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.