Emerging and Re-Emerging Warheads for Targeted Covalent

Perspective Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology Matthias Gehringer* and Stefan A. Laufer

J. Med. Chem. Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/27/19. For personal use only.

Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany ABSTRACT: Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting noncatalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of noncatalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheads beyond α,β-unsaturated amidesrecently used in the design of targeted covalent ligands. Promising reactive groups that have not yet demonstrated their utility in TCI development are also highlighted. Special emphasis is placed on the discussion of reactivity and of case studies illustrating applications in medicinal chemistry and chemical biology.

1. INTRODUCTION Covalent targeting has played a subordinate role in drug discovery for a long time. Although covalent modification of proteins1,2 and nucleobases3 is a key element in the regulation of biological systems, the systematic development of reactive drugs has been considered highly adventurous because of potential toxicity arising from promiscuous labeling, haptenization, and idiosyncratic drug reactions.4,5 Nevertheless, over 40 covalent modifier drugs are currently approved by the FDA.6 However, the mode of action of most of these compounds has been discovered serendipitously rather than resulting from rational design. A notable exception are inhibitors of amide-/ ester-bond cleaving hydrolases, where reversible and irreversible covalent bond formation has long been used as a design strategy to address catalytic nucleophiles such as activated serine or cysteine residues.7,8 During the past decade, covalent targeting has experienced a resurgence. So-called “targeted covalent inhibitors” (TCIs) addressing poorly conserved amino acids now provide the basis for a multitude of industrial drug discovery programs, especially in oncology.5 According to the definition from a seminal review by Juswinder Singh and colleagues, a TCI is “an inhibitor bearing a bond-forming functional group of low reactivity that, following binding to the target protein, is positioned to react rapidly with a specific noncatalytic residue at the target site.”5 Naturally, this concept can further be extended to other types of targeted covalent ligands beyond (enzyme) inhibitors. The renewed interest in covalent targeting is based on the perception that properly designed TCIs could offer multiple advantages, some of which would even result in a more favorable benefit−risk balance. Irreversible covalent inhibitors profit from nonequilibrium kinetics, potentially enabling full target occupancy, even if © XXXX American Chemical Society

reversible binding affinity is moderate. In contrast to reversible binders, irreversibly bound compounds do not compete with natural ligands or substrates. Their durable target engagement can further decouple pharmacodynamics from pharmacokinetics provided that the protein of interest has a sufficiently slow rate of resynthesis.6 The driving force and spatial requirements of covalent bond formation can be exploited to reduce the size of ligands without sacrificing potency and selectivity, thereby increasing “drug-likeness.” Considering that a substantial fraction of the drug candidates fail due to ADME issues,9 the above features promise significant benefits. In cases where permanent protein modification is undesired, covalentreversible chemistry can be used to harness potential advantages in terms of potency and selectivity while decreasing the risk of haptenization, inhibitor depletion by glutathione (GSH), and irreversible off-target modification.10 The residence times of covalent-reversible inhibitors strongly depend on reversible interactions stabilizing the covalent complex. Thus, they can be tuned to fit the target’s turnover rate.11 Ideally, the unmodified ligand would be released to reengage with the target after protein degradation or after nonspecific covalent binding to off-targets incapable of stabilizing the covalent complex. TCIs are typically generated by structure-based design from optimized reversible ligands, which are modified by attachment of an electrophilic covalent reactive group12 (CRG; often termed “warhead”) to address a proximal amino acid, most frequently cysteine. Alternative strategies that have recently been employed to identify covalent binders include fragmentReceived: July 23, 2018 Published: December 19, 2018 A

DOI: 10.1021/acs.jmedchem.8b01153 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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based13−16 or tethering approaches17 and DNA-encoded libraries featuring electrophilic ligands.18,19 In TCI design, binding kinetics require special consideration. Because a detailed perspective on the implications of covalent binding kinetics has recently been provided,20 only a brief overview shall be given at this place. TCI binding normally involves a two-step process in which an initial reversible binding event takes place, followed by the covalent bond-forming reaction. The first step is described by the equilibrium constant KI, which accounts for the ligand concentration required to achieve a half-maximal rate of covalent modification. The second step is characterized by kinact, the maximal potential rate of covalent modification/inactivation. The overall inhibitory efficiency is best described as the second-order rate constant of covalent target modification expressed by kinact/KI.21 As a result, covalent inhibition is time-dependent and prolonged exposure leads to an increase in target occupancy. Consequently, KD, IC50, or EC50 values are not the appropriate measures for comparing covalent ligands because they vary over time and do not reflect the relative contribution of KI and kinact to the observed overall effect. Notably, time-dependent inhibition result from other factors like compound aggregation or instability in buffer and time-dependence cannot be considered as an ultimate proof of covalent binding despite the presence of a reactive group. On the other hand, very slow off-rate ligands may also be misinterpreted as covalent binders in washout and jump dilution experiments.20 Cross-validation of the presumed covalent interaction by both mass spectrometry and X-ray crystallography should thus be performed. Ideally, the latter data would be supported by studies with the mutated target protein devoid of the reactive amino acid and with congeneric ligands lacking the CRG. The two-step binding mechanism of TCIs differs from the one of reagents used for protein labeling or bioconjugation. The latter are used for chemo- but not site-selective covalent modification and preferably address the most reactive and accessible side chains. In this case, the reaction can be described by a one-step mechanism20 without the requirement of specific reversible binding. Here, comparably reactive moieties relying solely on their intrinsic chemical reactivity toward the desired amino acids are used. Warheads to be utilized in TCIs require a more balanced reactivity profile. They should be reactive enough to form a covalent bond with the target residue in a proximity driven or templated manner, but only after the proper alignment of both reaction partners by the reversible binding event. Inherent reactivity should be reduced to a necessary minimum to prevent nonspecific off-target labeling or reaction with GSH. Because the reactivity of amino acid side chains varies as a function of their chemical environment, the reactivity of ideal CRGs should be readily adjustable to match the specific requirements of the target. Warheads intended for in vivo use, either in drugs or in chemical probes, should be chemically and (usually) metabolically stable and nontoxic. If chemical probes are intended for the investigation of specific targets or pathways in cells, some degree of chemical instability or toxicity might be tolerable. The latter class of ligands should be as selective as possible, which is not necessarily true for drugs. In contrast, reactive probes for activity-based protein profiling approaches (ABPP)22 require a balanced amount of promiscuity to enable the profiling of a defined set of targets. As a result of the time-dependent nature of covalent target engagement, even optimized compounds, which have demon-

