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

Executive Summary: PF-562271 HCl is a highly potent, reversible ATP-competitive inhibitor that selectively targets focal adhesion kinase (FAK, IC₅₀=1.5 nM) and proline-rich tyrosine kinase 2 (Pyk2, IC₅₀=14 nM) (APExBIO). It exhibits >10-fold selectivity for FAK over Pyk2 and >100-fold selectivity over most other kinases, except some cyclin-dependent kinases (CDKs). This tool compound suppresses FAK phosphorylation in vivo, inhibits tumor growth and metastasis in mouse models, and is insoluble in water and ethanol but highly soluble in DMSO at ≥26.35 mg/mL with gentle warming (Adams et al., 2025). PF-562271 HCl is integral to dissecting FAK/Pyk2 signaling in cancer research workflows.

Biological Rationale

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates cell adhesion, migration, and survival. Dysregulation of the FAK/Pyk2 signaling pathway is implicated in cancer progression, metastasis, and modulation of the tumor microenvironment (Adams et al., 2025). Proline-rich tyrosine kinase 2 (Pyk2), homologous to FAK with 48% sequence identity, also contributes to these cellular processes. The metastatic cascade involves the formation of pre-metastatic niches (PMNs) by primary tumor cells, with FAK signaling central to the recruitment of myeloid progenitor cells (MPCs) and the establishment of pro-tumorigenic microenvironments. Inhibiting FAK/Pyk2 disrupts these processes, offering a tractable target for anti-cancer strategies (Adams et al., 2025).

Mechanism of Action of PF-562271 HCl

PF-562271 HCl is the hydrochloride salt of PF-562271, designed as a potent, reversible, ATP-competitive inhibitor. It binds the ATP-binding site of FAK and Pyk2, blocking autophosphorylation and downstream signaling. The compound demonstrates an IC₅₀ of 1.5 nM for FAK and 14 nM for Pyk2, with approximately 10-fold selectivity for FAK. Selectivity over other kinases exceeds 100-fold, except for select CDKs. In vivo, PF-562271 HCl achieves inhibition of FAK phosphorylation at an EC₅₀ of 93 ng/mL. This blockade suppresses tumor cell adhesion, migration, and survival, thereby impairing the metastatic potential in preclinical models (APExBIO product data).

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK kinase activity with an IC₅₀ of 1.5 nM under cell-free conditions (pH 7.4, 25°C) (APExBIO).
• Pyk2 is inhibited with an IC₅₀ of 14 nM, confirming ~10-fold selectivity for FAK over Pyk2 (APExBIO).
• PF-562271 HCl achieves >100-fold selectivity against a panel of non-FAK, non-Pyk2 kinases, except for certain CDKs (Adams et al., 2025).
• In tumor-bearing mouse models, FAK phosphorylation is inhibited at EC₅₀=93 ng/mL (plasma, 37°C) (Adams et al., 2025).
• PF-562271 HCl reduces tumor growth and metastatic spread in vivo, correlating with decreased pre-metastatic niche formation (Adams et al., 2025).
• Compound is insoluble in water and ethanol, but soluble in DMSO (≥26.35 mg/mL, 25–37°C) (APExBIO).

Applications, Limits & Misconceptions

PF-562271 HCl is extensively used to dissect FAK/Pyk2 signaling pathways, model tumor microenvironment modulation, and support anti-cancer drug development. It enables precise inhibition in cell viability, proliferation, and cytotoxicity assays, as detailed in PF-562271 HCl (SKU A8345): Practical Solutions for FAK/Pyk2 in Cell Biology (this article provides more recent selectivity data and in vivo benchmarks than the linked piece). In comparison to PF-562271 HCl: Selective ATP-Competitive FAK/Pyk2 Inhibitor for Cancer Research, the current review clarifies solubility requirements and storage stability. For immunomodulation studies, see PF-562271 HCl: Decoding FAK/Pyk2 Inhibition in Immunomodulation, which this article extends with updated in vivo efficacy data.

Common Pitfalls or Misconceptions

• PF-562271 HCl is not effective when dissolved in water or ethanol; use only DMSO for stock preparation (≥26.35 mg/mL, gentle warming).
• Long-term storage of PF-562271 HCl solutions is not recommended; prepare fresh solutions for each experiment to ensure potency.
• While highly selective, the compound may inhibit certain cyclin-dependent kinases at higher concentrations.
• It is not a pan-kinase inhibitor; activity is largely restricted to FAK, Pyk2, and select CDKs.
• Not suitable for in vivo use in species or models where DMSO vehicle is contraindicated or poorly tolerated.

Workflow Integration & Parameters

PF-562271 HCl is supplied as a solid and should be stored at -20°C. Stock solutions are prepared in DMSO at concentrations ≥26.35 mg/mL, using gentle warming (25–37°C) as needed. For optimal stability, use solutions promptly after preparation. The compound is compatible with standard cell viability, proliferation, and cytotoxicity assay workflows. Inhibition of FAK autophosphorylation can be detected using Western blotting or ELISA at concentrations as low as 1–100 nM, depending on cell type and assay sensitivity. In vivo, dosing regimens should be optimized to achieve plasma levels near the established EC₅₀ of 93 ng/mL for FAK phosphorylation inhibition (Adams et al., 2025). For detailed implementation, see the manufacturer's PF-562271 HCl product page.

Conclusion & Outlook

PF-562271 HCl remains a benchmark ATP-competitive FAK/Pyk2 inhibitor for dissecting focal adhesion kinase signaling and tumor microenvironment modulation. Its nanomolar potency, selectivity, and robust in vivo performance enable high-confidence cancer biology studies. As FAK/Pyk2 signaling is further implicated in metastatic progression and immunomodulation, PF-562271 HCl will continue to be a valuable research tool. For sourcing and further technical data, APExBIO supplies PF-562271 HCl under SKU A8345 (product page).