Dioxazolones as Masked Isocyanate Electrophiles for Global Profiling of Lysine Reactivity and Ligandability

Due to the widespread distribution and critical roles of lysine residues, the development of new methods to characterize their reactivity and ligandability could significantly expand the landscape of druggable targets. Herein, we report a new class of five-membered heterocyclic electrophiles, dioxazolones, which function as masked isocyanate electrophiles for in-depth profiling of functional lysine residues. These probes demonstrated remarkable chemoselectivity and exceptional reaction kinetics across the proteome, enabling the identification of over 3000 covalently modifiable lysine residues. And up to 30% of them were undetected by previously reported probes. Leveraging fragment-based ligand discovery, we identified dioxazolone-based fragments that covalently engage previously uncharacterized or functional lysines, such as K33 of SLC25A5, K69 of CSNK2A2, and K97 of MAP2K1, modulating protein function and substantially expanding the content of ligandable lysines. Furthermore, we demonstrated the utility of the dioxazolone moiety as a latent electrophilic warhead in the design of Bruton’s tyrosine kinase (BTK) inhibitors, enabling covalent modification of the conserved catalytic lysine residue (K430) and offering a potential strategy to overcome drug resistance. These findings establish dioxazolones as versatile tools for lysine-targeted covalent chemistry.