Developing covalent affinity-based probes and inhibitors for sialyltransferases using a modular and direct-to-biology compatible chlorotriazine (ClTriZ) warhead platform

Covalent chemical probes are powerful tools for protein profiling and inhibitor development, but their synthesis often poses significant challenges. Here, we introduce chlorotriazine (ClTriZ) as a novel covalent warhead that enables efficient and modular synthesis of covalent probes via a fully water compatible, “double-click” direct-to-biology (D2B) platform. The ClTriZ warhead displays a labelling rate of kL = (9.0 ± 0.3) x 10-5 s−1, comparable to the established ligand-directed chemistry warheads, and when integrated into the platform supports rapid generation and screening of covalent probes and inhibitors directly with recombinant proteins or in cell lysates without purification. Applying this platform, we identified proximity-induced, context-dependent probes that selectively label multiple targets, including avidin, cereblon (CRBN) and sialyltransferases. We focused further on sialyltransferases, using the platform to rapidly iterate and identify highly ligand-specific probes that selectively label the endogenous sialyltransferase Lst in live Neisseria gonorrhoeae. Notably, these ClTriZ probes also enabled the first reported irreversible inhibition of a sialyltransferase, PmST1, with kinact / KI = 56.2 ± 6.1 M-1 s-1, overcoming the lack of covalent intermediates in SN2/SNi catalytic mechanisms, characteristic of this enzyme class. Overall, our platform provides a versatile and high-throughput route to develop covalent probes and inhibitors, with broad potential across diverse glycosyltransferases and beyond to other protein families.