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ScienceDirect Covalent protein modification: the current landscape of residue-specific electrophiles D Alexander Shannon and Eranthie Weerapana Functional amino acids that play critical roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chemical-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chemical probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid selectivity of these groups. Here, we survey the current landscape of electrophiles that covalently target various nucleophilic amino acids in proteins and highlight proteomic applications that have benefited from the unique properties of these electrophiles. Addresses Department of Chemistry, Boston College, Chestnut Hill, MA 02467, United States Corresponding author: Weerapana, Eranthie ([email protected])

Current Opinion in Chemical Biology 2015, 24:18–26 This review comes from a themed issue on Omics Edited by Benjamin F Cravatt and Thomas Kodadek

http://dx.doi.org/10.1016/j.cbpa.2014.10.021 1367-5931/# 2014 Elsevier Ltd. All right reserved.

Introduction Covalent modification of proteins is critical for various applications such as the development of irreversible inhibitors and activity-based protein profiling (ABPP). ABPP typically relies on the selective targeting of a single enzyme, or a functionally related family of proteins, with a chemical probe comprised of a protein-reactive electrophile and a reporter group [1]. Two of the earliest examples of ABPP were the use of fluorophosphonate (FP)-based probes for targeting serine hydrolases (SHs), and vinyl sulfone and epoxide-based probes for lysosomal cysteine proteases [2,3]. These early ABPP studies exploited existing knowledge of affinity labels specific for these enzyme classes. More recently, non-directed approaches that utilize electrophiles such as sulfonate esters and chloroacetamides have enabled the profiling of enzymes for which cognate affinity labels do not exist, Current Opinion in Chemical Biology 2015, 24:18–26

thereby expanding the enzyme classes amenable to ABPP [4,5]. These non-directed probes were shown to bind to their respective targets in an activity-based manner, thereby selectively modifying functionally relevant amino acids despite the overabundance of non-functional residues in the proteome [6]. This concept was intimately explored by measuring the reactivity of both functional and non-functional cysteine residues using a highly reactive iodoacetamide (IA) probe. Using a quantitative massspectrometry platform, termed isoTOP-ABPP, over a thousand cysteine residues in the proteome were ranked in order of nucleophilicity [7]. This analysis revealed that the subset of highly reactive cysteines was enriched in functional residues that were critical to catalysis and regulation. Therefore, using diverse electrophiles to covalently modify highly reactive amino acids in the proteome provides a means to identify novel functional loci across disparate protein families and extend the tools of ABPP to a larger subsection of the proteome. Amino acids such as serine, cysteine, lysine, tyrosine, threonine, aspartate, and glutamate have the potential to be nucleophilic depending on the protein microenvironment. Covalent modification of these residues relies on the judicious selection of an electrophile with the appropriate affinity toward the activated amino-acid side chain. This affinity is mediated primarily by the relative hardness/ softness of the nucleophile–electrophile pair. Diverse electrophiles have disparate amino-acid reactivity profiles, especially within the context of a proteome [6]. Here, we survey the current landscape of protein-reactive electrophiles, with a particular focus on their amino-acid selectivity. Recent reviews have highlighted chemical transformations that are amenable to protein labeling [8,9]; here, we update these available reactions and emphasize their utility for chemical proteomics.

Covalent modification of serine The SH enzyme family contains an activated serine within a catalytic dyad or triad and comprises approximately 1% of the human proteome [10]. The sensitivity of SHs to inhibition by fluorophosphates and FPs was well characterized, and the FP electrophile was adapted to generate ABPP tools to study the SH family [2]. FP probes have enabled the functional annotation of novel SHs, the discovery of selective inhibitors, and the characterization of dysregulated SH activities in diseases such as cancer [11]. The low reactivity of serine residues located www.sciencedirect.com

Protein-reactive electrophiles for chemical proteomics Shannon and Weerapana 19

Figure 1

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Serine-directed electrophiles for covalent protein modification.

