PF-562271 HCl: Potent ATP-Competitive FAK/Pyk2 Inhibitor for Cancer Research

Executive Summary: PF-562271 HCl is a highly selective, reversible inhibitor of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), with IC₅₀ values of 1.5 nM for FAK and 14 nM for Pyk2, demonstrating ~10-fold selectivity for FAK over Pyk2 and >100-fold selectivity over most kinases except select CDKs (APExBIO datasheet). It is effective at inhibiting FAK phosphorylation in vivo with an EC₅₀ of 93 ng/mL, resulting in suppressed tumor growth and metastasis in mouse models (Cancer Lett 2025). The compound is highly soluble in DMSO (≥26.35 mg/mL, gentle warming) but insoluble in water/ethanol, and must be stored at −20°C. PF-562271 HCl is widely used to interrogate FAK/Pyk2 signaling and the tumor microenvironment, supporting both mechanistic and translational oncology research (Strategy Article). APExBIO supplies PF-562271 HCl as a solid for research use only, with clear protocols for optimal stability.

Biological Rationale

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase central to cell adhesion, migration, and survival, especially in cancer progression (Cancer Lett 2025). Pyk2 is a homolog sharing 48% amino acid identity with FAK and is implicated in similar pathways. Dysregulation of FAK/Pyk2 signaling is linked to enhanced tumor cell motility, invasion, angiogenesis, and metastasis. Targeting these kinases with small-molecule inhibitors like PF-562271 HCl enables disruption of oncogenic signaling networks. This approach is especially valuable in gastric and other solid tumors where FAK is upregulated or hyperactivated, contributing to resistance against conventional and immunotherapies. Recent machine learning-driven analyses of gastric cancer highlight the importance of precise pathway interrogation to stratify risk and predict therapy response (Cancer Lett 2025).

Mechanism of Action of PF-562271 HCl

PF-562271 HCl is a potent ATP-competitive, reversible inhibitor of both FAK and Pyk2. It binds at the ATP-binding site of these kinases, preventing autophosphorylation (notably at Y397 in FAK) and downstream signaling. The inhibitor demonstrates a 1.5 nM IC₅₀ for FAK and a 14 nM IC₅₀ for Pyk2 in biochemical assays (APExBIO). Selectivity profiling shows over 100-fold selectivity for FAK/Pyk2 over other kinases, except for certain cyclin-dependent kinases (CDKs), where some overlap is observed. In cell-based and in vivo models, PF-562271 HCl blocks FAK phosphorylation, resulting in reduced cell migration, proliferation, and survival. In tumor-bearing mouse models, effective inhibition of FAK phosphorylation is achieved at an EC₅₀ of 93 ng/mL, correlating with significant suppression of tumor growth and metastasis (Cancer Lett 2025).

Evidence & Benchmarks

• PF-562271 HCl inhibits recombinant FAK with an IC₅₀ of 1.5 nM and Pyk2 with an IC₅₀ of 14 nM under standard biochemical assay conditions (pH 7.4, 25°C, ATP 100 μM) (APExBIO).
• Demonstrates >100-fold selectivity for FAK/Pyk2 over most other kinases, except select CDKs (e.g., CDK2/Cyclin A IC₅₀ ~200 nM) (APExBIO).
• In murine tumor models, PF-562271 HCl achieves FAK phosphorylation inhibition at 93 ng/mL (plasma), suppressing tumor growth and metastasis in vivo (Cancer Lett 2025).
• Highly soluble in DMSO at ≥26.35 mg/mL (25°C, gentle warming), insoluble in water/ethanol (APExBIO).
• Effective for dissecting FAK/Pyk2 signaling in cancer microenvironment and immunomodulation research (BCA-Protein Article).

Applications, Limits & Misconceptions

PF-562271 HCl is primarily used in preclinical research to dissect FAK/Pyk2-mediated signaling in cancer models, evaluate effects on tumor microenvironment, and explore anti-metastatic drug mechanisms. It supports studies on tumor cell migration, invasion, and response to immunotherapy-based combinations, as shown in gastric cancer radiopathomics research (Cancer Lett 2025).

• Enables precise inhibition of the focal adhesion kinase signaling pathway for mechanism-of-action and target validation studies.
• Serves as a benchmark tool for screening novel FAK/Pyk2 inhibitors and evaluating combination therapies.
• Widely adopted in microenvironment modulation and immunomodulatory research (Immunomodulation Article), extending the mechanistic depth beyond simple kinase inhibition.

For a more technical protocol-driven perspective, see our comparison to PF-562271 HCl: Precision FAK/Pyk2 Inhibitor for Cancer Research, which details laboratory workflow and troubleshooting. This article extends previous coverage by integrating recent clinical and machine learning findings in gastric cancer.

For readers interested in strategic and translational insights, PF-562271 HCl: Strategic Dissection of FAK/Pyk2 Signaling focuses on the translational impact in metastatic prostate cancer, while the present article provides updated selectivity and solubility benchmarks.

Common Pitfalls or Misconceptions

• PF-562271 HCl is not selective for FAK/Pyk2 over all kinases; cross-reactivity with some CDKs exists (IC₅₀ ~200–300 nM).
• Compound is insoluble in water or ethanol; improper solvent selection leads to precipitation and loss of potency.
• Long-term storage of PF-562271 HCl solutions (even in DMSO) at −20°C results in degradation; use solutions promptly post-preparation.
• Not suitable for direct clinical use; for research applications only as supplied by APExBIO.
• Does not inhibit immune checkpoint proteins (e.g., PD-1/PD-L1) directly; acts upstream via kinase signaling pathways.

Workflow Integration & Parameters

PF-562271 HCl is supplied as a solid by APExBIO (SKU: A8345) and should be stored at −20°C under desiccated conditions (PF-562271 HCl product page). For in vitro work, dissolve in DMSO to ≥26.35 mg/mL with gentle warming. Prepare working solutions fresh; avoid freeze-thaw cycles. Use at nanomolar concentrations for kinase assays and at concentrations corresponding to in vivo EC₅₀ (93 ng/mL) for murine models. Integrate with imaging, transcriptomics, or immunomodulatory platforms for advanced experimental workflows, as demonstrated in recent gastric cancer radiopathomics studies (Cancer Lett 2025). For advanced applications in tumor microenvironment modulation, see PF-562271 HCl: Beyond FAK/Pyk2 Inhibition in Tumor Microenvironment; this article clarifies solubility and selectivity parameters for reproducible integration.

Conclusion & Outlook

PF-562271 HCl is a validated, potent ATP-competitive inhibitor of FAK and Pyk2, providing high selectivity and robust in vivo efficacy. It is indispensable for dissecting FAK/Pyk2 signaling, benchmarking anti-cancer strategies, and exploring tumor microenvironment mechanisms. The compound's established role in preclinical models and synergy with machine learning-driven biomarker discovery suggest future potential in translational oncology. For detailed protocols and ordering, refer to the APExBIO PF-562271 HCl product page.