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

Executive Summary: PF-562271 HCl is an ATP-competitive and reversible inhibitor with nanomolar potency against focal adhesion kinase (FAK; IC₅₀ = 1.5 nM) and proline-rich tyrosine kinase 2 (Pyk2; IC₅₀ = 14 nM) (APExBIO). It shows high selectivity (>100-fold) over most other protein kinases except some cyclin-dependent kinases. PF-562271 HCl inhibits FAK phosphorylation in vivo (EC₅₀ = 93 ng/mL in tumor-bearing mice), suppressing tumor growth and metastasis (Wang et al., 2025). The compound is supplied as a solid, is soluble in DMSO (≥26.35 mg/mL), and is widely used in research targeting cancer cell adhesion, migration, and tumor microenvironment modulation (Related Article). For optimal performance, storage at -20°C and prompt solution use are recommended (APExBIO).

Biological Rationale

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates signals from integrins and growth factor receptors. Its activation regulates cell adhesion, migration, proliferation, and survival (Wang et al., 2025). FAK is overexpressed or hyperactivated in many solid tumors, correlating with poor prognosis and enhanced metastatic potential. Proline-rich tyrosine kinase 2 (Pyk2) is a FAK homolog sharing 48% amino acid identity and supports compensatory or parallel signaling in cancer cells. Both kinases modulate cytoskeletal dynamics, cell motility, and tumor-stromal interactions. Aberrant FAK/Pyk2 signaling is implicated in cancer immune evasion, tumor microenvironment modulation, and therapy resistance. Thus, selective inhibition of FAK/Pyk2 has emerged as a targeted strategy to dissect oncogenic pathways and sensitize tumors to immunotherapy and radiotherapy (Advanced FAK/Pyk2 Inhibitor Article; extends the mechanistic and translational context for PF-562271 HCl).

Mechanism of Action of PF-562271 HCl

PF-562271 HCl is an ATP-competitive, reversible inhibitor. It binds to the ATP-binding pocket of FAK and Pyk2, preventing phosphorylation of tyrosine residues critical for kinase activation. The compound exhibits an IC₅₀ of 1.5 nM for FAK and 14 nM for Pyk2 under standard in vitro kinase assay conditions at 25°C and pH 7.4 (APExBIO). Selectivity profiling reveals >100-fold preference for FAK over most other kinases, with the exception of some cyclin-dependent kinases (CDKs), where selectivity is reduced. In tumor models, PF-562271 HCl administration leads to rapid dephosphorylation of FAK at Y397, inhibition of downstream signaling events (e.g., reduced paxillin phosphorylation), and decreased cell migration and invasion. In vivo, the compound demonstrates an EC₅₀ of 93 ng/mL for FAK pathway inhibition in mouse xenograft models. This inhibition translates into reduced tumor growth, angiogenesis, and metastatic spread (Wang et al., 2025).

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK phosphorylation with an in vitro IC₅₀ of 1.5 nM and Pyk2 phosphorylation with an IC₅₀ of 14 nM (APExBIO).
• Demonstrates >100-fold selectivity for FAK over most other kinases except limited CDKs (APExBIO).
• Inhibits FAK phosphorylation in tumor-bearing mice with an EC₅₀ of 93 ng/mL (intravenous dosing, 4 h post-administration) (Wang et al., 2025).
• Suppresses tumor growth and metastasis in multiple xenograft models, including lung, breast, and prostate cancer (Precision FAK/Pyk2 Inhibitor Article).
• Widely used for dissecting FAK/Pyk2 signaling in tumor microenvironment and immune modulation studies (Mechanistic Roadmap Article).

Applications, Limits & Misconceptions

PF-562271 HCl is extensively employed in preclinical cancer research to study:

• FAK/Pyk2-mediated cell adhesion, migration, and invasion.
• Tumor growth and metastasis suppression in xenograft and syngeneic models.
• Tumor microenvironment modulation and immune cell infiltration studies.
• Synergy with radiotherapy and immunotherapies targeting PD-1 and TIGIT (Wang et al., 2025).

Its robust selectivity profile and nanomolar potency make it suitable for dissecting kinase pathway dependencies in cancer cells. However, researchers should be mindful of the following:

Common Pitfalls or Misconceptions

• PF-562271 HCl is not effective in water-based buffers; it is insoluble in water and ethanol, requiring DMSO for dissolution (APExBIO).
• The compound is not selective against all cyclin-dependent kinases (CDKs); off-target effects may arise at higher concentrations.
• Long-term storage of solutions is discouraged due to stability loss; fresh preparation is recommended (Practical Solutions Article; this article clarifies storage and stability best practices beyond prior workflow guides).
• Results obtained in murine models may not fully translate to human clinical contexts without further validation.
• PF-562271 HCl is not approved for clinical use; it is for research purposes only.

Workflow Integration & Parameters

PF-562271 HCl (SKU: A8345) is supplied as a solid and should be stored at -20°C. For optimal solubilization, dissolve in DMSO to concentrations ≥26.35 mg/mL using gentle warming. The compound remains stable as a solid under recommended storage but degrades in solution over time; use freshly prepared aliquots for each experiment. For cell-based assays, typical working concentrations range from 1–100 nM, depending on the cell type and endpoint. In vivo dosing regimens in mouse models frequently utilize intravenous or intraperitoneal administration, with pharmacodynamic endpoints measured at 4–24 hours post-dose (Translational Frontier Article; this article updates guidance with the latest stability and in vivo kinetic data). Always ensure DMSO vehicle controls are included. Refer to the PF-562271 HCl product page (APExBIO) for detailed handling instructions.

Conclusion & Outlook

PF-562271 HCl stands as a benchmark ATP-competitive FAK/Pyk2 inhibitor, enabling rigorous analysis of focal adhesion kinase signaling and tumor microenvironment dynamics. Its high selectivity, potency, and reproducible pharmacology make it a cornerstone for translational oncology, especially when integrated with immunotherapeutic or radiotherapeutic regimens (Wang et al., 2025). Continued application of PF-562271 HCl will further refine our understanding of kinase-driven tumor biology and support the development of next-generation anti-cancer strategies.