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

Executive Summary: PF-562271 HCl is a reversible, ATP-competitive inhibitor targeting 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 in vitro conditions (APExBIO). This compound demonstrates approximately 10-fold selectivity for FAK over Pyk2 and over 100-fold selectivity against most other kinases, except select cyclin-dependent kinases (CDKs) (Anichini et al. 2022). PF-562271 HCl is validated in vivo, showing FAK phosphorylation inhibition at an EC50 of 93 ng/mL and effective tumor growth suppression in mouse models. It is insoluble in water/ethanol but dissolves in DMSO at ≥26.35 mg/mL with warming, and is supplied as a solid for optimal stability. As a product of APExBIO, it is widely adopted in studies of kinase signaling, tumor microenvironment modulation, and anti-cancer drug development.

Biological Rationale

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates cell adhesion, migration, survival, and signal transduction in both normal and tumor tissues (Anichini et al. 2022). Aberrant FAK signaling is implicated in tumor progression, metastasis, and therapeutic resistance. Proline-rich tyrosine kinase 2 (Pyk2), a close FAK homolog sharing 48% sequence identity, also modulates cell motility and invasion. Inhibiting FAK/Pyk2 disrupts oncogenic signaling cascades, reduces tumor cell survival, and alters the tumor microenvironment. Targeting these kinases is a validated strategy for preclinical cancer research and for evaluating combinatorial immunotherapeutic approaches.

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 FAK, preventing autophosphorylation at Tyr397, which is essential for downstream signaling. The compound exhibits nanomolar potency (IC50 = 1.5 nM for FAK; 14 nM for Pyk2) and demonstrates rapid, reversible inhibition under cell-free and cellular conditions. Selectivity profiling indicates >100-fold selectivity over most other kinases, with notable exception for some CDKs where inhibition is less pronounced. In tumor-bearing mouse models, PF-562271 HCl reduces FAK phosphorylation with an EC50 of 93 ng/mL plasma concentration, correlating with impaired tumor growth and metastasis (Anichini et al. 2022).

Evidence & Benchmarks

• PF-562271 HCl inhibits FAK kinase activity with an in vitro IC50 of 1.5 nM, as determined by ATP-competitive kinase assay (APExBIO).
• Pyk2 kinase activity is inhibited with an IC50 of 14 nM, demonstrating ~10-fold selectivity for FAK over Pyk2 (Anichini et al. 2022).
• In mouse models, PF-562271 HCl suppresses FAK phosphorylation at a plasma EC50 of 93 ng/mL, resulting in measurable tumor growth inhibition (Anichini et al. 2022).
• Solubility is ≥26.35 mg/mL in DMSO at room temperature with gentle warming; insoluble in water and ethanol (APExBIO).
• PF-562271 HCl demonstrates >100-fold selectivity against a kinase panel except for certain CDKs, where selectivity is reduced (Anichini et al. 2022).

This article extends beyond the workflow optimization focus of 'Leveraging PF-562271 HCl (SKU A8345) for Reproducible FAK…' by providing direct mechanistic and benchmark data for translational oncology applications.

It also updates the strategic perspective outlined in 'PF-562271 HCl and the Translational Frontier: Mechanistic…' by enumerating current evidence benchmarks and clarifying selectivity profiles under controlled conditions.

Applications, Limits & Misconceptions

PF-562271 HCl is widely used in preclinical cancer models to interrogate FAK/Pyk2-dependent pathways, assess tumor cell invasion, and modulate tumor microenvironment interactions. The compound is a valuable tool for studying kinase-driven resistance mechanisms and for benchmarking new immunotherapeutic combinations. However, its selectivity profile necessitates consideration of potential off-target effects, especially involving CDKs at higher concentrations.

Common Pitfalls or Misconceptions

• PF-562271 HCl is not water-soluble; improper solvent use may cause precipitation and assay failure.
• It does not distinguish between FAK and Pyk2 at higher concentrations; selectivity diminishes above 100 nM.
• The inhibitor is not suitable for long-term solution storage; degradation may occur at room temperature or upon repeated freeze-thaw cycles.
• It is not an irreversible inhibitor; kinase activity can recover upon compound removal.
• Not all tumor types are equally sensitive; context-dependent resistance mechanisms may limit efficacy (Anichini et al. 2022).

This piece clarifies boundaries not fully addressed in 'PF-562271 HCl (SKU A8345): Scenario-Driven Solutions for…' by providing explicit guidance on solubility, selectivity, and storage constraints.

Workflow Integration & Parameters

For optimal results, PF-562271 HCl should be dissolved in DMSO at concentrations ≥26.35 mg/mL with gentle warming. Working solutions should be freshly prepared and used promptly. The solid form is recommended for long-term storage at -20°C. In cell-based assays, nanomolar dosing is required for FAK-specific inhibition, with typical effective concentrations ranging from 1 to 100 nM, depending on cell type and assay conditions (APExBIO). Assay controls should account for potential off-target effects at higher doses. Data reproducibility is enhanced by referencing established workflows as described in related articles. For more on integrating PF-562271 HCl into advanced experimental models, see 'Strategic Horizons in Translational Oncology: FAK/Pyk2 In…', which this article builds upon by providing updated, quantitative benchmarks and explicit workflow parameters.

Conclusion & Outlook

PF-562271 HCl remains a gold-standard ATP-competitive inhibitor for dissecting FAK/Pyk2 signaling in cancer research. Its nanomolar potency, robust selectivity, and validated in vivo activity make it indispensable for studies of tumor progression, metastasis, and microenvironmental crosstalk. Continued benchmarking and strategic integration with immunotherapeutic approaches will further expand its utility. As a flagship offering from APExBIO, PF-562271 HCl supports precision experimental design and translational oncology innovation. For detailed specifications and ordering, refer to the official product page.