A twist in the tale: shifting from covalent targeting of a tyrosine in JAK3 to a lysine in MK2.

While cysteine targeting in kinases is well established and widely used, covalent interactions with other amino acids remain much less explored. We aimed to develop covalent inhibitors targeting tyrosine residues in the protein kinases JAK3 and MK2 using structure-based design principles to generate small sets of ligands containing tyrosine-reactive sulfonyl fluoride and the less-explored fluorosulfate warheads. While the JAK3 inhibitors failed to achieve covalent binding, the fluorosulfate-bearing MK2 inhibitor 42, which had been designed as an allosteric binder, unexpectedly formed a bond with the "catalytic" lysine, additionally uncovering a unique interaction at the hinge region. This highlights the untapped potential of fluorosulfates and provides a rare example of the use of this electrophile for lysine targeting in kinases. Our results highlight the limitations of traditional design methods and support the integration of fragment/lead-like covalent library screening to discover unanticipated interactions.