Abstract PR-2: Discovery of p21-activated kinase inhibitor PF-03758309
Authors: Guo Chuangxing , Zhang Junhu , McAlpine Indrawan , Johnson Catherine , Marakovits Joseph , Dong Liming , Kephart Susan , Yang Anle , Tikhe Jayashree , Li Haitao , Guo Lisa , Bouzida Djamal , Deng Ya-Li , Knighton Dan , Piraino Joseph , Lee Joseph , Smeal Tod , Christensen Jamie , Kraynov Eugenia , Loi Cho-Ming , Younis Hasam , Dagostino Eleanor , Murray Brion W. 
Published: June 2012

The p21-activated kinase (PAK) family members are key effectors of Rho family GTPases, which act as regulatory switches that control such cellular processes as motility, proliferation, and cell survival. Some members of this family (such as Cdc42) have been shown to be required for Ras driven tumorigenesis. PAK4 is a key effector for Cdc42 and mediates downstream signals that control cell motility, proliferation and cell survival. The PAK family consists of PAK1,2,3 (‘group 1’) and PAK4,5,6 (‘group 2’). Both PAK4 and PAK1 have been shown to be required for Ras driven transformation. PAK4 has been shown to be oncogenic and able to drive anchorage independent growth when activated. PAK4 expression and activity is broadly up-regulated in solid tumors such as colon, ovarian and pancreatic cancers. Pfizer's PAK4 inhibitor program started with HTS of kinase focus library compounds. Multiple series were identified from the screening effort. Initial optimization mainly focused on kinase selectivity, ADME properties and ability to modulate target in vivo, leading to selection of pyrrolopyrazoles as the lead series. By hybridizing the pyrrolopyrazole core with aminopyrimidine series, we discovered potent PAK4 inhibitors with low to sub nM cellular activity. For the new hybrid series, the challenge was to attain good oral bioavailability. The observed poor absorption was likely due to high efflux nature of the template. Medicinal chemistry strategies, such as reducing molecular charge, lowering polar surface area and improving ligand efficiency, were applied to reduce efflux. As a result, two sub-series achieved excellent in vivo tumor growth inhibition when dosed orally. PF-03758309 has demonstrated excellent profile, leading to its selection as a clinical development candidate.

PF-3758309 is a potent ATP-competitive inhibitor of PAK4 kinase domain (Kd = 4.5 nM). In engineered cell assays, PF-3758309 inhibited PAK4 dependent phosphorylation of its substrate GEF-H1 (IC50 = 1 nM). It potently inhibits the anchorage independent growth of HCT116 cells (IC50 = 0.24 nM). PF-3758309 exhibited broad anti-proliferative activity across a panel of 67 cell lines (CRC/pancreatic/NSCLC): 66% IC50 &lt; 100 nM and 36% IC50 &lt; 10 nM. In a human xenograft tumor model, PF-3758309 demonstrated a dose-dependent inhibition of both GEF-H1 and PAK4 phosphorylation. PF-3758309 is highly efficacious in human xenograft tumor models. Five of seven models tested showed robust tumor growth inhibition (&gt;70%TGI at 15–20 mg/kg PO) by PF-3758309: HCT116, A549, MDAMB231, M24met, and Colo205. Broad kinase screening has demonstrated that this is a selective pan-PAK inhibitor with potential additional activities (e.g. AMPK). The pharmacodynamic and antitumor effects of PF-3758309 support its evaluation as an anticancer agent.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-2.