strated good selectivity in screening panels, can cause significant off-target labeling after extended exposure.23,24 On the other hand, relatively small, reactive, and presumably unselective fragments can possess an unexpected degree of selectivity when profiled against isolated proteins15 or cellular proteomes.13 The latter findings suggest that an increased reactivity might be exploited to achieve kinetic selectivity if the reactive moiety was completely neutralized by the target or metabolically deactivated after having engaged the target (vide infra). Certain highly reactive compounds such as αcyanoacrylamide-derived Michael acceptors can further be useful for covalent-reversible targeting approaches.25 Finally, the ideal balance of reversible binding affinity and specificity, inherent reactivity, and metabolic stability depends on the envisaged field of application and needs to be evaluated on a case by case basis. There has been a very high interest in novel TCIs within the last years, the tremendous amount of published work having been compiled in a plethora of recent reviews.5,6,26−39 Most of these reports focus primarily on noncatalytic cysteine residues addressed by α,β-unsaturated carbonyl compounds. Although cysteine-targeted Michael acceptors, most notably α,βunsaturated amides, are clearly the dominant CRGs in the realm of current TCIs, they are not intended to be a key subject in this essay. An overview of α,β-unsaturated carbonyl compounds has already been provided in a recent Perspective article by Kay Brummond, Daniel Harki, and co-workers, extensively discussing the chemistry and reactivity of Michael acceptors as well as their application in drug discovery and beyond.40 Instead, this Perspective focuses on less common warheads, recent innovations, and emerging concepts, highlighting case studies to illustrate their potential in medicinal chemistry and chemical biology. Reagents for protein modification, which have also been reviewed recently,41−47 are not included except for certain functional groups where utility of the underlying reactivity in TCI design is anticipated. Likewise, warheads that have exclusively been used to target highly reactive active site nucleophiles, e.g., catalytic serine, threonine, or cysteine residues in proteolytic enzymes, are not a major focus of this Perspective and may be found elsewhere.7,8,48 As an exception, few moieties engaging catalytic nucleophiles are discussed to highlight the underlying reactivity or design principles. It should further be pointed out that many warhead classes included in this article have previously found application in targeting the active sites of proteases and other hydrolase enzymes. Despite the enormous amount of work that has been published on acrylamide-derived TCIs, systematic studies evaluating warheads beyond α,β-unsaturated amides are comparably sparse and scattered. Although we aimed to include all relevant and recent information, this work should rather be considered a (personally biased) perspective on interesting chemistry and current developments and not a comprehensive review of the field. This article covers literature published before July 2018, therefore interesting work which appeared during the revision of the manuscript (e.g., cysteinetargeted cyanamides as Janus kinase 3 inhibitors,49 histidinetargeted linear alkyl bromides as 17β-hydroxysteroid dehydrogenase inhibitors,50 and methionine-targeted epoxides as bromodomain inhibitors51), is not discussed. The article is organized by reactive amino acid side chains. Thus, warheads that have been used to target different residues may occur in several sections. (Pseudo)irreversible CRGs are discussed first, B



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Figure 1. Kinetic selectivity of fumaric acid esters. Selectivity is povided by rapid bond formation with the target. Slightly slower ester cleavage deactivates the warhead, preventing even slower labeling of undesired proteins.

equivalently positioned, poorly conserved cysteine in the solvent-exposed front region of ErbB/HER family receptor tyrosine kinases or TEC family kinases like Bruton’s tyrosine kinase (BTK) via acrylic or butynoic amides.35,60,61 An analogous cysteine has been used to generate highly selective Janus kinase (JAK) 3 inhibitors with a promising potential in the treatment of inflammatory disorders.62 Besides TCIs, endogenous ligands such as cyclopentenone prostaglandins,63 but also a multitude of other natural α,β-unsaturated carbonyl compounds,40 covalently engage noncatalytic cysteines. As suggested by the differing pKa values, cysteine reactivity can vary over a wide range, and not all exposed cysteine residues may be readily amenable to covalent modification.64 Targeting less reactive cysteine residues is not always straightforward, and success may crucially depend on precise positioning and sufficient reactivity of the CRG but also on assistance by the CRG or neighboring groups in deprotonating the thiol. Moreover, nonessential cysteines are prone to mutation (see for example the EGFR C797S resistance mutant)65 which can be considered an Achilles’ heel of cysteine-targeted covalent drugs, especially in oncology. On the other hand, acquired cysteine residues (e.g., in the oncogenic K-Ras G12C or the p53 Y220C mutants) may open up new avenues for addressing challenging targets. 2.1. Cysteine Addition to Metabolically Labile Monomethyl Fumarates. Although targeted covalent inhibitors derived from α,β-unsaturated amides are not a major topic here, some recent “noncanonical” approaches merit further discussion. As mentioned, TCIs often feature excellent selectivity for their targets upon short-term treatment but extended exposure has been shown to erode selectivity.23,66 Slower covalent modification of off-targets can be missed by classical screening techniques typically investigating short-term effects (minutes to few hours). A possible solution to this issue is provided by covalent-reversible binders, such as the abovementioned α-cyanoacrylamides. Another interesting approach to minimize time-dependent off-target modification has recently been developed in Benjamin Cravatt’s laboratories. While investigating the cysteine labeling profile of the immunomodulatory drug dimethyl fumarate (DMF) in T cell proteomes, they found the active metabolite monomethyl fumarate (MMF) to be devoid of substantial cysteine

while covalent-reversible warheads may be found at the end of each section. A brief personal perspective on benefits and challenges of the respective CRG is given in each paragraph, and broader discussion may be found in the final Perspective section. Being a major subject of this article, inherent warhead reactivity and reaction rates are only compared directly if they were tested in the same assay. Otherwise, only a qualitative assessment is provided. Notably, there is still a heavy reliance on IC50/EC50 data in the field, although kinact/KI is the recommended measure for covalent binders. We have included kinetic data whenever it was available, but IC50 values are the basis for discussion if no such data has been reported.

2. TARGETING THE CYSTEINE SIDE CHAIN Sulfur is the only third-row element encoded in proteinogenic amino acids and has a distinct role in biological systems. The thioether group in methionine and the neutral cysteine thiol group are only moderately nucleophilic. However, nucleophilicity is increased by several orders of magnitude for the thiolate form of the cysteine side chain, making it the strongest nucleophile among the 20 canonical amino acids.52,53 Being highly polarizable, thiols and thiolates are soft bases according to Pearson’s hard and soft acids and bases (HSAB) concept.54 The increased nucleophilicity and polarizability of thiolates compared to alkoxides is owed to the size and the high energy of the 3sp3 lone pairs. The thiol group can easily be deprotonated (pKa ≈ 8.6 for cysteine), and the pKa can shift several orders of magnitude within proteins.55 While pKa values between 2.5 and 11.1 have been measured for cysteines’ thiols in proteins,56 noncatalytic cysteine residues typically feature pKa values in the range between 7.4 and 9.1. Catalytic cysteines, in contrast, are pKa-perturbed to favor the highly nucleophilic thiolate anion.55 Therefore, it is hardly surprising that cysteine has various roles in catalysis, e.g., in the active sites of cysteine proteases, ubiquitin ligases, or tyrosine phosphatases, but also in metal binding, structural stabilization (via the formation of disulfide bridges), and posttranslational/ redox regulation.57,58 Notwithstanding these key functions, cysteine is a relatively rare amino acid with a prevalence of only 1.9%.59 Hence, noncatalytic cysteine residues played a key role in recent TCI design efforts. For example, all the six currently approved targeted covalent kinase inhibitors address an C



