New Reversible Covalent Warheads: Cyanobenzothiazoles Recruiting DCAF16 for Targeted Protein Degradation

Protein N-terminal cysteines (NCys) are valuable for selective modification and posttranslational regulation, but their proteome-wide landscape remains unexplored. We constructed a library of electrophilic probes, including cyanobenzothiazole (CBT), to systematically map ligandable native NCys residues. Modifiable NCys sites were identified in proteins such as GFPT1 and CSTB, revealing accessible NCys in the human proteome. Integrating CBT into a BRD4-targeting ligand generated a reversible covalent degrader (DC₅₀ = 16.7 nM, D_max = 98%). Mechanistic studies showed that CBT modifies DCAF16 at Cys58, promoting ternary complex formation and enabling efficient ubiquitination and degradation. The platform achieved potent and selective degradation of challenging targets like EGFR^{L858R/T790M/C797S} and HER2 without hook effects, outperforming clinical inhibitors. This work provides the first proteome-wide map of ligandable NCys residues and establishes CBT as a versatile platform for covalent targeted protein degradation (TPD), opening new avenues for precision therapeutics.