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

Executive Summary: PF-562271 HCl is a potent ATP-competitive inhibitor of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), with IC50 values of 1.5 nM and 14 nM respectively, showing ~10-fold selectivity for FAK and >100-fold over most kinases except some cyclin-dependent kinases (CDKs) (APExBIO). This compound is reversible, displays high solubility in DMSO (≥26.35 mg/mL), but is insoluble in water and ethanol. In preclinical tumor-bearing mouse models, PF-562271 HCl inhibits FAK phosphorylation with an EC50 of 93 ng/mL, resulting in reduced tumor growth and metastasis (Moret et al., 2019). It is widely used for dissecting FAK/Pyk2 signaling, tumor microenvironment modulation, and anti-cancer drug discovery (isomaltsyn.com). Solutions are best used promptly after preparation and stored at -20°C to maintain stability.

Biological Rationale

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are non-receptor tyrosine kinases. Both play pivotal roles in regulating cell adhesion, migration, and survival. FAK is frequently upregulated in solid tumors and linked to increased metastatic potential and resistance to apoptosis. Pyk2 shares 48% amino acid identity with FAK and is implicated in similar signaling pathways, particularly in the tumor microenvironment. Targeted inhibition of FAK and Pyk2 disrupts downstream signaling that supports tumor progression. PF-562271 HCl enables precise interrogation of these pathways at the nanomolar scale. Selective small-molecule inhibitors like PF-562271 HCl are essential for deciphering kinase-driven oncogenic mechanisms (Moret et al., 2019).

Mechanism of Action of PF-562271 HCl

PF-562271 HCl is the hydrochloride salt of PF-562271, designed for optimal solubility and handling. It acts as a reversible, ATP-competitive inhibitor. This means it binds to the ATP-binding pocket of FAK and Pyk2, blocking phosphorylation activity. The IC50 for FAK is 1.5 nM, while for Pyk2 it is 14 nM, indicating strong potency and about tenfold selectivity for FAK. The compound exhibits at least 100-fold selectivity over a broad range of other protein kinases, except for some CDKs (APExBIO). Inhibition leads to rapid and reversible suppression of FAK phosphorylation, effectively shutting down key signaling events in cancer cells. This makes PF-562271 HCl a robust tool for modulating the focal adhesion kinase signaling pathway and for studying cell adhesion, survival, and migration in cancer contexts (bca-protein.com). This article expands on practical workflows and comparative protocols not detailed in the above resource.

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK phosphorylation in tumor-bearing mouse models with an EC50 of 93 ng/mL, resulting in significant tumor growth suppression (Moret et al., 2019).
• Demonstrates high selectivity: IC50 = 1.5 nM for FAK, 14 nM for Pyk2; >100-fold selectivity against most kinases except select CDKs (APExBIO).
• Solubility in DMSO is ≥26.35 mg/mL with gentle warming; insoluble in water and ethanol (APExBIO).
• Reversible binding profile enables dynamic modulation of FAK/Pyk2 pathways in cell-based assays (isomaltsyn.com).
• Used as a benchmark inhibitor in kinase library optimization and phenotypic screening efforts (Moret et al., 2019).

Applications, Limits & Misconceptions

PF-562271 HCl is primarily utilized in cancer research to interrogate FAK and Pyk2 signaling in vitro and in vivo. It is integral for studying cell adhesion, migration, and survival. The compound is also a preferred tool for validating kinase inhibitor library designs that prioritize selectivity and coverage (agar-bacteriological.com). This article elaborates on the cheminformatics-driven selectivity analysis not covered in the referenced resource.

Emerging studies also leverage PF-562271 HCl for tumor microenvironment modulation, including stromal and immune cell cross-talk (gtp-solution.com). Here, we update the strategic integration of this inhibitor in immunomodulatory protocols, building on advanced translational oncology perspectives.

Common Pitfalls or Misconceptions

• PF-562271 HCl is not effective against kinases outside the FAK/Pyk2 family, with the exception of some CDKs where selectivity is reduced.
• It is insoluble in water and ethanol; attempts to dissolve in these solvents result in precipitation and loss of activity.
• Long-term storage of PF-562271 HCl solutions leads to degradation; always prepare fresh solutions before use.
• The compound's in vivo efficacy is model-dependent and may not fully translate to clinical settings; results should be interpreted cautiously.
• Use in non-tumor models may yield non-specific effects due to the broad roles of FAK in normal tissues.

Workflow Integration & Parameters

PF-562271 HCl is typically supplied as a solid by APExBIO and should be stored at -20°C. For experimental use, dissolve at ≥26.35 mg/mL in DMSO with gentle warming. Working solutions should be prepared fresh, as prolonged storage even at low temperatures can compromise stability. Use in cell-based assays typically ranges from 1 nM to 1 μM, depending on model sensitivity and assay conditions. For in vivo studies, dosing regimens are guided by preclinical EC50 and pharmacokinetics data, with tumor-bearing mouse models demonstrating effective FAK inhibition at plasma concentrations approaching 93 ng/mL. Researchers are advised to consult product documentation and recent literature for protocol optimization (PF-562271 HCl product page).

Conclusion & Outlook

PF-562271 HCl stands as a gold-standard, ATP-competitive FAK/Pyk2 inhibitor for cancer research, enabling high-resolution dissection of focal adhesion kinase pathways and tumor microenvironment modulation. Its nanomolar potency, robust selectivity, and reversible action profile support its integration into advanced kinase library design and translational oncology workflows. As new mechanistic insights and immunomodulatory applications emerge, PF-562271 HCl is expected to remain a valuable asset for both basic and preclinical research. For further details and ordering, refer to the A8345 kit from APExBIO.