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

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.

Stability and Cell Permeability of Sulfonyl Fluorides in the Design of Lys‐Covalent Antagonists of Protein‐Protein Interactions

“…Indeed, recent reports are proposing a Lysinome or a Tyrosinome as an ensemble of targets that present targetable Lys or Tyr residues in proximity of their binding sites in enzymes [12] . Our recent application included a side by side investigation of the merits and pitfalls of aryl‐sulfonyl fluorides and aryl‐fluorosulfates as possible electrophiles to target Lys, Tyr, and His in protein–protein interactions [1b] . We found that when properly juxtaposed to a binding site Lys residue, aryl‐fluorosulfates approach the proper balance of stability, reactivity and cell permeability, similar to what is observed with Cys and acrylamides.…”

“…Typical of non‐covalent targeting agents, compound 1 displayed a ΔTm ∼5 °C (regardless of the pre‐incubation time of either 30 min or 2 h, Table 1), while putative covalent compounds showed significantly larger shifts (ΔTm of >20 °C; Table 1). [1b] However, some agents displayed a larger denaturation thermal shift only at the longer incubation time (namely agents 9 and 11 ), suggesting a slower reaction with the target. Compound 4 , compatible with its lack of stability in buffer (Table 1), did not induce large ΔTm values nor displayed low nanomolar affinity in the DELFIA assay (Table 1).…”

“…Thermal shift (ΔTm) data were obtained pre‐incubating protein and ligand for either 30 min (top value) or 2 h (bottom value) at room temperature. We had previously reported compound 12 [1b] . Values and standard errors represent the average of 4 independent measurements.…”

The design and development of covalent protein-protein interaction inhibitors for cancer treatment

“…The tetrapeptide mediates its interactions with various members of the IAP family, including the Xlinked inhibitor of apoptosis protein (XIAP). In addition, Lys-covalent inhibitors have been designed with a benzamide-sulfonyl fluoride warhead to target the BIR3 domain of XIAP [60,61].…”

“…In another study, the structure-based biophysical and biochemical approaches were used to develop different kinds of warheads which can easily form covalent adducts with alternative amino acids such as histidine, threonine, serine, and tyrosine [62]. It was found that aryl-fluoro sulfate electrophiles can form covalent bonds with Lys, Tyr, and His residues in PPIs [61]. With the tremendous progress and resurgence for covalent drugs, these successful cases present valuable and novel ideas for covalent PPI inhibitors and suggest the possibility for extending the target cysteinome range to other residues.…”