Chemo-Selective Cys–Pen Disulfide for Proximity-Induced Cysteine Cross-Linking

Disulfide-rich architectures are valuable pharmacological tools or therapeutics. Besides, a ligand-induced conjugate strategy offers potential advantages in potency, selectivity, and duration of action for novel covalent drugs. Combining the plentiful disulfide-rich architecture library and ligand-induced conjugate via thiol–disulfide interchange would supply great benefits for developing site specific covalent inhibitors. Cysteine–cysteine (Cys–Cys) disulfide bonds are intrinsically unstable in endogenous reductive environment, while cysteine–penicillamine (Cys–Pen) disulfide bonds show satisfactory stability. We envisioned the Cys–Pen disulfide as a potential ligand-induced covalent bonding warhead, and this disulfide could reconstruct with the protein cysteine in the vicinity of the peptide binding site to form a new disulfide. To evaluate our design, protein PLCγ1-c src homology 2 domain and RGS3-PDZ domain were tested as models. Both proteins were successfully modified by Cys–Pen disulfide and formed new disulfides between proteins and peptides. The new disulfide was then analyzed to confirm it was a newly formed disulfide bond between Pen of the ligand and a protein Cys near the ligand binding site. HDAC4 was then chosen as a model by utilizing its "CXXC" domain near its catalytic pocket. The designed Cys–Pen cyclic peptide inhibitor of HDAC4 showed satisfactory selectivity and inhibitory effect.