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

Executive Summary: PF-562271 HCl is an ATP-competitive, reversible inhibitor targeting focal adhesion kinase (FAK) and Pyk2 with high selectivity and nanomolar potency (IC₅₀ for FAK = 1.5 nM; Pyk2 = 14 nM) (APExBIO). The compound effectively reduces FAK phosphorylation in tumor xenograft models, inhibiting tumor proliferation and metastasis (EC₅₀ = 93 ng/mL) (Keller et al., 2023). PF-562271 HCl is insoluble in water but dissolves at ≥26.35 mg/mL in DMSO, and should be stored at -20°C for stability. It provides over 100-fold kinase selectivity relative to most non-FAK/Pyk2 kinases, except some CDKs. The inhibitor is widely adopted in preclinical oncology, enabling precise modulation of cell adhesion and migration signaling pathways (see related article).

Biological Rationale

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are non-receptor tyrosine kinases involved in cell adhesion, migration, proliferation, and survival (Keller et al., 2023). Dysregulation of FAK/Pyk2 signaling promotes tumor progression and metastasis in various solid tumors. Elevated FAK activity correlates with poor prognosis and resistance to targeted therapies, including HER2 inhibitors in breast cancer. Targeting these kinases disrupts integrin and growth factor signaling, impacting the tumor microenvironment and immune cell infiltration. PF-562271 HCl enables researchers to dissect these pathways with high precision due to its selectivity and potency. This article extends previous work by providing detailed workflow parameters and benchmarking evidence beyond what is found in Harnessing ATP-Competitive FAK/Pyk2 Inhibition: Strategic....

Mechanism of Action of PF-562271 HCl

PF-562271 HCl is an ATP-competitive, reversible inhibitor of FAK and Pyk2 (APExBIO). It binds to the ATP-binding pocket of the kinase domain, blocking autophosphorylation and downstream signaling. Potency is highest for FAK (IC₅₀ = 1.5 nM) and approximately 10-fold lower for Pyk2 (IC₅₀ = 14 nM). The compound exhibits >100-fold selectivity over most kinases except certain cyclin-dependent kinases. Upon administration, PF-562271 HCl reduces phosphorylation at FAK Tyr397, leading to impaired focal adhesion turnover and cell migration. This action translates into reduced tumor cell proliferation, survival, and metastatic potential. The inhibitor is effective in both in vitro cell lines and in vivo xenograft and transgenic mouse models. Compared to conventional FAK inhibitors, PF-562271 HCl offers superior selectivity and operational stability (see Q&A in related article).

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK autophosphorylation in tumor xenografts with an EC₅₀ of 93 ng/mL (Keller et al., 2023, https://doi.org/10.1186/s13046-022-02578-w).
• The compound demonstrates an IC₅₀ of 1.5 nM against FAK and 14 nM for Pyk2 in biochemical assays (APExBIO).
• PF-562271 HCl exhibits >100-fold selectivity for FAK/Pyk2 over most protein kinases, minimizing off-target effects (product datasheet, APExBIO).
• In vivo, PF-562271 HCl suppresses tumor growth and reduces metastasis in transgenic mouse models, with dose-dependent responses (Keller et al., 2023, https://doi.org/10.1186/s13046-022-02578-w).
• The inhibitor supports modulation of the tumor microenvironment, influencing immune cell infiltration and tumor-immune interactions (see mechanistic insights).

Applications, Limits & Misconceptions

PF-562271 HCl is widely applied in cancer biology for:

• Investigating FAK/Pyk2-driven tumor proliferation and metastasis.
• Modeling cell adhesion, migration, and invasion in vitro.
• Assessing therapeutic efficacy in xenograft and genetically engineered mouse models.
• Exploring tumor microenvironment modulation, including immune infiltration and stromal interactions.
• Combining with HER2-targeted therapies to study resistance mechanisms in breast cancer (Keller et al., 2023).

Compared to PF-562271 HCl: Unraveling FAK/Pyk2 Inhibition in Tumor-Im..., this article provides updated benchmarks and clarifies solubility and storage constraints.

Common Pitfalls or Misconceptions

• PF-562271 HCl is not effective in models lacking FAK/Pyk2 expression or where tumor growth is independent of these kinases.
• The compound should not be dissolved in water or ethanol due to poor solubility; DMSO (≥26.35 mg/mL) is required (APExBIO).
• Prolonged storage above -20°C may degrade compound potency and selectivity.
• Off-target effects may occur at high concentrations, especially for cyclin-dependent kinases.
• Results may not fully translate to human clinical settings without further validation.

Workflow Integration & Parameters

• Preparation: Dissolve PF-562271 HCl in DMSO at concentrations up to 26.35 mg/mL with gentle warming. Avoid water and ethanol as solvents (APExBIO).
• Storage: Store aliquots at -20°C. Minimize freeze-thaw cycles to preserve integrity.
• Assay Use: Typical in vitro concentrations range from 10 nM to 1 µM, depending on cell type and endpoint.
• In vivo Dosing: Refer to published protocols for xenograft and mouse models; effective doses suppress FAK phosphorylation at EC₅₀ ~93 ng/mL (Keller et al., 2023).
• Controls: Include vehicle (DMSO) and, where possible, a non-FAK/Pyk2-selective kinase inhibitor for specificity assessment.

For troubleshooting and reproducible protocol optimization, see PF-562271 HCl (SKU A8345): Resolving FAK/Pyk2 Inhibition ..., which addresses scenario-driven Q&A for assay consistency. This article extends that discussion by providing updated evidence and workflow recommendations.

Conclusion & Outlook

PF-562271 HCl is a powerful, selective ATP-competitive inhibitor of FAK and Pyk2, enabling rigorous mechanistic interrogation of cell adhesion signaling in cancer models. Its nanomolar potency, selectivity profile, and validated benchmarks underpin its widespread adoption in oncology research. For optimal performance, strict adherence to solubility and storage guidelines is essential. While PF-562271 HCl has advanced preclinical understanding of FAK/Pyk2 biology, further studies in clinical models are warranted. For more details and ordering, visit the APExBIO PF-562271 HCl product page.