Developing a high-throughput drug screening assay for focal adhesion kinase (FAK) inhibitors
Focal adhesion kinase (FAK) is a promising cancer drug target due to its demonstrated role in cancer invasion and metastasis. Small molecule FAK inhibitors have been shown to decrease tumor growth and metastasis in several preclinical models. However, traditional FAK inhibitors focus on targeting its kinase domain, leaving FAK’s scaffolding of oncogenic drivers intact. Indeed, the proto-oncogene c-Src’s activation of FAK at FAK’s focal adhesion targeting (FAT) region through phosphorylation of tyrosine-925 (Y925) has been implicated in the promotion of cancer invasion and metastasis. Therefore, developing cancer therapeutics that inhibit the activation of FAK by c-Src is expected to disrupt FAK’s ability to promote cancer invasion and metastasis. The purpose of this project is to develop a novel kinase assay to efficiently screen thousands of molecules and enable the discovery of specific molecules that inhibit c-Src’s phosphorylation of FAK FAT Y925. This assay measures phosphorylation activity and its inhibition by small molecule drugs via a luciferase/luciferin-based reaction, whereby luminescence is emitted when phosphorylation has occurred. In order to verify the specificity of FAK Y925 phosphorylation by c-Src in the kinase assay, site-directed mutagenesis of non-specific phosphorylation sites (c-Src Y416E and Y527F, FAK FAT Y1007F) was performed to block these sites from being phosphorylated on both c-Src and FAT, ensuring that c-Src’s primary phosphorylation target in the kinase assay is FAK Y925. c-Src and FAT were successfully mutated at non-specific phosphorylation sites Y416 and Y527 (c-Src) and Y1007 (FAT) for their use in the kinase assay. FAT Y925 was successfully mutated to serve as an assay control. FAK FAT and c-Src kinase domain were successfully purified in their wild type forms, as well as single-mutant FAT Y1007F. Double mutant FAT and c-Src purification was performed as well. Virtual screening was performed to identify potential drug candidates that can block c-Src from phosphorylating FAK Y925 for use in this high-throughput screening assay. The optimization of this high-throughput drug screening assay will allow for the rapid and robust identification of c-Src/FAK Y925 inhibitors. The discovery of these compounds will enable further optimization for their use as targeted therapies for invasive and metastatic cancers.