PROJECT SUMMARY/ABSTRACTFocal adhesion kinase (FAK) or protein tyrosine kinase 2 (PTK2) is a 125 kDa non-receptor tyrosine kinaseand scaffolding protein that is overexpressed or amplified in many cancers including ovarian breast colorectalmelanoma glioblastoma and pancreatic cancers. FAK is involved in multiple biological pathways that cancontribute to cancer progression including cell migration invasion lymphangiogenesis anti-apoptosismetastasis epithelial-mesenchymal transition (EMT) and signaling pathways mediated by PI3K/AKT and -catenin. FAK has been validated as a target in cancer therapy by genetic manipulation and FAK knockdownresults in robust activation of apoptosis in cancer cells with minimal effects in normal cells. Many ATP-competitive FAK kinase domain inhibitors have been reported; however these are only cytostatic and/ormoderately selective for FAK and have shown limited efficacy in clinical trials. Kinase-independent roles of FAKare not blocked by kinase inhibition; in particular the scaffolding function of the C-terminal focal adhesiontargeting (FAT) domain. The FAK FAT domain is the major modulator of FAK-dependent anti-apoptosis and isthe domain regulated by the endogenous dominant-negative FAK isoform termed FAK-related non-kinase(FRNK). Specifically the FAT-paxillin protein-protein interaction (PPI) localizes FAK to the focal adhesion andmutation of residues at the FAT domain interface perturbs focal adhesion turnover cell adhesion migration andinvasion. Thus a major unmet need is the discovery of small molecule chemical probes that block FAK FATdomain scaffolding. In order to target the FAK FAT domain we have previously explored fragment screening bySPR/NMR and hydrocarbon-stapled peptides. These approaches led to FAT fragment-based hits withmicromolar affinity and the lead stapled peptide probe UA-1907 (KD = 1 M) that induces apoptosis in melanomacells. For this project we will develop novel drug screening assays leveraging chemical probe UA-1907 to identifysmall molecule FAT ligands that disrupt the anti-apoptotic scaffolding functions of FAK. Specifically we will: 1)design a TR-FRET biochemical assay to screen a 35000 compound PPI-biased library for hits that inhibit UA-1907 binding to FAT; 2) confirm biochemical hits as FAT ligands using SPR and HSQC-NMR approaches; and3) develop a NanoBiT cellular assay measuring FAK-paxillin binding to validate hits. In all we expect this projectto identify the first described small molecule-based inhibitors of the FAT-paxillin interaction that will serve as thebasis for future medicinal chemistry optimization studies.