Kinase mediated tyrosine phosphorylation is a crucial component of cellular signaling cascades. Precise regulatory control over kinase activity must be maintained during signaling as evidenced by numerous human diseases that arise upon dysregulation of protein kinases. Given the crucial role of kinases in cell signaling, these enzymes are key targets in treating numerous diseases. Traditional approaches for therapeutic targeting of kinases have involved small molecules that exert their effect directly on the kinase active site. Given the increasing instances of resistance to such drugs there is a need for devising new ways to target kinase activity. In our work on the Tec family tyrosine kinases (immunological kinases involved in signaling downstream of antigen receptors), we have characterized a number of specific allosteric sites and interactions that modulate the chemistry of the active site. These allosteric pockets are sometimes 30 angstrom removed from the active site or involve domains far from the kinase domain in primary sequence. The current application lays out specific aims to identify and characterize allosteric small molecule modulators of the Tec family kinases, Itk and Btk. In addition, we continue to move our understanding of Tec structure forward by solving the crystal structure of full length Btk. This will be the first strucure of any full length Tec kinase and the insight gained will facilitate further development of allosteric regulators for this family. We have established strong collaborations with immunologists that have extensive T- and B-cell signaling expertise (in particular as related to Itk and Btk). These colleagues are eager to test our small molecule reagents in their cellular and animal systems. Thus, this project spans basic structural biology and enzymology to small molecule discovery to physiological assays that will ultimately reveal the utility of the reagents we are developing to treat human disease.