Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy

Abstract

We have recently investigated the reactivity of aryl-fluorosulfates as warheads to form covalent adducts with Lys, Tyr, and His residues. However, the rate of reaction of aryl-fluorosulfates seemed relatively slow, putting into question their effectiveness to form covalent adducts in cell. Unlike the previously reported agents that targeted a relatively remote Lys residue with respect to the target’s binding site, the current agents were designed to more directly juxtapose an aryl-fluorosulfate with a Lys residue that is located within the binding pocket of the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP). We found that such new agents can effectively and rapidly form a covalent adduct with XIAP-BIR3 in vitro and in cell, approaching the rate of reaction, cellular permeability, and stability that are similar to what attained by acrylamides when targeting Cys residues. Our studies further validate aryl-fluorosulfates as valuable Lys-targeting electrophiles, for the design of inhibitors of both enzymes and protein–protein interactions.

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

“…To qualitatively evaluate the stability and subsequent reactivity of a variety of aryl‐sulfonyl fluorides, a library of agents that are synthetically accessible was prepared based on previous studies from our laboratory [1–2] . We demonstrated that incorporation of benzamide‐ or benzylamine‐sulfonyl fluorides in position X in the general tetra peptide of sequence A X PF‐CONH 2 (Table 1) resulted in agents that can covalently interact with Lys311 in the BIR3 domain of XIAP (Figure 1A) or Tyr311 in a Lys311Tyr BIR3 mutant (Figure 1B).…”

“…[2] Here, expanding on our previous work, we further identified properly substituted aryl-fluoro sulfates (in particular those in agents 7-11) that could be potentially used as pharmacological tools in targeting Lys or Tyr in antagonists of PPIs as well as in small molecules enzyme inhibitors. [19] Hence, together with our previous similar observations with aryl-fluoro sulfates, [2] the studies significantly widen the covalent druggable proteome from the Cysteinome [7o-q] to other more frequently occurring residues such as Lys, or Tyr. [1b,9a,20] In targeting PPIs, in particular, we envision that aryl-fluorosulfates, [2] and/or the substituted aryl-sulfonyl fluorides as reported here (agents 7-11), could be relatively easily incorporated into current drug discovery strategies, including fragment-, structure-, and/or NMR-based approaches, [15,17t,21] phage display, or DNA encoded libraries, [12b,22] aimed at deriving, potent, selective, and cell permeable agents that can form the basis for further lead optimizations and drug development studies.…”

“…Several manuscripts are emerging that report on targeting of Lys residues in active sites of proteins by introduction of appropriately placed electrophiles on existing ligands [9b,c, 10] . Our own recent studies [1–2] and others [11] focused on targeting Lys, Tyr, or His surface residues located at protein‐protein interfaces, suggesting that in principle the target space of covalent antagonists of PPIs could be expanded to include these residues. 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] .…”

“…Recently we compared the reactivity of aryl‐sulfonyl fluorides [1] and aryl‐fluorosulfates [2] electrophilic “warheads” when inserted in binding peptides targeting proteins of the Inhibitors of Apoptosis Proteins family (IAPs) [3] . These recent studies revealed that while benzamide‐sulfonyl fluorides can react readily with lysine, or tyrosine, they are unstable in aqueous buffer at physiological pH values.…”

“…On the contrary, we found that properly placed aryl‐fluorosulfates could also react readily with lysine targets and were very stable in buffer and in plasma. Indeed, unlike for aryl‐fluorosulfates containing agents that target XIAP, [2] we could not clearly detect the formation of a stable adduct in cell with covalent agents containing a benzamide‐sulfonyl fluoride electrophiles, presumably due to their instability [1] . Here, we explored if further substitutions of aryl‐sulfonyl fluorides placed at the X position of agents NMe‐Ala‐X‐Pro‐Phe‐CONH 2 could result in compounds with increased aqueous stability and retained reactivity for their intended target (residue Lys311 of the BIR3 domain of XIAP).…”