outside of a prototypical catalytic triad/dyad, coupled with the high affinity of FP toward hydroxyl nucleophiles over other reactive groups such as thiols and amines, renders the serine-FP reaction (Figure 1a) highly effective for proteomic applications. Diphenyl phosphonates (Figure 1b) have been used as covalent inhibitors and ABPP probes for the serine-protease subfamily of the SHs [12]. Most recently, a library of peptide-based diphenyl phosphonates contained members selective for many serine proteases, including chymotrypsin, cathepsin G and urokinase-type plasminogen activator (uPA) [13]. Additionally, diphenyl phophoramidate probes (Figure 1c) retain serine protease reactivity and selectivity while being amenable to strictly solid-phase synthesis techniques [14]. These electrophiles have significantly lower reactivity relative to FP, but incorporation of peptide-based binding groups direct these probes to protease active sites for covalent adduction. b-Lactams and b-lactones (Figure 1d) constitute another important class of serine-reactive electrophiles due to the well-characterized covalent modification of the serine nucleophile in penicillin binding proteins (PBPs) by b-lactams, and the abundance of the b-lactone motif in a variety of electrophilic antibiotic natural products. b-Lactam and b-lactone probe libraries were synthesized and evaluated within bacterial proteomes [15,16], resulting in labeling of various SHs, including PBPs and the ATP-dependent caseinolytic protease Clp. However, labeling was also observed for proteins with active-site www.sciencedirect.com

cysteines, such as b-ketoacyl-(acyl carrier protein) synthase III (KASIII), suggesting that these electrophiles are not selective for serine nucleophiles. More recently, it was shown that a related electrophile, the b-sultam, did not target serine-containing PBPs as expected, but instead reacted with an activated threonine residue in azoreductases [17]. The pan-serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), is widely utilized in protease cocktails, yet the proteome-wide reactivity of the sulfonyl-fluoride electrophile (Figure 1e) was poorly characterized. Sulfonyl-fluoride derivatives were shown to covalently label multiple serine protease sub-classes, demonstrating utility as a serine-reactive electrophile [18]. However, proteome-wide evaluation of protein labeling showed additional targeting of activated tyrosine residues, particularly within glutathione S-transferases (GSTs) [19]. Coumarin and isocoumarin-based compounds (Figure 1f) inhibit serine proteases, driven by nucleophilic attack on the lactone carbonyl group by the active-site serine. 4-Chloro-isocoumarin probes were identified to be highly selective for cathepsin G, elastase, and, to a lesser extent, uPA [20,21]. Furthermore, 4-chloro-isocoumarins capable of enhancing Toxoplasma gondii invasion by inhibiting the SH, palmitoyl protein thioesterase-1 (PPT1) were identified [22]. Despite the observed reactivity with activated serine residues, these electrophiles have been shown to react with other nucleophiles such as the thiol group in Current Opinion in Chemical Biology 2015, 24:18–26

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cysteine proteases and the active-site threonine in proteasome subunits [20]. Other chemotypes that covalently modify serine include carbamates and heterocyclic ureas. Carbamate-based inhibitors (Figure 1g) exist for numerous SHs [23], and recently, the balance between affinity and reactivity of this electrophile was explored. A series of carbamates with varying leaving groups, such as O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) were shown to be highly selective for SHs in vivo, although the NHS leaving group showed non-specific reactivity in vitro. An HFIP-containing carbamate showed exceptional selectivity for two SHs, monoacylglycerol lipase (MAGL) and a-b hydrolase-6 (ABHD6), and was further developed into a fluorescent activity-based imaging probe (JW912) [24]. The labeling of SHs by Nheterocyclic urea compounds, spurred the development of 1,2,3-triazole-urea ABPP probes (Figure 1h), which covalently carbamoylate active-site serine residues [25]. A triazole-urea probe selective for lysophospholipases LYPLA1 and LYPLA2 was used to monitor in vivo target engagement by a panel of reversible inhibitors [26]. This application is facilitated by the attenuated reactivity of this electrophile, relative to FP, and further underscores the necessity of having access to a panel of electrophiles with tunable reactivity toward a target nucleophile.

Covalent modification of cysteine The cysteine thiol is highly reactive due to the polarizability and electron-rich nature of sulfur, enabling a variety of functions, including nucleophilic and redox catalysis, metal binding and allosteric regulation [27]. These functional cysteines are found on diverse proteins such as proteases, oxidoreductases and kinases; therefore, cysteine-targeted electrophiles can be utilized to expand the protein families amenable to ABPP. Electrophilic ketones such as acyloxymethyl ketone (AOMK) (Figure 2a) and chloromethyl ketone (CMK) (Figure 2b), constitute some of the earliest cysteine-reactive electrophiles used to profile cysteine proteases. These probes were instrumental in revealing the role of individual caspases in the activation and progression of apoptosis [28,29]. The presence of the electrophilic carbonyl group suggests potential cross reactivity with other nucleophiles such as amines; however, selectivity for cysteines can be achieved by embedding the electrophile within a high-affinity peptide ligand for proteases. Notably, these electrophiles have been adapted to generate imaging agents for cysteine proteases in vivo [30], highlighting the selectivity that can be achieved even within a highly complex biological system. Epoxides (Figure 2c), with structures similar to the broadspectrum cysteine protease inhibitor E-64, can target