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Figure 2. Ibrutinib-derived fumarate esters and analogous probes equipped with a click handle.

reactivity,67 a finding that might be attributed to the decreased intrinsic reactivity of monomethyl fumarate but that also reflects the presumably lower cell permeability of MMF. On the basis of this observation, the hypothesis arose that replacement of a conventional α,β-unsaturated amide in a cysteine-targeted inhibitor by a methyl fumarate residue could furnish compounds that rapidly react with their targets while the slower inactivation by esterases prevents off-target modification (Figure 1). To test this hypothesis, the approved covalent Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib68 (1, Figure 2), which is known to react with off-targets in a time-dependent manner,23 was chosen for a first proof of concept study. Ibrutinib features a piperidin-3-yl-linked acrylamide warhead that labels BTK at Cys481.69,70 The latter was replaced by several amide-linked fumarates (3−7). Some of the studied compounds (2 and 5−7) were further equipped with an alkyne to provide a chemical handle enabling the attachment of tags for enrichment and target identification. Both ibrutinib-derived probe 2 and its methyl fumarate analogue 5 labeled BTK in Ramos cell lysates. As expected, fumarate 5 reacted faster with cysteine and possessed the higher reactivity toward the cellular proteome. When incubated with HEK293T cells stably expressing human carboxylesterase (hCES) 1, ibrutinib remained unaffected while the methylfumarate-derived inhibitor 3 was rapidly converted to the unreactive acid 4. This effect was not observed when using HEK293T cells stably expressing methionine aminopeptidase (MetAP) 2 as a control. A significant reduction in time-dependent proteome labeling accompanied by a modest decrease in BTK activity (ca. 10-fold) was observed for probe 5 when incubated over 24 h with a 6:1 coculture of Ramos and HEK293T cell lines, which was supposed to mimic the hCES1 activity in tumor xenografts. In contrast, the labeling profile of the ibrutinibderived probe 2 remained unaffected under these conditions. Competition experiments employing gel-based or MS proteomics71 confirmed that BTK engagement by inhibitor 3 was only marginally affected by hCES1, supporting the notion that kinase modification occurs at a higher rate than enzymatic ester hydrolysis. Only BTK and the kinase TEC, sharing an equivalently positioned cysteine,62 reacted with 3 in a hCES1insensitive manner while 1 retained its proteomic profile in the presence of the esterase. Subsequent profiling of probe 5 and its isopropyl ester analogue 6 in rodents (20 mg/kg ip) demonstrated BTK engagement in vivo, although both compounds were poorly stable in mouse plasma (t1/2 = < 2 min). Pretreatment with the covalent CES inhibitor JZL184,72 however, increased the plasma stability to 25.5 and 352 min for 5 and 6. In contrast, probe 2 and the free acid 7 did not require CES blockage to

obtain reasonable plasma stability (t1/2 = 168 and 129 min). Both 5 and 6 demonstrated significantly reduced off-target labeling in different tissues while substantial reactivity toward BTK was maintained. Although these results are qualitative in nature, they underline the increased BTK selectivity of the fumaric acid esters in vivo. Although the metabolic inactivation of the reactive species to promote kinetic selectivity is a promising concept, the generalizability of this approach remains to be shown. Alternative esters might be required to fine-tune stability against esterases, and those will need to fit the respective binding pockets. As suggested by the authors of the above study, alternative warheads with limited metabolic stability such as acrylates or thioacrylates may have the potential to address these issues.24 However, optimization of such compounds for clinical use, especially when considering oral administration will further complicate the situation due to the prolonged and more complex kinetics of uptake and distribution as well as first-pass metabolism. Moreover, proteins with a high turnover constitute a challenge to this conceptual framework as they require sustained exposure for pharmacological efficacy. Nevertheless, this study demonstrates that kinetic selectivity is achievable with metabolically labile warheads and it will be interesting to see whether related concepts will expand our current toolbox for TCI design. 2.2. Cysteine Addition to Allenamides. Recently, allenamides have been suggested as bioisosteres of α,βunsaturated amides for protein labeling purposes and inhibitor design. Although the reactivity of allenamides (general structure 8, Figure 3) toward thiols has been known for a long time,73,74 Teck-Peng Loh and colleagues were the first to

Figure 3. Mechanism of cysteine addition to allenamides. The prevalence of the mesomeric structure 9a rationalizes the formation of the nonconjugated product. An alternative mechanism involving attack of the neutral thiol to form a zwitterionic species followed by proton transfer was proposed by Loh and co-workers. D



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Figure 4. Osimertinib-derived allenamides as EGFR inhibitors.

Figure 5. Propiolonitriles as potential TCI warheads: (A) 3-Aryl and 3-alkyl propiolonitriles. (B) Mechanism of cysteine addition and thiol exchange.

investigate these reagents for cysteine labeling applications.75 They found a high intrinsic reactivity toward the thiol(ate) of isolated cysteine as well as for terminal and internal cysteine residues in peptides and proteins at pH 8. However, neither were reaction rates determined nor was a qualitative comparison of reactivity between aniline-derived allenamides and alkylamine-derived analogues provided. No labeling of other nucleophilic amino acids was observed. Upon reaction with cysteine at the sp-hybridized carbon center, only the thermodynamically disfavored nonconjugated product 10a was formed, which is probably due to prevalence of the mesomeric structure 9a favoring protonation in the α-position. Notably, a similar regioselectivity was recently observed for selenocysteine conjugation.76 Compared to their keto and ester analogues, terminal allenamides have a much lower tendency to undergo [3 + 2] cycloaddition reactions and cysteine labeling was shown to be irreversible even in the presence of a 100-fold excess of glutathione or dithiothreitol (DTT). While the Loh group only tested this CRG for protein labeling, a subsequent study by Yujun Zhao and colleagues exploited the allenamide moiety as a bioisosteric replacement of the acrylamide functionality in the approved EGFR inhibitor osimertinib (11, Figure 4).77 The allenamide warhead was well tolerated in terms of potency, and analogue 12 was slightly more active in blocking the EGFR T790M/L858R mutant compared to the parent compound (IC50 = 1.4 nM vs 3.9 nM for EGFRT790M/L858R). However, the compound partially eroded osimertinib’s selectivity over the wild-type enzyme (IC50 = 28 nM vs 142 nM for EGFRwt). Further optimization furnished low nanomolar inhibitors with increased (up to 43fold for compound 13) or decreased (only 6-fold for compound 14) selectivity over the wild-type enzyme. Selectivity in the enzyme assay, however, did not translate into cellular selectivity for NCI-H1975 (EGFRL858R/T790M) versus A549 (EGFRwt) cells. Target engagement in NCIH1975 cells was suggested by Western blot, but neither was covalent binding unambiguously demonstrated nor were off-

