Tom van der Wel;Riet Hilhorst;Hans den Dulk;Tim van den Hooven;Nienke M. Prins;Joost A. P. M. Wijnakker;Bogdan I. Florea;Eelke B. Lenselink;Gerard J. P. van Westen;Rob Ruijtenbeek;Herman S. Overkleeft;Allard Kaptein;Tjeerd Barf;Mario van der Stelt
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.