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cysteine proteases [31]. Epoxides are very mild electrophiles, and in the absence of the peptide backbone to direct these probes to proteases, no covalent labeling is observed [6]. Importantly, epoxides have been shown to target amino acids outside of cysteine. For example, the spiroepoxide-containing natural product fumagillin, covalently modifies a histidine residue in methionine aminopeptidase (MetAP) [32]. A library of spiroepoxide probes applied to a chemical-genetic screen for inhibitors of breast cancer-cell proliferation identified an inhibitor of phosphoglycerate mutase 1 (PGAM1) [33]. Labeling of PGAM1 occurs on a lysine residue [34], further illustrating the promiscuous amino-acid selectivity of epoxide derivatives. Sulfonate-ester (SE) electrophiles (Figure 2d) were the first to be used for non-directed ABPP studies, whereby a library of SEs with variable leaving groups showed diverse protein labeling [4,35]. More recently, a peptide-based phenyl-SE library identified a covalent modifier of the active-site cysteine of GST omega 1 (GSTO1) [36] showing the potential of this electrophile to target cysteine. However, a global analysis of SE targets showed modification of cysteine, tyrosine, aspartate and glutamate side chains [6]. Therefore, SEs are equally promiscuous as the epoxides, but are more reactive and show significant protein labeling in the absence of a directing ligand [6]. Chloracetamides (CA) and iodoacetamides (IA) (Figure 2e) are widely utilized in numerous ABPP applications. CA has been incorporated into libraries based on peptide [5,37,38], triazine [39], and piperidine [40] scaffolds, leading to the identification of covalent modifiers for nitrilases [37,38], protein disulfide isomerase (PDI) [39], and GSTO1 [40]. The more reactive IA covalently modifies 1000 cysteines in the proteome [7], enabling the global profiling of changes in cysteine reactivity. This highly reactive electrophile has been instrumental in identifying bacterial redox modulators [41], cellular targets of electrophilic lipids [42], and zinc-binding cysteines within complex proteomes [43]. These studies highlight the utility of a highly-reactive, yet residue-selective electrophile to gain insight into modulators of amino-acid nucleophilicity. IA has been shown to react with other nucleophiles, such as the amine group of lysine [44], albeit at high millimolar concentrations [7]. Due to the reduced reactivity of CA, reactivity with lysine has not been observed [6,44]. Maleimides (Figure 2f) constitute another class of cysteine-reactive electrophiles with comparable reactivity to IA. Only 35% overlap was observed across the protein targets for maleimide and IA [45], underscoring the fact that electrophiles that share similar reactivity and amino-acid selectivity can still target diverse subsets of the proteome. Recently, a panel of activated aryl halides was evaluated, inspired by the PPARg inhibitor GW9662, which contains a cysteine-reactive p-chloronitrobenzene (CNB) www.sciencedirect.com

electrophile (Figure 2g). CNB reacts via a nucleophilic aromatic substitution (SNAr) mechanism, and consequently, relocation of the electron-withdrawing nitro substituent tunes the reactivity in a predictable manner [46]. The CNB-based probe, RB-2-cb, covalently modified a reactive cysteine residue on b-tubulin [39], demonstrating the utility of this highly tunable electrophile in ABPP applications. The bioorthogonality of terminal alkynes has recently been questioned due to the observed covalent modification of cysteines by propargyl amides [47,48,49]. Ubiquitin propargylated at the C-terminus (Ub-Prg) inhibits a number of deubiquitinating enzymes (DUBs) through covalent modification of the active-site cysteine nucleophile [47] (Figure 2h). Similarly, incorporating propargylamine at the C-terminus of SUMO results in covalent adduction to active-site cysteines of cognate proteases [48]. This reactivity appears selective for the DUBs and other ubiquitin-modifying enzymes; replacing the electrophile of a caspase-1 inhibitor with a terminal alkyne showed no covalent labeling. Reactivity was reinstated upon increasing the peptide fragments to 16 or more residues, suggesting that large surface-area interactions with high affinity are necessary [47]. Other common cysteine-reactive electrophiles include Michael acceptors, for which preferential reactivity has been observed for cysteine over other nucleophiles such as lysine [50,51]. Recently, a panel of acrylamides (Figure 2i), vinylsulfonamides, aminomethyl methyl acrylates, and methyl vinylsulfones (Figure 2j) were evaluated for thiol reactivity for fragment-screening applications [52]. Of these, the acrylamide reactivity was significantly modulated by the attached substituent, resulting in >2000-fold variation in reactivity. In contrast, the other three electrophiles showed much smaller variations, thereby highlighting the need to take substituent effects on electrophile reactivity into consideration. Lastly, it is important to note that Michael adducts with cysteine are considered reversible, and a recent study with substituted acrylonitrile-based Michael acceptors demonstrated that this reversibility can be tuned in a predictable manner [53]. Such reversible covalent interactions can obviate concerns related to off-target protein modification [54].

