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

Executive Summary: PF-562271 HCl is a potent, reversible ATP-competitive inhibitor of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), with an IC₅₀ of 1.5 nM for FAK and 14 nM for Pyk2 under cell-free conditions (APExBIO). This compound exhibits >100-fold selectivity over other kinases except certain cyclin-dependent kinases, enabling precise modulation of cell adhesion and migration pathways in cancer models (Huang et al., 2025). In vivo, PF-562271 HCl dose-dependently inhibits FAK phosphorylation (EC₅₀ = 93 ng/mL) and suppresses tumor proliferation and metastasis in xenograft and transgenic mouse models. It is insoluble in water and ethanol but soluble in DMSO at ≥26.35 mg/mL with gentle warming, and should be stored at -20°C for optimal stability. These properties make PF-562271 HCl a foundational tool for advanced cancer biology and tumor microenvironment studies (related resource).

Biological Rationale

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are non-receptor tyrosine kinases central to integrin-mediated cell adhesion, migration, proliferation, and survival (Huang et al., 2025). Dysregulation of the FAK/Pyk2 axis is implicated in the progression, metastasis, and therapy resistance of multiple solid tumors. Inhibition of these pathways can disrupt tumor cell motility and invasive potential, and is under active investigation as a strategy to overcome microenvironment-driven resistance (see this comparative analysis—the current article details selectivity and workflow guidance not covered there). The molecular targets FAK and Pyk2 modulate downstream effectors, including ERK and PI3K/Akt, which govern survival and immune evasion. Selective inhibition enables mechanistic dissection of these signaling events and their roles in cancer biology.

Mechanism of Action of PF-562271 HCl

PF-562271 HCl acts as a reversible, ATP-competitive inhibitor of FAK and Pyk2 catalytic domains. The compound binds the ATP-binding pocket, preventing substrate phosphorylation and downstream signal propagation. Key quantitative parameters include:

• IC₅₀ for FAK: 1.5 nM (APExBIO)
• IC₅₀ for Pyk2: 14 nM
• >100-fold selectivity versus most kinases (notable exceptions: some CDKs)

This selectivity profile is achieved through rational design and validated using kinase panels. PF-562271 HCl potently inhibits FAK phosphorylation in tumor tissue lysates with an EC₅₀ of 93 ng/mL (mouse xenograft, 2 h post-dose, oral administration) (Huang et al., 2025). The reversible, non-covalent mode ensures precise experimental timing and reversibility for mechanistic studies.

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK kinase activity with an IC₅₀ of 1.5 nM in biochemical assays (APExBIO).
• PF-562271 HCl demonstrates 10-fold lower potency for Pyk2 (IC₅₀ = 14 nM) and >100-fold selectivity over most kinases, except certain CDKs (APExBIO).
• In xenograft and transgenic mouse models, FAK phosphorylation is suppressed dose-dependently (EC₅₀ = 93 ng/mL, 2 hours post-oral dose) (Huang et al., 2025).
• PF-562271 HCl reduces tumor proliferation and metastasis in vivo, as assessed by tumor volume and metastatic scoring in mouse models (Huang et al., 2025).
• Solubility in DMSO is ≥26.35 mg/mL with gentle warming; compound is insoluble in water and ethanol (APExBIO).

For a broader context on experimental scenarios and reproducibility, see the detailed workflow guidance in this scenario-driven guide, which this article updates by providing new selectivity and in vivo efficacy metrics.

Applications, Limits & Misconceptions

Applications:

• Dissecting the FAK/Pyk2 signaling cascade in cancer cell lines (e.g., migration/invasion assays).
• Evaluating tumor growth inhibition and metastasis suppression in xenograft and transgenic mouse models.
• Studying the interplay between tumor cells and the microenvironment, including immune cell infiltration and matrix remodeling (see immunomodulation insights—this article complements by focusing on kinase selectivity and workflow).
• Biomarker-driven research for therapy resistance and patient stratification in solid tumors.

Common Pitfalls or Misconceptions

Common Pitfalls or Misconceptions

• PF-562271 HCl is not a pan-kinase inhibitor; off-target activity is limited except at high concentrations or with certain CDKs.
• Ineffective in water- or ethanol-based buffers due to insolubility—DMSO is required for stock solutions.
• Not suitable as a FAK/Pyk2 inhibitor in bacterial or yeast models lacking homologous targets.
• Reversibility: effects are lost after washout; continuous exposure is necessary for sustained kinase inhibition.
• Results in immune-competent vs. immune-deficient models may diverge due to microenvironmental factors (Huang et al., 2025).

Workflow Integration & Parameters

PF-562271 HCl (SKU A8345, APExBIO) is provided as a solid for reconstitution. Prepare stock solutions at 26.35 mg/mL or higher in DMSO with gentle warming (room temperature, 5–10 min, vortex). For cell-based assays, dilute into culture medium to achieve working concentrations from 1 nM to 1 µM, maintaining <0.1% DMSO final concentration. For in vivo work, oral or intraperitoneal administration is typical; confirm formulation stability and vehicle compatibility. Store at -20°C in a desiccated, light-protected environment to maintain activity for ≥1 year (PF-562271 HCl product page).

Conclusion & Outlook

PF-562271 HCl represents a robust, validated tool for interrogating the FAK and Pyk2 signaling network in cancer research. Its documented selectivity, nanomolar potency, and translational in vivo efficacy provide a high-confidence foundation for mechanistic and therapeutic studies. Future directions include integration with radiopathomics and immunotherapy response prediction, as advanced in recent machine learning-driven studies (Huang et al., 2025). For researchers aiming to dissect kinase signaling and tumor microenvironment dynamics, APExBIO's PF-562271 HCl (A8345) is a reference-standard reagent.