targets assessed. Allenamide 12 showed poor oral exposure in mice, and both 12 and 14 possessed an over 10-fold lower stability in fetal bovine serum compared to osimertinib. The decreased stability is likely to result from the higher intrinsic reactivity of the allenamide warhead compared to osimertinib’s acrylamide. Pseudo-first-order rates for the reaction of compound 14 with glutathione (k′ = 0.303 min−1) were shown to be 7−28-fold higher than for the approved acrylamide-derived kinase inhibitors afatinib (k′ = 0.044 min−1), osimertinib (k′ = 0.011 min−1), and ibrutinib (k′ = 0.011 min−1). Although terminal allenamides offer certain advantages such as good accessibility, irreversibility, compact size, and a well-defined geometry, their utility in medicinal chemistry remains thus limited by the high intrinsic reactivity. In analogy to α,β-unsaturated amides,12 a decrease in reactivity might be achieved by employing alkylamine-derived amides. In certain cases, for example when poorly nucleophilic cysteines78 need to be addressed, the higher reactivity may provide a benefit, assuming that the reversibly binding part of the inhibitor confers sufficient kinetic selectivity to minimize offtarget labeling. 2.3. Cysteine Addition to Linear 3-Aryl and Alkyl Propiolonitriles. Alain Wagner and co-workers have recently established 3-aryl propiolonitriles (general structure 15, Figure 5A) as reagents for selective cysteine modification in peptide mixtures.79 The obtained vinyl thioether adducts were stable against thiols over a wide pH range (from 0−14), reducing agents (tris(2-carboxyethyl)phosphine (TCEP) and DTT), and a reasonable stability was also demonstrated in plasma and living cells. In contrast, structurally related aryl alkynones undergo thiol exchange via addition of a second thiol and subsequent elimination of one thiolate (Figure 5B).80 Reactivity of unsubstituted 15 is presumably too high for medicinal chemistry applications. However, reactivity could be decreased by substituents with a +M effect in the para-position (and vice versa, with substituents featuring a −M effect) and dropped substantially when ortho-substituents were introE



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Figure 6. 6-Ethynylthienopyrimidine as covalent ErbB kinase inhibitors. (A) 6-Ethynylthieno[3,2-d]pyrimidine and 6-ethynylthieno[2,3d]pyrimidine-derived inhibitors. (B) Suggested mechanism of cysteine addition.

Consistent with a mechanism in which the thiol(ate) attacks the triple bond generating a transient negative charge, the 6ethynylthieno[3,2-d]pyrimidine-derived compounds were more reactive due to better resonance stabilization of the latter. However, labeling was generally slow. While the acrylamide-derived control compound canertinib (CI-1033)93 completely labeled EGFR within less than 3 h, only 67% of covalent modification was observed for the terminal alkyne 17 after 20 h as determined by MS. Interestingly, the attachment of a 2-(R)-pyrrolidine residue (18) significantly accelerated covalent inactivation, leading to 83% labeling after 3 h and full labeling after 20 h, respectively. This acceleration was neither observed with an analogous compound featuring a (R)-2aminoethyl substituent (19) nor with a weakly basic 2-pyrazine derivative (20). The rate-enhancing effect of methylene-linked amino groups has been described previously for propiolamideand acrylamide-derived EGFR inhibitors.94,95 In the suggested mechanism, the basic amine increases the nucleophilicity of the thiol group by hydrogen bonding/deprotonation (Figure 6B). The distinct effect of the (R)-pyrrolidine residue compared to the primary propargylic amine in 19 might be rationalized by the spatial requirements for proton abstraction. An X-ray crystal structure confirmed covalent binding of compound 17 to Cys803 in ErbB4 (Figure 7) while EGFR labeling at Cys797 was proven by MS. Key compound 18, a potent EGFR, ErbB2, and ErbB4 inhibitor (IC50 = 7, 13, and 66 nM, respectively) with cellular IC50 values in the midnanomolar range featured >100-fold selectivity against 30 from 31 kinases in a small panel. Remarkably, it did not inhibit interleukin-2-inducible Tcell kinase (ITK) possessing an equivalently positioned cysteine. Dosed orally at 30 and 100 mg/kg b.i.d., compound 18 was an effective inhibitor of tumor growth in a murine BT474 cell xenograft model while compound 19 showed slightly lower activity in the same model system. In several follow-up studies, the same team investigated the effects of substituents at the 4-position of the pyrrolidine ring,96,97 the stereochemistry97 at the pyrrolidine substituent, and modifications at the aniline headgroup.98 Although some improvements of ADMET properties were achieved, no major advances in terms of biochemical and cellular potency and

duced. Moreover, the analogous 3-cyclohexylpropiolonitrile (16) reacted about 10 times slower compared to unsubstituted 15, however, the obtained reaction product also proved to be slightly less stable. Although no applications in a medicinal chemistry or chemical biology context have been discussed so far, this linear electrophile could become very useful when precise and rigid spatial placement of the warhead is required (provided that the binding cavity can host this elongate functionality). Still, it remains to be seen if this CRG can be attenuated to an appropriate intrinsic reactivity to become useful for chemical probe development. 2.4. Cysteine Addition to Alkenyl or AlkynylSubstituted Heteroarenes. Electron-deficient heteroaryl groups with (substituted) vinyl or ethynyl residues constitute a structural motif frequently found in patent applications claiming covalent inhibitors. These moieties are supposed to react with thiols via a conjugate addition, in which the intermediate negative charge is stabilized by the heteroarene. This reaction type has been known for a long time. For example, 4-vinylpyridins have been used to modify cysteines since the 1960s81,82 and chemical reactions exploiting alkenylated or alkynylated aromatic heterocycles for nucleophilic additions are not uncommon (e.g., refs 83−88). Vinylpyridines have also been applied in cysteine-specific protein labeling.89,90 and their reactivity can be readily modulated.91 In this light, it is surprising that only a few and relatively limited systematic studies evaluating this chemistry for cysteine-targeted inhibitors have been published so far. An early isolated series of publications on the application of such CRGs in medicinal chemistry was reported by David Uehling and co-workers from GSK in 2008/2009.92 They serendipitously found a noncatalytic cysteine of ErbB kinases to react with ethynylthienopyrimidine-based inhibitors. Although the ethynyl moieties were attached to the thiophene ring, which is usually electron-rich, covalent bond formation with Cys803 in ErbB4 and Cys797 in EGFR was observed. In a subsequent SAR study, the influence of a terminal substituent at the ethynyl moiety was evaluated for both a 6-ethynylthieno[3,2-d]pyrimidine (17−20, Figure 6A) and a 6-ethynylthieno[2,3-d]pyrimidine series (exemplified by compound 21). F