Covalent modification of lysine Selective modification of functional lysine residues is a significant challenge due to the abundance of non-functional lysines and N-terminal protein amines. Nonspecific primary amine labeling can be achieved using reductive alkylation with aldehydes; this can be performed under mild conditions using easily-separable iridium catalysts [55]. Alternatively, labeling with 2-acetylphenylboronic acid forms a stable iminoboronate (Figure 3a) that can be reversed at low pH [56]. Similar Current Opinion in Chemical Biology 2015, 24:18–26

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non-specific lysine labeling reagents include succinimidyl esters, sulfonyl chlorides, isocyanates, and thioisocyanates. A successful lysine-directed ABPP application is the use of an acyl-phosphate probe to target a conserved lysine in the ATP-binding pocket of protein kinases [52]. ATP or ADP analogs embedding an acyl-phosphate electrophile (Figure 3b) covalently modify 75% of the human protein kinases. The acyl-phosphate is highly reactive, resulting in complete proteome labeling within 5 min. Although a valuable tool for kinase profiling, this electrophile has limitations for general ABPP applications, including sensitivity to hydrolysis and poor cell permeability and in vivo stability. Recent exploration of aryl halides as protein-reactive electrophiles discovered the lysine-reactive dichlorotriazine (Figure 3c) that functions through an SNAr mechanism. Labeling of lysine residues annotated as sites of acetylation, as well as active-site residues and ATP-binding sites was observed [46]. Although the dichlorotriazine is more hydrolytically stable relative to the acyl-phosphates, it still displays high proteome reactivity, therefore a milder form of this electrophile would be more Current Opinion in Chemical Biology 2015, 24:18–26

suitable for ABPP applications. Most recently, the N-phenylsulfonamide-containing Toll-like receptor 4 (TLR4) inhibitor TAK-242, was demonstrated to covalently modify Lys64 on human serum albumin (HSA) [57]. Although the general reactivity of the N-phenylsulfonamides (Figure 3d) is poorly explored, it may have potential utility as a lysine-reactive electrophile.

Covalent modification of tyrosine Electrophiles that label tyrosine residues include the SE electrophile (Figure 4a), which targets an active-site proton donor in corticosteroid-11-b-dehydrogenase 1 [6]. However, as mentioned previously, SE is highly promiscuous and targets cysteine, aspartate and glutamate residues in addition to tyrosine. Similarly, sulfonylfluorides (Figure 4b) react with highly conserved tyrosines in multiple GSTs, but also target the activated serine in serine proteases [19]. Recently, 3-fluorosialyl fluoride (DFSA; Figure 4c) was shown to modify a tyrosine within sialidase active sites [58]. However, this electrophile is specific to the sialic-acid scaffold, and is therefore not amenable as a general tool for profiling tyrosine reactivity. Promising candidates for global tyrosine labeling are cyclic diazodicarboxamides such www.sciencedirect.com

Protein-reactive electrophiles for chemical proteomics Shannon and Weerapana 23

Figure 4

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as 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD; Figure 4d) [59,60], which functions via an ene-type reaction that is amenable to aqueous conditions.

