a protein tyrosine kinase (PTK) that has been shown to have elevated activity in several cancers, including cancers of the breast, colon, lung and others, compared to normal tissues. The overall purpose of our research is to develop new chemotherapeutic agents, specifically targeted to pp60c-src, to be used in the treatment of tumors that possess elevated activity of this PTK. Because of the activity differential between tumor and normal tissue, we feel that inhibitors of pp60c-src will have low general toxicity and great therapeutic potential. Since the natural substrates of PTKs are proteins our work is aimed at the development of peptide-based inhibitors. The major hypothesis to be tested in this proposal is that by understanding the modes of binding of peptide inhibitors of our target enzyme, we can develop active-site directed, small molecule peptidomimetic inhibitors that bind to the enzyme with increased affinity and enhanced bioavailability. Our strategy is to develop tight binding peptides, determine their conformations both free in solution and when bound to the enzyme, and from this information design peptidomimetic inhibitors. We have developed a cyclic decapeptide which serves as a "lead" peptide which is a competitive inhibitor of pp60c-src, Kii+640 nM, and which is very selective for this enzyme versus other PTKs and control enzymes. In this proposal our efforts will be divided into two major areas, (1) further understanding the nature of the interactions of or peptides with the active site of the enzyme using additional analogues as well as NMR and molecular modeling, and (2) usage of this information to design non-peptidic inhibitors. From NMR studies carried out to date and the structure of the insulin receptor kinase, we have developed hypotheses of the modes of binding of the peptide and have designed compounds o test this hypothesis. Further NMR studies will provide more detailed structural information that will be used in the design of peptidomimetic inhibitors. Additionally, we are developing mechanism-based (suicide) inhibitor groups to be incorporated into our peptide mimetics. Feedback from the biological testing as well as the structural studies will enable continuing refinement of our inhibitors. Those compounds taken to pre-clinical animal testing will be synthesized in multi-gram quantities in our laboratories.