Discovery and characterization of PAMs. MS-751 cells were labeled with either heavy or light amino acids, lysed, and incubated with ×50 free KPT-7523 or DMSO, respectively, for 2 hours. The lysates were then incubated with KPT-7523 resin overnight. Samples were combined and washed, and then either digested on beads or run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), separated by molecular weight ranges, and then digested. Proteins were quantified by mass spectroscopy and analyzed. Both the (A) on-bead digestion and (B) in-gel digestion revealed PAK4 (red circle) as the major target of KPT-7523, with minor targets identified as red dots. (C) Cell lysates from MS-751, HeLa, and U2OS cells were treated with ×50 free KPT-7523, free KPT-7523–polyethylene glycol, or DMSO for 2 hours. The lysates were then passed over KPT-7523 resin overnight at 4°C. The resin was washed with buffer and run on SDS-PAGE for WB with PAK4. PAK4 specifically bound to KPT-7523 resin in all 3 cell lines tested. (D) MDA-MB-231 cells were treated with either DMSO or 10 µM KPT-7523 for 48 hours and then collected by lysing. The lysates were incubated with either KPT-7523 resin, KPT-9101 resin (inactive compound), or untagged resin overnight at 4°C. Resin was washed and run on SDS-PAGE and probed for PAK1-6. KPT-7523 resin bound specifically to PAK4. KPT-9101 and untagged resin did not bind to PAK4. (E) PAK4 regulatory (1-290 aa) and kinase (291-591 aa) domains were produced and purified from E coli. The purified protein was incubated with KPT-7523 resin overnight, washed, and then run on SDS-PAGE. PAK4 antibody measured specific binding of PAK4 kinase domain to KPT-7523. (F) Structure of the clinical candidate KPT-9274.