Covalent modification of threonine Numerous electrophilic small molecules inhibit the proteasome by targeting the N-terminal threonine active-site nucleophile. Peptide-based boronic-acid inhibitors (Figure 4e) date back to the late 1990s [61], spurring the development of bortezomib, a clinically approved drug for multiple myeloma. More recently, boronic-acid inhibitors were developed for autotaxin (ATX), a secreted nucleotide pyrophosphatase/phosphodiesterase containing a threonine nucleophile [62]. Interestingly, the preference of boron for hard oxygen over soft sulfur nucleophiles provides selectivity for ATX over other cysteine-containing phosphatases. This underscores the importance of selecting for electrophiles that minimize cross reactivity with functionally related proteins. Additional covalent modifiers of the proteasome include vinyl sulfones, epoxyketones (Figure 4f), and alpha-ketoaldehydes (Figure 4g) [63,64]. For example, peptidebased epoxyketones were used to screen for specificity of proteasome inhibitors, a rapidly growing area of interest due to the clinical success of bortezomib [63]. It is important to note that the threonine reactivity of the boronic acids, the epoxyketones and alpha-keto-aldehydes is promoted by the presence of a proximal N-terminal amine, which stabilizes the boronic-acid adduct [65], and affords a stable 6-membered morpholine www.sciencedirect.com

ring product with the other two electrophiles [63,64]. Therefore, although applicable to the proteasome subunits, these chemistries may be less conducive for profiling internal threonine residues. The aforementioned b-sultams (Figure 4h) can modify internal threonine residues [17], but the generality of this method is yet to be evaluated.

Covalent modification of aspartate and glutamate Retaining glucosidases contain a nucleophilic carboxylate residue within the active site, which is covalently modified by the 1,2-epoxide-containing natural product, cyclophellitol. ABPP probes for glucosidases, such as glucocerebrosidase, comprise epoxide and aziridine-containing cyclophellitol mimics (Figure 4i) [66,67]. The SE electrophile (Figure 4j) can also modify aspartates and glutamates that act as active-site proton donors/acceptors in dienoyl-CoA isomerase and long-chain specific acylCoA dehydrogenase [6]. However, as indicated previously, both these electrophiles are promiscuous in terms of amino-acid selectivity and are not specific to carboxylate side chains.

Summary and future directions When designing covalent modifiers for proteomic applications such as ABPP, both the amino-acid selectivity and the general reactivity of an electrophile need to be considered. Once a potentially nucleophilic residue is identified within the protein(s) of interest, an electrophile Current Opinion in Chemical Biology 2015, 24:18–26

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with innate reactivity toward that amino acid is needed. In this review, we have surveyed the amino-acid selectivity of the current landscape of electrophiles. In particular, we highlight highly selective electrophiles that only react with a single residue, as well as those that promiscuously react with numerous side chains. In some cases, the promiscuous reactivity of the electrophile can be overcome with a tight-binding ligand to direct the probe toward a specific nucleophilic residue proximal to the ligand-binding site. In addition to the amino-acid selectivity, the general reactivity of the electrophile needs to be considered, since this reactivity dictates the binding affinity that needs to be achieved by the directing elements of the probe. As detailed in this review, the use of a mild electrophile necessitates a tight-binding ligand to position the electrophile for optimal nucleophilic attack. In contrast, highly reactive electrophiles will modify proteins in the absence of a directing group, albeit with preferential reactivity toward more reactive functional residues. Given the expansion of chemical proteomic applications such as ABPP, it is critical to continuously grow the current armory of electrophiles so as to span the spectrum of selectivity and reactivity. The availability of an array of diverse electrophiles will allow for the judicious and rational selection of an optimal reactive group for each application.

Acknowledgements

7.

Weerapana E, Wang C, Simon GM, Richter F, Khare S, Dillon MB, Bachovchin DA, Mowen K, Baker D, Cravatt BF: Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 2010, 468:790-795.

8. Basle E, Joubert N, Pucheault M: Protein chemical modification  on endogenous amino acids. Chem Biol 2010, 17:213-227. This is a recent review that summarizes chemical transformations that are feasible on protein scaffolds. 9. 

Takaoka Y, Ojida A, Hamachi I: Protein organic chemistry and applications for labeling and engineering in live-cell systems. Angew Chem Int Ed Engl 2013, 52:4088-4106. This is a recent review that summarizes chemical transformations that are feasible on protein scaffolds.

