AbstractThe N‐terminal FERM domain of focal adhesion kinase (FAK) contributes to FAK scaffolding and interacts with HER2, an oncogene and receptor tyrosine kinase. The interaction between HER2 and FAK drives resistance to FAK‐kinase domain inhibitors through FAK Y397 transphosphorylation and FAK re‐activation upon inhibition. As such, FAK FERM remains an attractive drug discovery target. In this report, we detail an alternative approach to targeting FAK through virtual screening‐based discovery of chemical probes that target FAK FERM. We validated the binding interface between HER2 and FAK using site‐directed mutagenesis and GST pull‐down experiments. We assessed the ligandability of key‐binding residues of HER2 and FAK utilizing computational tools. We developed a virtual screening method to screen ~200,000 compounds against the FAK FERM domain, identifying 20 virtual chemical probes. We performed GST pull‐down screening on these compounds, discovering two hits, VS4 and VS14, with nanomolar IC50s in disrupting HER2‐FAK. We performed further testing, including molecular docking, immunofluorescence, phosphorylation, and cellular invasion assays to evaluate the compounds’ biological effects. One probe, VS14, was identified with the ability to block both auto‐ and transphosphorylation of Y397. In all, these studies identify two new probes that target FAK FERM, enabling future investigation of this domain.
