This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.As key regulators of signal transduction pathways, protein kinases are misregulated in a variety of human pathologies. Accordingly protein kinases have long been considered excellent targets for inhibition by small molecules. However, the number of small molecules that target clinically relevant kinases is still quite low. This results predominantly from two considerations; 1) most kinase inhibitors are competitive with ATP and 2) the extent of evolutionary conservation of the ATP binding domain within protein kinases. This means that compounds identified as exquisitely potent inhibitors of a particular kinase in vitro may inhibit a broad number of kinases in vivo. Demonstrating in vivo specificity, a prerequisite for any translational therapeutic, remains a significant obstacle. Pak1 represents an attractive target as it is directly implicated in malignancy and its mechanism of activation allows for the isolation of ATP noncompetitive inhibitors. Paks are members of the Ste20 superfamily of kinases, and complement Ste20 deletions in S.cerevisiae. I have engineered a strain of S.cerevisiae in which growth is directly linked to the kinase activity of ectopic Pak1. I propose to use this platform to screen the small molecule libraries at the CCET for compounds that inhibit Pak1-dependent growth in vivo and to use the power of genetics to counter-select against compounds that target unrelated proteins. I propose confirm specificity by testing promising compounds identified from this platform against recombinant Pak1 in vitro and to further transition these compounds cell culture systems to evaluate their ability to inhibit in higher systems.
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