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while 23 was unreactive. Unfortunately, only indirect evidence for the covalent engagement of Cys98 was provided and no validation by MS or X-ray crystallography performed. To this end, the authors showed that only 22, but not 23, decreased [H3]-histamine binding after washout, an effect that was not observed when performing the same experiment with the hH4 C98S mutant. However, even at the highest concentration of 10 μM (incubation for 1 h), 22 only partially blocked [H3]histamine binding after washout, indicating a slow reaction and incomplete covalent labeling. Interestingly, the affinities of both compounds did not significantly differ between the wildtype receptor and the C98S mutant, suggesting reversible binding to be the main contributor to the latter. 2.5. Cysteine Addition to Nonactivated Terminal Alkynes. Propargylamines such as selegiline and rasagiline are well-known as mechanism-based covalent monoamine oxidase (MAO)-B inhibitors in the treatment of Parkinson’s disease,100,101 and terminal alkynes have further found application as activity-based probes for CYP enzymes.102,103 In these compounds, however, the respective alkynes undergo oxidative activation to generate electrophiles, which can covalently engage their protein targets. While strain activated alkynes are known to react with cysteine residues,104,105 the nonactivated terminal alkynes employed in CuAAC click reactions106 have been assumed to be inert in biological samples. In this light, recent reports on protein-templated cysteine addition to nonactivated, and thus poorly electrophilic, terminal alkynes are quite remarkable (exemplified by general structure 24, Figure 9A). In 2013, the groups of Huib Ovaa107 and Henning Mootz108 independently discovered this reactivity while employing propargylamide-labeled ubiquitin (Ub), SUMO, or peptides derived thereof, intended for clickchemistry-based labeling. The active site cysteine of deubiquitinating isopeptidases (DUBs) and SUMO proteases selectively attacked the triple bond at the 2-position forming the Markovnikov vinyl thioether adduct. Interestingly, N-but3-ynyl amide (24a) and even N-hex-5-ynyl amide homologues (24b) were also reactive while the allylic amide 24c showed poor reactivity (Figure 9B). Introduction of a terminal methyl group (24d) or geminal 2,2-dimethyl substitution (24e) at the propargyl residue prevented cysteine addition.107 The approach could also be applied to other cysteine proteases such as caspase-1107 or more recently the viral leader protease Lbpro.109 Although these results partially question the biological inertness of terminal alkynes used in click-chemistry labeling applications, the underlying reaction seems to be quite specific and, so far, limited to certain cysteine proteases. It has been proposed that this reaction requires the stabilization of the intermediary carbanion by the hydrogen bond donors in the oxyanion hole.110 In the SUMO-protease Senp1, however, mutation of key residues in the catalytic triad (i.e., H533A and D550A) and the oxyanion hole (Q597A) did not prevent the formation of the covalent adduct.108 Further mechanistic investigations will be required to elucidate the structural requirements promoting these reactions and it remains to be seen, if noncatalytic cysteines could covalently trap propargylamides and similar terminal alkynes in certain settings. 2.6. Cysteine Targeting by Nucleophilic Aromatic Substitution (SNAr) Reactions. Although nucleophilic aromatic substitution (SNAr) reactions have a long history in covalent protein targeting,111 they are still largely underdeveloped in the field of TCIs. In this type of reaction, covalent labeling is effected by a nucleophile, e.g., a cysteine

Figure 7. X-ray crystal structure of compound 17 in complex with the ErbB4 kinase domain (PDB 2R4B). The terminal alkyne moiety has reacted with Cys803 to form a vinyl thioether adduct. The N1-atom of the pyrimidine ring is further anchored to the backbone NH of Met799 in the hinge region via a hydrogen bond. The pyrimidine N3atom is engaged in a water-mediated hydrogen bond to the side chain of Thr860 preceding the conserved DFG motif.

covalent labeling efficiency could be made. Moreover, it should be pointed out that neither biochemical nor cellular inhibitory potencies showed a clear correlation with the propensity to covalently modify the target in vitro, accentuating that the comparatively slow cysteine addition was not the key driver of biological activity. Since no further studies with this inhibitor class have been reported since 2009, it can be assumed that their development was discontinued. Nevertheless, the provided data shows that heteroarene-derived Michael-type acceptors are compatible with a physiological environment and can in principle be used as CRGs in drug discovery. Systematic studies, however, will be required to explore the tunability and versatility of this type of warhead. Moreover, when alkynylated heteroaryl groups are employed as acceptors, the stability of the adducts toward a second thioether addition and accompanying thiol exchange reactions (see section 2.3) will necessitate further investigation. More recently, the group of Rob Leurs modified reversible 2-aminopyrimidine-based antagonists of the human histamine H4 receptor with a 2-vinyl substituent to covalently address Cys98 located in the transmembrane domain (TM) 3.99 Both VUF14480 (22, Figure 8), the key compound of this study,

Figure 8. 2-Vinylpyrimidine-derived H4 receptor ligand VUF14480 and the unreactive analogue VUF14481.

and its nonelectrophilic analogue VUF14481 (23), bound the hH4 receptor with similar (apparent) affinities in the upper nanomolar range. Both compounds acted as partial agonists and potencies in a GTPγS-binding assay were comparable to the respective affinities but time-dependence was not determined in either assay system. As expected, 22 reacted with GSH and cysteine ethyl ester to form covalent adducts, G



Perspective

Figure 9. Nonactivated terminal alkynes as cysteine traps. (A) Reaction of C-terminally propargylated ubiquitin 24 with the active site cysteine in DUBs. (B) Reactive (top) and nonreactive (bottom) analogues.