10. Long JZ, Cravatt BF: The metabolic serine hydrolases and their functions in mammalian physiology and disease. Chem Rev 2011, 111:6022-6063. 11. Simon GM, Cravatt BF: Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J Biol Chem 2010, 285:11051-11055. 12. Mahrus S, Craik CS: Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem Biol 2005, 12:567-577. 13. Serim S, Mayer SV, Verhelst SH: Tuning activity-based probe selectivity for serine proteases by on-resin ‘click’ construction of peptide diphenyl phosphonates. Org Biomol Chem 2013, 11:5714-5721. 14. Haedke UR, Frommel SC, Hansen F, Hahne H, Kuster B, Bogyo M, Verhelst SH: Phosphoramidates as novel activity-based probes for serine proteases. Chembiochem 2014, 15:1106-1110. 15. Bottcher T, Sieber SA: Beta-lactones as privileged structures for the active-site labeling of versatile bacterial enzyme classes. Angew Chem Int Ed Engl 2008, 47:4600-4603.

This work was financially supported by the Smith Family Foundation, the Damon Runyon Cancer Research Foundation (DRR-18-12) and Boston College.

16. Staub I, Sieber SA: Beta-lactams as selective chemical probes for the in vivo labeling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence. J Am Chem Soc 2008, 130:13400-13409.

References and recommended reading

17. Kolb R, Bach NC, Sieber SA: beta-Sultams exhibit discrete binding preferences for diverse bacterial enzymes with nucleophilic residues. Chem Commun (Camb) 2014, 50:427-429.

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 

Cravatt BF, Wright AT, Kozarich JW: Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu Rev Biochem 2008, 77:383-414. This is a comprehensive review of activity-based protein profiling (ABPP), which summarizes the technology and its applications.

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Liu Y, Patricelli MP, Cravatt BF: Activity-based protein profiling: the serine hydrolases. Proc Natl Acad Sci U S A 1999, 96:1469414699.

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Bogyo M, Verhelst S, Bellingard-Dubouchaud V, Toba S, Greenbaum D: Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem Biol 2000, 7:27-38.

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Adam GC, Sorensen EJ, Cravatt BF: Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat Biotechnol 2002, 20:805-809.

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Barglow KT, Cravatt BF: Discovering disease-associated enzymes by proteome reactivity profiling. Chem Biol 2004, 11:1523-1531.

Weerapana E, Simon GM, Cravatt BF: Disparate proteome reactivity profiles of carbon electrophiles. Nat Chem Biol 2008, 4:405-407. This paper analyzes the amino-acid selectivity of chloroacetamide and sulfonate ester electrophiles, demonstrating that carbon-based electrophiles can have either highly selective or promiscuous amino-acid specificities.

6. 

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18. Shannon DA, Gu C, McLaughlin CJ, Kaiser M, van der Hoorn RA, Weerapana E: Sulfonyl fluoride analogues as activity-based probes for serine proteases. Chembiochem 2012, 13:2327-2330. 19. Gu C, Shannon DA, Colby T, Wang Z, Shabab M, Kumari S, Villamor JG, McLaughlin CJ, Weerapana E, Kaiser M, Cravatt BF  et al.: Chemical proteomics with sulfonyl fluoride probes reveals selective labeling of functional tyrosines in glutathione transferases. Chem Biol 2013, 20:541-548. The sulfonyl fluoride electrophile of the common serine protease inhbitor AEBSF also reacts with catalytically important H-site tyrosine residues in various GST-family enzymes. 20. Bihel F, Que´le´ver G, Lelouard H, Petit A, Alve`s da Costa C, Pourquie´ O, Checler F, Thellend A, Pierre P, Kraus JL: Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new beta-amyloid peptide production inhibitors and their activities on various classes of protease. Bioorg Med Chem 2003, 11:3141-3152. 21. Haedke U, Go¨tz M, Baer P, Verhelst SH: Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases. Bioorg Med Chem 2012, 20:633-640. 22. Child MA, Hall CI, Beck JR, Ofori LO, Albrow VE, Garland M, Bowyer PW, Bradley PJ, Powers JC, Boothroyd JC, Weerapana E et al.: Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nat Chem Biol 2013, 9:651-656. 23. Bachovchin DA, Ji T, Li W, Simon GM, Blankman JL, Adibekian A, Hoover H, Niessen S, Cravatt BF: Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening. Proc Natl Acad Sci U S A 2010, 107:20941-20946.

