PF-3758309 (PF-03758309) (IC50=1.3 nM), a pyrrolopyrazole inhibitor of PAK4, has effective ATP-competition.

PAK5:18.1 nM(Ki), PAK6:17.1 nM(Ki), PAK4:1.3 nM, PAK4:18.7 nM(Ki), PAK3:99 nM

PF-3758309 (Kd = 2.7 nM), an effective inhibitor of PAK4 （Ki=18.7 nM）, has ATP competition. PF-3758309 (IC50 = 1.3 nM) inhibits phosphorylation of the PAK4 substrate GEF-H1 in cells. In the tumor cell, PF-3758309(IC50 = 4.7 nM) grows in a linesanchorage-independent manner. In HCT116 cells, PF-3758309 inhibits endogenous pGEF-H1 accumulation. In A549 cells, PF-3758309 effectively inhibits cellular proliferation in IC50 of 20 nM and anchorage-independent growth in IC50 of 27 nM.

In the HCT116 tumor model, PF-3758309 act as an antiproliferative inducing apoptosis. In the sensitive model, PF-3758309 (EC50=0.4 nM) blocks the growth of multiple human tumor xenografts.

In phospho-GEF-H1 cellular assay, TR-293-KDG cells are incubated for 3 hours with PF-3758309. TR-293-KDG cells were captured on an anti-HA antibody-coated plate, which is detected with an anti-phospho-S810-GEF-H1 antibody, and quantified with a horseradish peroxidase-goat anti-rabbit antibody conjugate. TR-293-KDG cells are constructed from HEK293 cells, which is transfected with tetracycline-inducible PAK4-kinase domain (amino acids 291-591) and expressed HA-tagged GEFH1ΔDH (amino acids 210-921).

HEK293T, HCT116, and SKOV3 cells are plated on 384-well plates for 24 hours culture. PF-3758309( ~1 μM) is added to the cell culture incubating 72 hours. After drug treatment,cell proliferation and other indicators of metabolism are measured.

Nude mice with Xenograft tumors were orally fed with PF-3758309（7.5-30 mg/kg BID）dissolved in 0.5% methylcellulose.