Figure 10. SNAr-based covalent ligands. (A) Classical mechanism of the SNAr reaction with cystein. (B) Selected examples of early SNAr-based ligands and reagents. Leaving groups are highlighted in red.

and can be found elsewhere.113 Yet, it is important to note that the reactivity of SNAr electrophiles can be readily adjusted by varying the electronic nature of substituents, and the leaving group. In early applications, SNAr warheads have been exploited in inhibitors like the 2-chloroquinoxaline L-764406 (25, Figure 10B),115 the 2-chlorobenzamide GW9662 (26)116 or the 2sulfonylpyridine GSK3787 (27),117 which target peroxisome proliferator-activated receptors (PPARs) by labeling different noncatalytic cysteines. 2-Sulfonyloxadiazoles, as exemplified by 28, have been used to covalently block the catalytic serine of AmpC β-lactamases118 or as maleimide replacements for the generation of serum-stable antibody conjugates.119 Other heteroarylsulfone-derived SNAr reagents such as MSBT (2(methanesulfonyl)benzothiazole, 29)120 have been employed as cysteine capping reagents and for protein conjugation.121 The SNAr reactivities of perfluoroarenes122,123 and dichloro-stetrazine124 have been further exploited for peptide stapling. Notably, the cysteine reactivity of highly activated SNAr electrophiles has been correlated with their skin sensitization potential.125 In 2011, a team of researchers around Kiplin Guy identified methylsulfonyl nitrobenzoates exemplified by MLS000389544

thiol(ate), displacing a leaving group from an electron-deficient aryl ring. Classically, SNAr reactions have been considered to proceed via a stepwise addition−elimination mechanism (aryne, radical and SN1-type mechanisms are not considered here) involving negatively charged intermediates termed Meisenheimer- or σ-complexes, which are stabilized by the electron-withdrawing group(s) (Figure 10A).112,113 More recent studies suggest that many SNAr-reactions rather proceed in a concerted manner via Meisenheimer-like transition states.114 The reactivity of (hetero)arenes in SNAr reactions increases with the electron deficiency of the aromatic system. Reactivity can be readily increased by adding electronwithdrawing (typically −M) substituents or heteroatoms, which are ideally positioned to the ortho- or para-position of the leaving group. In most cases, the rate of SNAr-reactions is determined by the attack of the nucleophile while elimination of the leaving group is faster.113 Therefore, the SNAr reactivity of aryl halides typically ranks F ≫ Cl ≈ Br > I, roughly correlating with the increasing polarization of the Cδ+−Xδ− bond. However, different rankings might be observed because the loss of the nucleofuge can also become rate limiting under certain conditions. A detailed discussion of all factors influencing SNAr reactions is beyond the scope of this article H



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(30) as low micromolar irreversible inhibitors of the thyroid hormone receptor (TR) β/steroid coactivator (SRC) 2 interaction by high throughput screening (HTS) of a library of 500000 compounds.126 These compounds selectively labeled Cys298 as determined by MS, the same cysteine that had previously been shown to react with Michael acceptors formed in situ from β-aminoketones.127 It is noteworthy that nucleophilic displacement of the methylsulfonyl residue was selective in the proximity of an activated ester moiety as an alternative reaction site. As one would expect, removal of the nitro group resulting in a less electrophilic analogue was detrimental to activity. However, the replacement of the methylsulfonyl group by chloride or fluoride also furnished poorly active compounds, which is more surprising because sulfones are typically less reactive than fluorides in SNAr reactions.113 This finding might reflect the involvement of the methylsulfonyl residue in reversible binding or point to the complex interplay of different factors on SNAr reactivity. In 2015, John Kuriyan and co-workers reported the sulfonyltetrazole 31 (Figure 11), the NBD-dye 32, and the

Figure 12. Electron-deficient (hetero)aryl probes used for evaluation of SNAr-based labeling in proteomes. (A) General structures. (B) Selected compounds preferably labeling cysteine or lysine.

promiscuous labeling reflecting the highly electron-deficient nature of this heteroarene. Intriguingly, however, 34a and 35a affected distinct sets of proteins, and peptide mapping showed that 34a preferentially labeled cysteine thiols while 35a had an unexpected preference for the lysine side chain. Although this behavior could not be fully rationalized, complementary experiments showed that 35a preferentially reacts with cysteine in solution, emphasizing that solution reactivity is not necessarily predictive of reactivity within protein binding sites. A recent study from Walter Fast and co-workers investigated 4-halopyridines as quiescent SNAr electrophiles.131 They found protonation of the pyridine nitrogen atom to be a critical factor for the reactivity of this compound class. To this end, the reaction with GSH at physiological pH was compared between 4-chloropyridine (36, Figure 13A) and its charged Nmethylated analogue (37, Figure 13B). Earlier data from Flanagan et al. had already demonstrated that 2-chloropyridine reacts only slowly with GSH at pH 7.4, with a rate constant in the same range as observed for N-methylacrylamide.12 Similarly, 36 showed very low reactivity comparable to that of ampicillin, styrene oxide, and acrylamide. In contrast, the Nmethylated analogue’s reactivity increased by more than 3 orders of magnitude, thus being in the same range as the one of iodoacetamide. Arguing with the classical SNAr mechanism, the accelerating effect would be attributed to better stabilization of the intermediate adduct being a neutral dihydropyridine instead of a negatively charged σ-complex in this case (Figure 13B). Against the initial expectation that 4-chloropyridine would be more reactive than the respective bromo and iododerivative, reaction rates with thiophenol increased in the order Br > I > Cl (4-fluoropyridines were not included in this study). The same ranking was obtained when the 4-halopyridines were assayed for their capability of inactivating dimethylarginine dimethylaminohydrolase (DDAH) 1 by reacting with the catalytic cysteine residue. For further investigations, a series of disubstituted 4-halopyridines with various substituents at ortho- or meta-position was prepared (general structure 38, Figure 13C). All of these compounds showed no or little reactivity in the GSH assay, even if an electron-withdrawing substituent was present in the C3-position. Profiling this structurally heterogeneous series against DDAH revealed no clear correlation between kinact/KI and the substitution pattern. However, in a set of 2-methylpyridines with an increasing number of fluorine substituents at the methyl group (pKa values ranging from 4.7 to 0.4), a correlation between the pKa

Figure 11. Covalent inhibitors antagonizing the interaction of ZAP-70 and Syk with ITAMs. Leaving groups are highlighted in red.

sulfonylpyrimidine carboxamide 33 as low molecular weight inhibitors antagonizing the interaction between the tandem Src homology (SH) 2 domains of the nonreceptor tyrosine kinases ZAP-70 and Syk and doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs).128,129 These compounds, which were identified by HTS, inhibited the SH2− ITAM interaction at low micromolar concentrations in a timedependent manner. X-ray and MS experiments confirmed the specific binding to single cysteines when no excess of the reagents was used. The X-ray structures of compounds 31 (PDB 4XZ0) and 32 (PDB 4XZ1) in complex with the ZAPtSH2 domain did not show any specific interactions with the protein and switching from TCEP- to DTT-containing buffer completely abolished inhibition by all three compounds. Therefore, it can be assumed that the high intrinsic reactivity of these electrophiles precludes application in cells or in vivo. A reactivity analysis of electron-deficient halogenated (hetero)aryl fragments in cellular proteomes was reported in 2014 by Eranthie Weerapana and co-workers.130 By using an alkyne-tagged series of chloronitrobenzoic amides (general structure 34, Figure 12A), they found that selectivity critically depends on the positioning of the nitro group. As expected, a meta relationship between the nitro moiety and chloride leaving group gave an unreactive compound, whereas compound 34a (Figure 12B) with the nitro group in the para-position was the most reactive. Dichloropyridines and -pyrimidines (general structure 35) also showed limited reactivity, while an analogous triazine (35a) caused more I



