Establishing selectivity of FAK-paxillin PPI inhibitor using pulldown proteomics and a focal adhesion protein selectivity panel.

UA-2012 (and related non-myristoylated analog UA-1907) is a lead alpha-helical cyclic peptide which inhibits the focal adhesion kinase (FAK)-paxillin protein-protein interaction (PPI) and is being evaluated for the treatment of cutaneous melanoma. However, the development of an empirical approach to measure PPI inhibitor selectivity remains an important need. We report the development of a pulldown-MS proteomic approach, including a custom synthesized non-myristoylated UA-1907-agarose probe, to evaluate the binding selectivity of candidate FAK PPI inhibitors. Melanoma lysates were probed with UA-1907-conjugated agarose beads and eluted associated proteins were analyzed through untagged mass-spectroscopy proteomics. The identified proteins led to the development of a custom focal adhesion (FA) selectivity panel comprised of recombinant VinT, VinH, PARVA, PARVB, Talin-1 Rod 8, and the FAK FAT domain. Surface plasmon resonance (SPR) screening of these FA proteins against UA-1907 determined that only the FAK-FAT domain has a nanomolar binding affinity (KD) for UA-1907, whereas other FA proteins have no binding. Overall, we report the development of a customized pulldown-MS approach to characterize PPI drug selectivity that has utility in the FAK drug discovery field.