Discovery of PPI inhibitors for the FAK FAT domain

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PROJECT SUMMARY/ABSTRACT Focal adhesion kinase (FAK), or protein tyrosine kinase 2 (PTK2), is a 125 kDa non-receptor tyrosine kinase and scaffolding protein that is overexpressed or amplified in many cancers, including ovarian, breast, colorectal, melanoma, glioblastoma, and pancreatic cancers. FAK is involved in multiple biological pathways that can contribute to cancer progression, including cell migration, invasion, lymphangiogenesis, anti-apoptosis, metastasis, epithelial-mesenchymal transition (EMT), and signaling pathways mediated by PI3K/AKT and β- catenin. FAK has been validated as a target in cancer therapy by genetic manipulation, and FAK knockdown results 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/or moderately selective for FAK, and have shown limited efficacy in clinical trials. Kinase-independent roles of FAK are not blocked by kinase inhibition; in particular, the scaffolding function of the C-terminal focal adhesion targeting (FAT) domain. The FAK FAT domain is the major modulator of FAK-dependent anti-apoptosis and is the 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, and mutation of residues at the FAT domain interface perturbs focal adhesion turnover, cell adhesion, migration, and invasion. Thus, a major unmet need is the discovery of small molecule chemical probes that block FAK FAT domain scaffolding. In order to target the FAK FAT domain, we have previously explored fragment screening by SPR/NMR and hydrocarbon-stapled peptides. These approaches led to FAT fragment-based hits with micromolar affinity and the lead stapled peptide probe, UA-1907 (KD = 1 µM) that induces apoptosis in melanoma cells. For this project, we will develop novel drug screening assays leveraging chemical probe UA-1907 to identify small 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 35,000 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; and 3) develop a NanoBiT cellular assay measuring FAK-paxillin binding to validate hits. In all, we expect this project to identify the first described small molecule-based inhibitors of the FAT-paxillin interaction that will serve as the basis for future medicinal chemistry optimization studies.