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Figure 13. 4-Halopyridines as quiescent SNAr electrophiles. (A) SNAr-reaction with 4-chloropyridine according to the classical mechanism. An anionic Meisenheimer intermediate is formed. (B) Analogous mechanism of the reaction with N-methyl-4-chloropyridine. A neutral dihydropyridine species is formed as the intermediate. (C) General structure of the investigated compounds. (D) Alkyne-tagged probes used for proteomic analysis.

of the protonated pyridine and reactivity was observed. Counterintuitively, more electron-deficient compounds reacted more slowly. This suggests that the DDAH enzyme, which possesses a proximal aspartate (Asp66), would stabilize the protonated pyridine, thereby enhancing its reactivity. However, no X-ray crystal structures or mutation studies were provided to fortify this hypothesis. Further experiments were performed with alkyne-tagged probes 39 and 40 (Figure 13D) in soluble Escherichia coli proteomes. Gel-based analysis showed that 39 only modifies a few proteins and the number even decreases when cell lysates are denatured prior to exposure. In contrast, N-methylated analogue 40 labeled much more proteins and the number increased substantially in denatured lysates. These observations suggest that the neutral 4-halopyridines require activation by the protein environment for becoming reactive, while the charged N-methyl-4-halopyridines can promiscuously label surface-exposed nucleophiles, which are more abundant in the denatured samples. In a recently published drug discovery campaign, Robin Fairhurst and co-workers from Novartis aimed to identify covalent inhibitors targeting a rare cysteine (Cys552)35 located in the hinge region of the fibroblast growth factor receptor (FGFR) 4 tyrosine kinase.132 A high throughput screen revealed compound 41 (Figure 14A) as a nanomolar inhibitor (IC50 = 32 nM) of wild-type FGFR4 sparing the FGFR4 C552A mutant. The compound did not hit the other FGFR family members (FGFR1−3), which are devoid of this cysteine. The high activity of this structurally simple molecule can be attributed to displacement of the 6-pyridyl chlorine atom by the cysteine’s thiol. Covalent modification at Cys552 was confirmed by MS and X-ray crystallography (Figure 14B). The compound possessed a 170-fold lower rate constant (kinact/KI = 3.0 × 104 M−1s−1) than an acrylamide-based inhibitor from the same study targeting Cys477 common to all FGFR family members. Although 41 selectively labeled Cys552 under the conditions used in MS and X-ray experiments, a 10-fold excess also modified Cys477 indicating at least some promiscuity. Interestingly, the latter modification seems to leave kinase activity unaffected, highlighting the fact that enzymatic or competition-based kinase assays are typically not suited for the identification of nonactive site binders. Although the authors recognized the potential of this compound for optimization in terms of reactivity, potency, and selectivity, the inhibitor was dropped in favor of a covalent-reversibly binding aldehyde (vide infra, section 2.12) owing to the short FGFR4 resynthesis half-life ( Br > Cl > F). αHaloacetamides are quite versatile and can be used to label various nucleophiles (typically Cys but also Lys, His, Tyr, activated Ser, and Thr etc.). Iodoacetamides, for example, are highly reactive and known as cysteine capping reagents.155 Iodoacetamide-derived clickable probes have recently been used to identify hyper-reactive cysteines by means of chemical proteomics.156 In contrast, α-bromoacetamides, which feature a slightly lower reactivity,12 are far less common. αChloroacetamides, which are the most frequently employed haloacetamides in covalent ligand design, possess further reduced reactivities. Their stability against GSH at pH 7.4 is in the same range as the one of α,β-unsaturated amides,12 and a linear chloroacetamide-derived alkyne probe showed only moderate levels of labeling in soluble mouse liver proteomes.157 In the case of aniline-derived α-haloacetamides, GSH reaction rates correlate with the Hammett parameter of the aryl substituent, i.e., electron-donating substituents decrease reactivity and vice versa.158 Reactivity of αhaloacetamides can be further decreased by adding steric bulk in proximity to the reaction site. Therefore, the reaction rates of α-bromopropionamides are lower than the ones of αchloroacetamides and α-chloropropionamides are even significantly less reactive than for example N-benzyl acrylamide (Figure 19C).12 In support of the low and specific reactivity of α-chloropropionamides, a recent ABPP-based study identified (S)-CW3554 (53, Figure 19D) as an irreversible inhibitor of the protein disulfide isomerase A1 (PDIA1) with good selectivity in HEK293 cells.159 Interestingly, the analogous (R)-α-chloropropionamide specifically labeled a distinct protein, the aldehyde dehydrogenase ALDH2, highlighting the role of the stereochemistry at the α-position of the warhead. However, it should be noted that both enzymes contain strongly nucleophilic active site cysteines and the potential of α-chloropropionamides in TCI design remains to be demonstrated.160,161 As an example of cysteine targeted alkyl halides devoid of an α-carbonyl group, 1,4-disubstituted 5-chloromethyl-1,2,3-triazoles have recently been identified by Alexander Adibekian and co-workers as inhibitors of the O6-methylguanine-DNAmethyltransferase (MGMT), a noncatalytic DNA repair protein that possesses an active site cysteine.162 The approach was inspired by N-heterocyclic carbene ligands, in which the reactive metal center is shielded by bulky substituents. Analogously, it was hypothesized that the reactivity of the 5chloromethyl substituent can be modulated sterically by substituents at the N1- and the C4-position of the triazole

ring. Ideally, these substituents would at the same time confer selectivity for a given target. A promiscuous clickable probe (54) was first synthesized and reacted with the proteome of MCF7 cancer cell lysates. MGMT was identified as one among the multiple labeled proteins. A small library of triazoles was synthesized by uncatalyzed or ruthenium-promoted azide− alkyne cycloaddition163 and analyzed in a competition assay against probe 54. In this screening, analogue AA-CW236 (55) was identified as a highly potent MGMT inhibitor (KI = 24 nM), albeit with slow inactivation kinetics (kinact = 0.03 min−1) (Figure 20). The compound did neither cross-react with any of

Figure 20. 5-Chloromethyl-1,2,3-triazoles as covalent MGMT inhibitors.

