Development of a high-throughput TR-FRET assay to identify inhibitors of the FAK-paxillin protein-protein interaction.

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase and scaffolding protein that is primarily regulated by integrin signaling. FAK signaling increases cell motility in both normal and cancer cells, and FAK is often overexpressed and/or dysregulated in many types of cancer. FAK has three different domains: an N-terminal FERM domain, a central kinase domain (the traditional target for drug discovery), and a C-terminal focal adhesion targeting (FAT) domain. The FAT domain represents an alternative approach to targeting FAK, and our aim is to identify novel small molecules that will inhibit FAT protein-protein interactions (PPI), which may have implications for cancer and fibrosis treatment. Here, we describe the development and validation of a robust high-throughput screening (HTS) assay suitable for identifying inhibitors of the FAT:paxillin PPI. The 384-well low volume assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology and uses the high affinity biotin-PEG-1907 stapled peptide to mimic paxillin. We also present the development of a TR-FRET counterscreen assay using CD47 and SIRPα to detect nonspecific inhibitors, as well as an orthogonal surface plasmon resonance (SPR) binding assay. We employed the FAT: biotin-PEG-1907 assay to screen a 31,636-compound small molecule library. Primary positives (hits) from HTS were confirmed in concentration-response primary and counterscreen assays and validated in the SPR binding assay. We discovered 4 inhibitors of the FAT:paxillin PPI using this approach and established a framework for small molecule drug discovery efforts targeting the FAT domain of FAK.