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

Executive Summary: PF-562271 HCl, offered by APExBIO, is a potent, reversible, and ATP-competitive 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 under cell-free conditions (APExBIO product page). It achieves over 100-fold selectivity against unrelated kinases except some CDKs, enabling precise perturbation of FAK/Pyk2-driven signaling in cancer and metastasis models (Moret et al., 2019). In xenograft and transgenic mouse models, PF-562271 HCl dose-dependently inhibits tumor proliferation with a reported EC₅₀ of 93 ng/mL for FAK phosphorylation. The compound is a solid with a molecular weight of 543.95 and optimal solubility in DMSO (≥26.35 mg/mL at 25°C), but is insoluble in water and ethanol. Proper storage at −20°C is recommended to maintain compound integrity.

Biological Rationale

Focal adhesion kinase (FAK; PTK2) and proline-rich tyrosine kinase 2 (Pyk2; PTK2B) are non-receptor tyrosine kinases that regulate cell adhesion, migration, proliferation, and survival. Aberrant FAK/Pyk2 signaling is implicated in tumorigenesis, metastasis, and the modulation of the tumor microenvironment (Moret et al., 2019). Targeted inhibition of these kinases enables delineation of their roles in cancer cell behavior and tumor progression. PF-562271 HCl is widely used to dissect the mechanistic contributions of FAK/Pyk2 in solid tumor models (Related Article), extending our understanding of cell adhesion kinase pathways beyond what is achievable with genetic knockdown or less selective inhibitors.

Mechanism of Action of PF-562271 HCl

PF-562271 HCl is an ATP-competitive, reversible inhibitor that binds the kinase domains of FAK and Pyk2. The compound prevents ATP from accessing the catalytic site, thereby blocking autophosphorylation and downstream signaling. With an IC₅₀ of 1.5 nM for FAK and 14 nM for Pyk2, PF-562271 HCl demonstrates approximately 10-fold selectivity for FAK over Pyk2 and >100-fold selectivity against the majority of off-target kinases (APExBIO). However, selectivity is reduced for certain cyclin-dependent kinases (CDKs). In cellular contexts, this inhibition translates to blocked phosphorylation of FAK at Tyr397 and reduced migration, invasion, and proliferation of cancer cells. The compound’s reversibility allows for dynamic modulation of signaling in dose- and time-dependent studies.

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK with an IC₅₀ of 1.5 nM in cell-free kinase assays (APExBIO).
• Pyk2 inhibition by PF-562271 HCl yields an IC₅₀ of 14 nM under identical conditions (APExBIO).
• >100-fold selectivity is observed against non-target kinases except some CDKs (see Table 1 in Moret et al., 2019).
• Dose-dependent suppression of FAK phosphorylation in tumor xenografts achieves EC₅₀ = 93 ng/mL (Moret et al., 2019).
• In vivo, PF-562271 HCl reduces tumor size and metastatic burden in transgenic mouse models (see Supporting Data, Moret et al., 2019).

This article provides a comprehensive, protocol-driven context for PF-562271 HCl, extending the mechanistic specificity discussed in PF-562271 HCl: Mechanistic Precision and Translational Progress by focusing on selectivity, solubility, and workflow optimization.

Applications, Limits & Misconceptions

PF-562271 HCl is used extensively in cancer biology to study:

• FAK/Pyk2 pathway modulation in solid tumor models and cell lines.
• Inhibition of tumor growth and metastasis in xenograft and genetically engineered mouse models.
• Dissection of tumor microenvironment dynamics, including stromal and immune cell interactions (Advanced Strategies for Tumor Microenvironment; this article integrates chemical stability and selectivity data absent from that article).
• Phenotypic screens and drug combination studies, leveraging its narrow kinase inhibition profile.

Common Pitfalls or Misconceptions

• PF-562271 HCl is not a pan-kinase inhibitor; it is highly selective for FAK/Pyk2 and only weakly inhibits most other kinases (Moret et al., 2019).
• Compound is insoluble in water and ethanol; DMSO is required for stock solution preparation (APExBIO).
• Not suitable for long-term storage at room temperature; must be stored at −20°C for stability.
• Partial off-target effects may occur on certain CDKs; verify kinase panel data for specific applications.
• Reversibility enables washout and time-course studies, but requires careful dosing for sustained inhibition.

Compared to PF-562271 HCl (SKU A8345): Scenario-Driven Solutions, this article emphasizes quantitative selectivity and bioanalytical benchmarks rather than experimental scenarios.

Workflow Integration & Parameters

PF-562271 HCl (SKU A8345) is supplied as a solid compound by APExBIO. Stock solutions should be prepared in DMSO at ≥26.35 mg/mL with gentle warming. The compound is insoluble in water and ethanol. For cell-based assays, working concentrations are typically in the 1–1000 nM range, with vehicle controls recommended. Storage at −20°C in tightly sealed vials maintains stability for >12 months. Analytical validation by LC-MS and NMR is available from APExBIO. For detailed mechanistic and translational guidance, see Precision FAK/Pyk2 Inhibition in Advanced Tumor Models; this dossier updates workflow integration with explicit solubility and storage parameters.

Conclusion & Outlook

PF-562271 HCl is a benchmark tool for selective, reversible inhibition of FAK and Pyk2 in cancer research. Its well-characterized selectivity, potency, and pharmacological profile enable robust interrogation of adhesion kinase signaling in vitro and in vivo. Proper application—including attention to solubility, storage, and dosing—facilitates reproducible results and supports translational studies targeting tumor progression and metastasis. Continued chemical library optimization, as discussed by Moret et al. (2019), reinforces the value of highly selective kinase inhibitors like PF-562271 HCl in the era of precision oncology.