Focal adhesion kinase (FAK) is a member of a family of nonreceptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migrationThe ability of cells to respond appropriately to environmental cues is critical to maintaining cellular, tissue, and organism homeostasis. One such environmental cue is derived from cellular adhesion to the extracellular matrix. The loss of adhesiondependent cellular regulation can lead to increased cellular proliferation, decreased cell death, changes in cellular differentiation status, and altered cellular migratory capacity, all of which are critical components of cell carcinogenesis and metastatic progression.The FAK 4 family kinases (which include FAK and Pyk2) regulate cell adhesion, migration, and proliferation in a variety of cell types (for review see Refs. 1-3). Adhesion of cells to the extracellular matrix is mediated by heterodimeric transmembrane integrin receptors located within sites of close opposition to the underlying matrix called focal adhesions. Integrin engagement and clustering stimulates FAK phosphorylation on Tyr 397 , creating a high affinity binding site for Src and Src family kinases. The FAK⅐Src complex phosphorylates many components of the focal adhesion, resulting in changes in adhesion dynamics and the initiation of signaling cascades. In addition to FAK catalytic activity, FAK also functions as a scaffold to organize structural and signaling proteins within focal adhesions.The importance of FAK as a regulator of normal cellular function is underscored by the number of cancers reported to have alterations in FAK expression and/or activity, including colon, breast, thyroid, prostate, cervical, ovarian, head and neck, oral, liver, stomach, sarcoma, glioblastoma, and melanoma (4, 5). Additionally, alterations in FAK expression and/or activity have been associated with tumorigenesis and increased metastatic potential (4, 5). Currently, it is unclear how the catalytic and/or scaffolding function of FAK contributes to tumor progression. To date studies of FAK function have relied on the expression of dominant interfering mutants or elimination of FAK expression by genetic knock-out, antisense oligonucleotide expression, or small interfering RNA.Herein, we report the biochemical and cellular characterization of a novel small molecule inhibitor, PF-573,228 (here after referred to as PF-228), that targets FAK catalytic activity. The inhibitor interacts with FAK in the ATP-binding pocket and effectively blocks the catalytic activity of recombinant FAK protein or endogenous FAK expressed in a variety of normal and cancer cell lines. Treatment of cells with PF-228 blocked FAK phosphorylation on Tyr 397 and concomitantly reduced the tyrosine phosphorylation of paxillin, a recognized downstream effector of FAK signaling. Drug treatment of normal and cancer cells resulted in decreased cell migration and inhibited adhesion turnover, biological activities previously ascribed to FAK. Interestingly, inhibition of FAK activi...

Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC 50 of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cellbased assay measuring phospho-FAK with an IC 50 of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100Â selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC 50 of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATPcompetitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.

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CP-673,451 is a potent inhibitor of platelet-derived growth factor β-receptor (PDGFR-β) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-β in cells (IC50 = 1 nmol/L) being more than 450-fold selective for PDGFR-β versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-β inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-β phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-β phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC50 of 120 ng/mL in plasma at Cmax. In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. × 5, p.o., corresponding to 5.5 ng/mL at Cmax). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. × 10 days dosing routinely inhibited tumor growth (ED50 ≤ 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-β phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.

Supplementary Data from Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271