Proteome‐Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents

Abstract

In this study,wepresent ahighly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells.O ur method is based on alkynylation of cysteines in complex proteomes using a" clickable" alkynyl benziodoxolone bearing an azide group.T his reaction proceeds fast, under mild physiological conditions,and with avery high degree of chemoselectivity.T he formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalizedw ith TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, ad iarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase Igamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery. Despitetheirrelativelylowabundanceinproteins,cysteinesare vital for cellular biochemistry.F or example,c ysteines form disulfide bridges,a re actively involved in enzyme catalysis as nucleophiles and can be posttranslationally oxidized or modified through palmitoylation, prenylation or nitrosylation.[1] Many of the so-called functional cysteines display pronounced hyperreactivity and these reactive hot spots can be precisely identified in complex proteomes with broad-range electrophilic probes.[2] Such probes represent particularly useful tools for competitive activity-based protein profiling [3] (ABPP) of targets of covalently binding cysteinereactive drugs and metabolites,asimpressively demonstrated in recent publications.[4] Over the past few years,abroad variety of novel methods for broad-spectrum, yet chemoselective targeting of cysteiner esidues have been reported. [5] However,w hile most of these transformations deliver excellent results on peptides and recombinant proteins,o nly very few of them have been shown to be also effective in labeling cysteines in cells or cellular lysates and therefore suitable for proteomics applications.I odoacetamide (IAA) is widely accepted as gold standard among MS-compatible cysteinereactive chemical probes, [6] but it displays rather low chemical stability in aqueous buffers and it only reacts with afraction of functional cysteines. [7] Moreover,i odoacetamide also modifies lysines and the formed artifacts lead to misassignment of lysine ubiquitination sites by mass spectrometry.[8] Thus,there is aclear need for new electrophilic probes complementary to IAA that would enable labeling of so far "inaccessible" subsets of proteomic cysteines,which would then allow more comprehensive target profiling.Herein, we present discovery and an in-depth evaluation of a" clickable" alkynyl benziodoxolone as ahighly efficient and chemoselective cysteinereactive probe with an iodoacetamide-complementaryp r...

Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension

“…Contrary to AspA controls, 12 , 13 the removal of thiols on cell surfaces with maleimide 26 , 22 iodoacetamide 27 , 12 and the most powerful hypervalent iodine reagent 28 ( 29 ) failed to inactivate ETP 6 ( Figure 4 C). Similarly, the presence of 10% serum 22 caused only a minor ∼25% reduction of ETP uptake ( Figures 4 C and S5 ).…”

“…This poor responsiveness to inhibitors and activators such as cytochalasin B, DTT, Ellman’s reagent, TFRC siRNA, or serum indicated that the unique reactivity of ETPs is decisive for function. High reactivity of ETPs in both oxidized and reduced form allows for covalent capture by nonactivated thiols and disulfides in cellular targets 29 that are otherwise beyond reach ( Table 1 , entries a–d, f, h–i). Moreover, the possibility of repeated disulfide-exchange cycles in neutral water suggested that ETPs can change targets during uptake ( Table 1 , entries a–d, e–g).…”

Cyclic Hypervalent Iodine Reagents for Atom‐Transfer Reactions: Beyond Trifluoromethylation

“…In 2015, Adibekian, Waser,a nd co-workers reported an efficient method for the proteomic profiling of cysteine residues in complex proteomes in both cell lysates and living cells by using azide-functionalized alkynyl benziodoxolone 100 (Scheme 20). [51] Theobtained thioalkyne adducts could be easily functionalized by copper-catalyzed cycloaddition of the azide with alkynes (CuAAC), either with fluorophores or biotin. Benziodoxolone 100 (JW-RF-010) was both more efficient and more selective than state-of-the-art reagents for cysteine functionalization, such as N-iodoacetamides,a nd allowed the identification of ad ifferent set of proteins.T he method was used for the identification of the targets of curcumin in HeLa cells.…”

Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization

“…Michael additions to α,β-unsaturated carbonyl compounds in maleimides,6 sulfone reagents7 and recently developed carbonylacrylic reagents8 are the most prominent examples. Other methods rely on SN 2 -reactions of haloalkyl bonds such as iodoacetamides,9 on alkynylation10 and fluoroalkylation11 with hypervalent iodine reagents, on nucleophilic aromatic substitution of perfluoroaromatic compounds12 or on Pd-mediated cross-coupling 13. The level of reactivity for cysteine in these methods is predetermined by the chemical nature of the reactive group itself.…”