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24. Chang JW, Cognetta AB, Niphakis MJ, Cravatt BF: Proteome wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition. ACS Chem Biol 2013, 8:1590-1599. This paper studied the selectivity of a panel of carbamate electrophiles with varying reactivity and incorporate the most selective of these carbamates into an imaging probe. Notably, background labeling of nonserine hydrolases observed in vitro was absent in vivo, emphasizing the need to test electrophiles in a complex living system whenever possible. 25. Adibekian A, Martin BR, Wang C, Hsu KL, Bachovchin DA,  Niessen S, Hoover H, Cravatt BF: Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors. Nat Chem Biol 2011, 7:469-478. This paper explores the effectiveness of triazole ureas as serine-reactive electrophiles with mild reactivity and high selectivity 26. Adibekian A, Martin BR, Chang JW, Hsu KL, Tsuboi K, Bachovchin DA, Speers AE, Brown SJ, Spicer T, FernandezVega V, Ferguson J et al.: Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes. J Am Chem Soc 2012, 134:10345-10348. 27. Pace NJ, Weerapana E: Diverse functional roles of reactive cysteines. ACS Chem Biol 2013, 8:283-296. 28. Faleiro L, Kobayashi R, Fearnhead H, Lazebnik Y: Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J 1997, 16:2271-2281. 29. Berger AB, Witte MD, Denault JB, Sadaghiani AM, Sexton KM, Salvesen GS, Bogyo M: Identification of early intermediates of caspase activation using selective inhibitors and activitybased probes. Mol Cell 2006, 23:509-521. 30. Blum G, von Degenfeld G, Merchant MJ, Blau HM, Bogyo M:  Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. Nat Chem Biol 2007, 3:668-677. This paper highlights the potential application of protein-reactive electrophiles as in vivo imaging probes for enzyme activity. 31. Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M: Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem Biol 2000, 7:569-581. 32. Lowther WT, McMillen DA, Orville AM, Matthews BW: The antiangiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase. Proc Natl Acad Sci U S A 1998, 95:12153-12157. 33. Evans MJ, Saghatelian A, Sorensen EJ, Cravatt BF: Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nat Biotechnol 2005, 23:1303-1307. 34. Evans MJ, Morris GM, Wu J, Olson AJ, Sorensen EJ, Cravatt BF: Mechanistic and structural requirements for active site labeling of phosphoglycerate mutase by spiroepoxides. Mol Biosyst 2007, 3:495-506. 35. Adam GC, Cravatt BF, Sorensen EJ: Profiling the specific  reactivity of the proteome with non-directed activity-based probes. Chem Biol 2001, 8:81-95. This paper introduces the concept of using non-directed probes for ABPP by exploiting the inherent reactivity of functional amino acids in the proteome. 36. Pace NJ, Pimental DR, Weerapana E: An inhibitor of glutathione S-transferase omega 1 that selectively targets apoptotic cells. Angew Chem Int Ed Engl 2012, 51:8365-8368. 37. Barglow KT, Cravatt BF: Substrate mimicry in an activity-based probe that targets the nitrilase family of enzymes. Angew Chem Int Ed Engl 2006, 45:7408-7411.

40. Couvertier SM, Weerapana E: Cysteine-reactive chemical probes based on a modular 4-aminopiperidine scaffold. MedChemComm 2014, 5:358-362. 41. Deng X, Weerapana E, Ulanovskaya O, Sun F, Liang H, Ji Q, Ye Y, Fu Y, Zhou L, Li J, Zhang H et al.: Proteome-wide quantification and characterization of oxidation-sensitive cysteines in pathogenic bacteria. Cell Host Microbe 2013, 13:358-370. 42. Wang C, Weerapana E, Blewett MM, Cravatt BF: A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. Nat Methods 2014, 11:79-85. 43. Pace NJ, Weerapana E: A competitive chemical-proteomic platform to identify zinc-binding cysteines. ACS Chem Biol 2014, 9:258-265. 44. Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L, Mann M: Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 2008, 5:459-460. 45. Wong HL, Liebler DC: Mitochondrial protein targets of thiol reactive electrophiles. Chem Res Toxicol 2008, 21:796-804. This paper provides a detailed comparison of the protein targets of iodoacetamide and maleimide and demonstrate that these two electrophiles target two different subsets of the proteome. 46. Shannon DA, Banerjee R, Webster ER, Bak DW, Wang C,  Weerapana E: Investigating the proteome reactivity and selectivity of aryl halides. J Am Chem Soc 2014, 136:3330-3333. This study evaluated the proteome reactivity of a panel of aryl halides and identified chloronitrobenzenes as cysteine reactive and dichlorotriazine as lysine reactive. 47. Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D et al.: On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc 2013, 135:2867-2870. 48. Sommer S, Weikart ND, Linne U, Mootz HD: Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne addition. Bioorg Med Chem 2013, 21:2511-2517. 49. Arkona C, Rademann J: Propargyl amides as irreversible  inhibitors of cysteine proteases — a lesson on the biological reactivity of alkynes. Angew Chem Int Ed Engl 2013, 52:8210-8212. This paper provides insight into the cysteine reactivity of terminal alkynes. 50. Doorn JA, Petersen DR: Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2nonenal and 4-oxo-2-nonenal. Chem Res Toxicol 2002, 15:1445-1450. 51. LoPachin RM, Gavin T, Petersen DR, Barber DS: Molecular mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic targets and adduct formation. Chem Res Toxicol 2009, 22:1499-1508. 52. Kathman SG, Xu Z, Statsyuk AV: A fragment-based method to  discover irreversible covalent inhibitors of cysteine proteases. J Med Chem 2014, 57:4969-4974. This paper evaluated substituent effects on the reactivity of a panel of Michael acceptors, demonstrating that the reactivity of certain electrophiles can be significantly modulated by adjacent functionality. 53. Krishnan S, Miller RM, Tian B, Mullins RD, Jacobson MP,  Taunton J: Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis. J Am Chem Soc 2014, 136:12624-12630. This paper applies computational methods to design reversible Michael acceptors.

