XO44 is a cell-permeable, broad-spectrum covalent kinase probe. Identified by CAS 2088112-70-1, it has the formula C25H27FN8O3S and was designed for chemical proteomics rather than therapeutic use.
The molecule combines a kinase-binding aminopyrimidine scaffold with a phenylsulfonyl fluoride electrophile. A piperazine linker, cyclopropylpyrazole substituent, and terminal alkyne complete the probe design. The alkyne provides a handle for post-labeling conjugation without serving as the kinase-reactive group.
Application of XO44
PF-6808472 is used in competitive kinase-engagement workflows in intact cells. Cells are treated with a test inhibitor and then exposed to the probe. After lysis, labeled proteins can be connected to an azide reporter or affinity tag by click chemistry, enriched, and quantified by mass spectrometry. Reduced labeling indicates that the test compound blocked access to a kinase ATP site. Dose-response and vehicle controls help distinguish specific engagement from changes in protein abundance or sample recovery.
In Vitro
The original Pfizer XO44 study reported covalent labeling of up to 133 endogenous kinases in live Jurkat cells despite competition from intracellular ATP. Fifty were not recovered by a kinobead workflow applied to Jurkat cell lysates, demonstrating the complementary coverage of intact-cell labeling. Detection depends on cell type, target expression, probe concentration, incubation time, and analytical sensitivity.
X-ray crystallography resolved the XO44 structure bound to the human EGFR kinase domain at 1.60 Å resolution. The complex, deposited as PDB 5U8L, confirms covalent modification of the conserved catalytic lysine, Lys745, by the sulfonyl fluoride group.
In Vivo
The alternate catalogue form PF-06808472 refers to the same probe. Foundational work did not report animal or human pharmacology. Experiments in live Jurkat cells are in cellulo studies and should not be described as in vivo evidence. Animal use would require separate validation of exposure, tissue penetration, tolerability, and recovery of labeled kinases. Its established role is intracellular chemoproteomic profiling, not organism-level kinase inhibition.
Biochemical and Physiological Actions
The XO44 chemical structure first binds noncovalently within the ATP pocket, positioning the sulfonyl fluoride near the conserved catalytic lysine. Nucleophilic attack by the lysine side chain then forms a stable covalent adduct. This proximity-driven reaction enables broad kinase capture while limiting indiscriminate labeling by the electrophile.
The terminal alkyne in the XO44 molecular structure supports copper-catalyzed azide-alkyne cycloaddition after lysis. Probe signal reflects ATP-site accessibility, target abundance, and competition from ATP or another ligand; it is not a direct measure of catalytic activity or proof that every detected kinase is active.
Features and Benefits of XO44
- Live-cell kinase engagement profiling before sample disruption.
- Covalent capture of up to 133 endogenous kinases in Jurkat cells.
- Click-compatible alkyne handle for enrichment and mass-spectrometric analysis.
- Binding mode validated by the EGFR–XO44 crystal structure PDB 5U8L.
- Verified formula C25H27FN8O3S and XO44 molecular weight of 538.60 g/mol.
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