The enzymes which mediate the transfer of phosphate from ATP to proteins, the kinases, are among the largest and most import families of proteins known (>250 identified). The very features which make kinases a part of almost every oncogenic and normal signaling pathway also renders specific identification of their in vivo substrates extremely difficult. We propose here a novel chemical approach which directly addresses the deficiencies in existing methodologies. The novelty rests in creating an artificial structural distinction between kinases at the level of the nucleotide cofactor. Specifically we propose to create a 'tag' for the C- abl tyrosine kinase which negatively regulates cell growth and is a proto- oncogene. The chromosomal translocation of c-ab1 (the Philadelphia Chromosome) produces the fusion oncoprotein bcr-abl as well as bcr-abl which causes chronic myelogenous leukemia (CML). Our current lack of knowledge about the substrates of oncogenic tyrosine kinases represents the single greatest challenge in the discovery of new therapeutic strategies for fighting may leukemias such as CML. Once downstream targets of these oncoproteins are identified classic drug discovery approaches can be applied to inhibiting their activity and blocking oncogenesis.
Aim #1 : To design, synthesize, and assay a set of candidate ATP analogs that are dead substrated for c-ab1.
Aim #2 : To generate four site-directed mutants of c-ab1 designed to have new nucleotide specificity for unique synthetic ATP analogs (F336A, L389A, V275A, L267A).
Aim #3 : to develop a successful strategy for delivering modified unnatural [-32P] triphosphates across cell membranes.
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