The Src kinase family encompasses eight non-receptor protein tyrosine kinases in mammals that regulate signaling pathways in virtually every cell type. Src-family kinases (SFKs) share a common regulatory mechanism that requires two intramolecular interactions to maintain the inactive state. These involve binding of the SH3 domain and a PPII helix in the SH2-kinase linker and interaction of the SH2 domain and a phosphotyrosine residue in the C-terminal tail. To compare the activation dynamics of the individual SFKs, a synthetic SFK SH3 domain-binding peptide (VSL12) was used to probe the sensitivity of SFKs to SH3-based activation. Surface plasmon resonance was used to confirm equivalent binding of the VSL12 peptide to the SH3 domains and near-full-length kinases. SFKs were tested with VSL12 in a kinetic kinase assay to measure the changes in the rate of activity induced by SH3:linker displacement. All SFKs tested were susceptible to activation, but to varying degrees. Further, autophosphorylation of the c-Src and Hck activation loops prior to VSL12-induced activation revealed that c-Src can achieve a higher level of activation if primed before SH3 domain displacement. These results suggest that distinct activation thresholds may exist for individual SFKs. To apply these findings in an inhibitor discovery setting, the interaction of c-Src with focal adhesion kinase (FAK) was studied. FAK activates c-Src by binding to its SH2 and SH3 domains and disrupting their regulatory influence on the kinase domain. The c-Src:FAK complex plays a major role in the migration and invasion of both normal and cancer cells, making it an attractive target for drug discovery. To test the idea that FAK binding induces allosteric changes in the kinase domain active site, a screening assay was developed to target these changes with small molecule inhibitors. Assay conditions were identified where c-Src activity was dependent on a phosphopeptide encompassing the FAK SH3- and SH2-binding motifs for c-Src. A focused library of kinase-biased inhibitors was screened to identify compounds displaying selectivity for the c-Src:pFAK peptide complex. An aminopyrimidinyl carbamate, WH-4-124-2, was discovered that showed five-fold selectivity for the complex. Molecular docking studies of this inhibitor on the kinase domain of Lck bound to imatinib suggest that WH-4-124-2 is a “Type II” kinase inhibitor that prefers the unphosphorylated, “DFG-out” conformation of the kinase. Selective inhibitors of a specific FAK-induced c-Src conformation may provide a unique approach to selective targeting of this key cancer cell signaling pathway
