Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.


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Reproducibility of experiments was confirmed by the use of separately measured (biological) replicates and/or appropriate controls. In vitro biochemical and cellular experiments were performed at least in two independent experiments. Attempts at replication were successful. The exact number of replicates per data point is indicated in figure legends.
LC-MS measurements for chemical proteomics experiments were randomized. Randomization was not applicable for other experiments. All biological and biochemical experiments were carried out with appropriate internal negative and/or positive controls as indicated.
The investigators were not blinded, because collection or analysis of the presented data was not prone to bias. All experiments are precise (and generally quantitative) measurements of enzyme activity, protein labeling, protein expression or phoshoprotein levels and are not based on subjective assessments. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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A numerical value for number of cells or percentage (with statistics) is provided. The used antibodies were validated by commercial parties (PamGene) the suppliers (PerkinElmer, Sigma Aldrich, Thermo Fisher, Abcam, Cell Signaling Technology) and/or previous literature reports for the used species and applications.
None of the cell lines were authenticated.
All of the cell lines were negative for mycoplasma infection during all of our routine checks.
None of the cell lines are present in the ICLAC register as commonly misidentified.
Phagocytosis assay: Post-infection with GFP-expression E. coli, HL-60 neutrophils were resuspended and transferred to Eppendorf tubes, washed in FACS buffer (1 mL, 500 g, 3 min) and fixed in 1% PFA in PBS (15 min, 4°C, in the dark), followed by two washing steps in PBS and resuspension in FACS buffer to a density of approximately 500 cells/#L.
The cell population consisted only of non-differentiated and differentiated HL-60 cells and the percentage of differentiated cells is indicated in the corresponding figures.