Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth

Abstract

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K d = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC 50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC 50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC 50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

Activity-Dependent Rapid Local RhoA Synthesis Is Required for Hippocampal Synaptic Plasticity

“…est concentration of the PAK inhibitor, which blocks PAK1/4 but not PAK2/3 (Murray et al, 2010); and (2) TEA-induced PAK phosphorylation was completely abolished only by the highest concentration of the PAK inhibitor, which suggests an additional contribution of PAK2/3 to the effect of TEA on PAK activation. Although previous work indicated that TBS-induced LTP consolidation did not require Rac/PAK signaling (Rex et al, 2009), we used a different PAK inhibitor (IPA-3), which does not interact with PAK4.…”

“…Two concentrations of PF-3758309 were used (50 and 500 nM), based on its different affinities for PAK1/4 versus PAK2/3 isoforms (Murray et al, 2010). Stimulation of PAK phosphorylation by TEA was significantly prevented by PF-3758309, but the effect was statistically significant only at the highest concentration (Fig.…”

FRAX597, a Small Molecule Inhibitor of the p21-activated Kinases, Inhibits Tumorigenesis of Neurofibromatosis Type 2 (NF2)-associated Schwannomas

“…In addition, we employed another PAK inhibitor from Pfizer (PF-3758309), which potently suppresses both group I (PAK1, PAK2, and PAK3) and II (PAK4, PAK5, and PAK6) PAKs (44). Importantly, while both FRAX597 and PF-3758309 display some limited off-target effects on other kinases, these off-target effects are largely non-overlapping (45).…”

Inhibition or Ablation of p21-activated Kinase (PAK1) Disrupts Glucose Homeostatic Mechanisms in Vivo

“…As such, therapeutic targeting of PAK1 in efforts to induce apoptosis of tumor cells has been advocated (3)(4)(5). PAK1 is one of six family members, categorized into two groups: PAK1-3 belong to group I and contain a unique autoinhibitory region, distinguishing them from Group II members PAK4 -6 (6).…”