Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chain

Abstract

Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.

Global profiling of lysine reactivity and ligandability in the human proteome

“…Considering its nucleophilic side chain and prevalence in proteins, lysine is an attractive candidate amino acid for covalent ligand development. pKa-perturbed lysine residues play important functional roles in proteins 54,55 , and electrophilic compounds have been found to target lysines in diverse types of proteins (e.g., metabolic enzymes, such as PGAM1 56 , hormone-binding proteins, such as TTR 57 , lipid kinases, such as PI3Ks 18 , and adaptor proteins, such as MCL-1 13 ). Nonetheless, our understanding of lysine reactivity and ligandability across the human proteome remains limited.…”

“…Among proteinaceous amino acids, lysine represents a potentially attractive candidate for covalent ligand development, as the lysine ε-amine is intrinsically nucleophilic, and lysines are found at many functional sites, including enzyme active sites 11,12 and at interfaces mediating protein-protein interactions 13 . Lysines also frequently serve as sites for post-translational regulation of protein structure and function through, for instance, acetylation 14 , methylation 15,16 , and ubiquitylation 17 .…”

“…Lysines also frequently serve as sites for post-translational regulation of protein structure and function through, for instance, acetylation 14 , methylation 15,16 , and ubiquitylation 17 . Individual lysine residues within functional protein pockets are also susceptible to modification by electrophilic small molecules, including natural products, such as Wortmannin 18 , which targets a lysine in the active sites of PI3K kinases, activated esters that react with a lysine in transthyretin (TTR) 19 , and boronic acid carbonyl antagonists of the apoptosis regulatory protein MCL-1 13 . Additional electrophiles that have been shown to react with proteinaceous lysine residues include dichlorotriazines 20,21 , imidoesters 22 , 2-acetyl- or 2-formyl-benzeneboronic acids 13,23 , isothiocyanates 24,25 , pyrazolecarboxamidines 26,27 , sulfonyl fluorides 28,29 , and vinyl sulfonamides 30 .…”

Progress in targeting the BCL-2 family of proteins

“…The compound induced conformational changes and the allosteric inhibition of BH3-domain interaction with MCL-1. Another study by Akçay et al modified a small molecule inhibitor of MCL-1 to include a strategically placed boronic acid group, which reversibly reacts with Lys234 [51]. Covalent binding to this lysine, unique to MCL-1, allowed specific increase in binding to MCL-1 over other BCL-2 family members.…”

Stilbene Boronic Acids Form a Covalent Bond with Human Transthyretin and Inhibit Its Aggregation

“…42–45 Boronic acid-based fluorogenic probes have been developed for sensing both saccharides 46 and reactive oxygen species, 47,48 as well as for molecular recognition 49 and protein conjugation. 50,51 These applications arise from the ability of boronic acids to form a covalent bond with a Lewis base that is reversible under physiological conditions. 52,53 Notably, boronic acids are benign, as their metabolic byproduct, boric acid, is present in a normal diet.…”