38. Barglow KT, Saikatendu KS, Bracey MH, Huey R, Morris GM, Olson AJ, Stevens RC, Cravatt BF: Functional proteomic and structural insights into molecular recognition in the nitrilase family enzymes. Biochemistry 2008, 47:13514-13523.

54. Serafimova IM, Pufall MA, Krishnan S, Duda K, Cohen MS,  Maglathlin RL, McFarland JM, Miller RM, Frodin M, Taunton J: Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat Chem Biol 2012, 8:471-476. This paper highlights the feasibility and the advantages of reversible covalent inhibitors over irreversible binders.

39. Banerjee R, Pace NJ, Brown DR, Weerapana E: 1,3,5-Triazine as a modular scaffold for covalent inhibitors with streamlined target identification. J Am Chem Soc 2013, 135:2497-2500.

55. McFarland JM, Francis MB: Reductive alkylation of proteins using iridium catalyzed transfer hydrogenation. J Am Chem Soc 2005, 127:13490-13491.

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26 Omics

56. Cal PM, Vicente JB, Pires E, Coelho AV, Veiros LF, Cordeiro C, Gois PM: Iminoboronates: a new strategy for reversible protein modification. J Am Chem Soc 2012, 134:10299-10305. 57. Asano S, Patterson JT, Gaj T, Barbas CF: Site-selective labeling  of a lysine residue in human serum albumin. Angew Chem Int Ed Engl 2014 http://dx.doi.org/10.1002/anie.201405924. (in press). This paper shows that a derivative of N-phenylsulfonamide TAK-242 covalently modifies a lysine on human serum albumin. If this reactivity can be applied more generally, it could be an extremely useful tool in lysine ABPP, as there are many possibilities for modifying the TAK-242 scaffold to alter protein-target selectivity. 58. Tsai CS, Yen HY, Lin MI, Tsai TI, Wang SY, Huang WI, Hsu TL, Cheng YS, Fang JM, Wong CH: Cell-permeable probe for identification and imaging of sialidases. Proc Natl Acad Sci U S A 2013, 110:2466-2471. 59. Ban H, Gavrilyuk J, Barbas CF 3rd: Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J Am Chem Soc 2010, 132:1523-1525. 60. Hu QY, Allan M, Adamo R, Quinn D, Zhai HL, Wu GX, Clark K, Zhou J, Ortiz S, Wang B, Danieli E et al.: Synthesis of a welldefined glycoconjugate vaccine by a tyrosine-selective conjugation strategy. Chem Sci 2013, 4:3827-3832. 61. Adams J, Behnke M, Chen S, Cruickshank AA, Dick LR, Grenier L, Klunder JM, Ma YT, Plamondon L, Stein RL: Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg Med Chem Lett 1998, 8:333-338. 62. Albers HM, Dong A, van Meeteren LA, Egan DA, Sunkara M, van Tilburg EW, Schuurman K, van Tellingen O, Morris AJ, Smyth SS,

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