Covalent Inhibitors of Protein–Protein Interactions Targeting Lysine, Tyrosine, or Histidine Residues

TLDR

Our studies reveal new avenues for designing potent covalent protein–protein interaction antagonists. Using XIAP as a model, we evaluated warheads incorporated into binding peptides for covalent adduct formation with Lys, Tyr, His, Ser, and Thr, and assessed aqueous/plasma stability, cell permeability, and cellular efficacy. We reported Lys‑covalent agents targeting XIAP BIR3 with a benzamide‑sulfonyl fluoride warhead and identified aryl‑fluoro sulfates as the most suitable electrophiles for Lys, Tyr, and His, due to their cell permeability and stability in aqueous buffer and plasma.

Abstract

We have recently reported a series of Lys-covalent agents targeting the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) using a benzamide-sulfonyl fluoride warhead. Using XIAP as a model system, we further investigated a variety of additional warheads that can be easily incorporated into binding peptides and analyzed their ability to form covalent adducts with lysine and other amino acids, including tyrosine, histidine, serine, and threonine, using biochemical and biophysical assays. Moreover, we tested aqueous, plasma stability, cell permeability, and cellular efficacy of the most effective agents. These studies identified aryl-fluoro sulfates as likely the most suitable electrophiles to effectively form covalent adducts with Lys, Tyr, and His residues, given that these agents were cell permeable and stable in aqueous buffer and in plasma. Our studies contain a number of general findings that open new possible avenues for the design of potent covalent protein–protein interaction antagonists.