the targets of probe 54 at a concentration of 300 nM nor label the MGMT C145A mutant. Furthermore, MS-based proteomics indicated a clean selectivity profile. The compound, in combination with the DNA alkylation agent temozolomide, significantly increased the O6-alkylguanine levels in MCF7 cells compared to temozolomide alone, indicating a sensitization to the chemotherapeutic drug. Because of the low inactivation rates, however, it is not unlikely that the bulk of the observed effects are promoted by the potent reversible interaction. Overall, alkyl halides, especially α-haloacetamides, seem well suited as warheads for noncatalytic cysteine residues. Key to the modulation of their reactivity is the nature of the leaving group and the steric environment, especially the substituent at the α-position, which needs to be tolerated by the binding site. Replacing halogens with alternative and tunable leaving groups such as (activated) esters or sulfonates may offer further options to achieve optimally balanced reactivity. 2.9. Cysteine Alkylation by Epoxides and Other Three-Membered Heterocycles. Epoxides (oxiranes) have a long history as warheads to target different types of proteases7,164 and glycosidases.165,166 In contrast, other threemembered heterocycles such as aziridines167 and thiiranes168,169 have received less attention. The reactivity of these heterocycles arises from their ring strain. Epoxides react with nucleophiles via SN2 mechanisms and their reactivity should be influenced by similar factors as discussed in the previous section. In the absence of an acid catalyst, nucleophilic attack preferably occurs at the sterically less hindered position. Epoxides are used as CRGs in approved drugs, e.g., the epoxyketone-based proteasome inhibitor carfilzomib (56, Figure 21)170 or the antibiotic fosfomycin (57).171 Fosfomycin is a covalent modifier of bacterial UDP-Nacetylglucosamine enolpyruvyl transferase (MurA) targeting a nonessential cysteine (Cys115 in E. coli).172,173 Despite being structurally simple, this orally available drug can be safely administered in multigram doses, illustrating the tolerability of epoxide-derived compounds.171 Moreover, epoxides can behave unreactive even if a proximal cysteine is present in N



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Figure 21. Examples of epoxide-containing drugs.

Figure 22. α-Acyl epoxides as warheads for putatively covalent EGFR inhibitors.

MS-based GSH binding assay, epoxide 60a did not show any adduct formation after 1 h, while a 36% conversion was found for acrylamide 59. Irreversible binding of the epoxides was suggested by washout experiments, however, no further analysis of binding kinetics was performed and no confirmation of covalent target modification by MS or X-ray crystallography reported. Therefore, it cannot unambiguously be concluded if covalent binding took place. Notably, Daniel Rauh and coworkers observed only very weak activity of the analogous epoxide 63 on the cSRC S345C mutant sharing an equivalently positioned cysteine.177 In a similar approach from our own group, an epoxide moiety was used to invert the isoform selectivity profile of ruxolitinib (64, Figure 23), an approved JAK1/JAK2/tyrosine kinase (TYK) 2 inhibitor.178 In search of selective JAK3 inhibitors, molecular modeling proposed that replacing ruxolitinib’s (R)-3-cyclopentyl propanenitrile side chain by a propylene oxide moiety could address JAK3 Cys909, which is unique within the JAK family. We prepared a series of ruxolitinib-derived triazoles and epoxy analogue 65 appeared

the target’s binding site (e.g., trapoxin A in complex with HDAC 8174) and some approved drugs, e.g., ixabepilone (58),175 feature a bystander epoxide, suggesting that this structural motif cannot be considered a safety issue per se. Styrene oxide has recently been shown to possess a similarly low reactivity as acrylamide or ampicillin toward GSH.131 On a proteomic scale, terminal alkyl and spiro-epoxides have shown little or no protein labeling.157 However, despite these facts, epoxides are often regarded with skepticism by medicinal chemists, which probably traces back to the well-known toxic effects of certain epoxy-metabolites as well as their DNA alkylating potential.6 Despite the wealth of published literature, systematic studies on the factors determining the reactivity of epoxides and other three-membered heterocycles in a complex biological environment remain sparse. Another reason for the low abundance of epoxides in TCI discovery campaigns is their facile hydrolysis by epoxide hydrolases, and factors determining metabolic stability of this compound class are not fully understood. On the other hand, this liability might be used to confer kinetic selectivity in a similar manner as the fumaric acid esters discussed in section 2.1 do. Moreover, the small size and the possibility of being attacked at two proximal positions make epoxides quite versatile CRGs. Two of the few examples for the application of epoxides in the rational targeting of noncatalytic cysteine residues are highlighted below. A publication from 2010 by Alessio Lodola and co-workers describes a series of α-acyl epoxides as inhibitors of wild-type EGFR.176 Notably, other electrophiles such as activated phenyl esters, carbamates, nitriles, and heterocycles containing an electrophilic sulfur atom were also evaluated in this study. Starting from the covalent inhibitor PD168393 (59, Figure 22), replacement of the acrylamide moiety by three different epoxide-containing residues furnished highly potent compounds (IC50 = 0.5, 0.5, and 1.2 nM for 60a, 60b, and 61, respectively) with cellular activities in the low nanomolar range. In contrast, the β-naphthylamine-derived control compound 62 did not show any activity in the A431 cell assay, suggesting the absence of general cytotoxicity. In an LC-

Figure 23. Ruxolitinib-derived triazoles with a propylene oxide warhead as selective JAK3 inhibitors. O



Perspective

Figure 24. K-Ras G12D or G12C-targeted covalent inhibitors. Key compound 66a features an aziridine warhead.

to be a potent JAK3 inhibitor (IC50 = 35 nM) with a high selectivity (70−160-fold) in the JAK family. Because the other JAK family members possess a serine at the position equivalent to Cys909, the selectivity shift, which was not observed for nonreactive control compounds, suggests a covalent interaction with Cys909. However, covalent modification was not unambiguously proven since we discontinued this series in favor of tricyclic covalent-reversible JAK3 inhibitors.179 Aziridines have been used for targeting active site carboxylates in glycosidases,180 but reports on their application as CRGs in chemical biology and drug discovery remain sparse. Their avoidance can probably be traced back to the wellknown DNA-alkylating properties of aziridinium ions, which are famous as the active species generated from N-lost derived chemotherapeutics.181 Nevertheless, a recent example employing an aziridine as TCI warhead was provided by the groups of Kevan Shokat and John Burke.182 With the aim of covalently addressing the oncogenic K-Ras G12D mutant via the aspartate, different electrophilic head groups were attached to an optimized irreversible ligand of K-Ras G12C (general structure 66, Figure 24). Despite the reactivity of the representative aziridine 66a toward carboxylate groups in solution, negligible modification of K-Ras G12D was observed. In contrast, the oncogenic K-Ras G12C mutant was fully labeled. Binding was independently confirmed by differential scanning calorimetry and by hydrogen−deuterium exchange MS (HDX-MS). X-ray-crystallography and MS finally revealed the exact binding mode (Figure 25). It is worth mentioning that the attack of the thiol group occurred at the sterically more hindered α-carbon atom. 2.10. Cysteine Targeting by Nitroalkyl Groups As Masked Electrophiles. The covalent complex formation between 3-nitropropionate (3-NP, 67a, Figure 26) and Mycobacterium tuberculosis isocitrate lyase (ICL) was recently investigated by the group of Andrew Murkin. They found this compound to irreversibly inhibit ICL without the requirement for cofactors or prior redox-activation of the nitro group.183 After 20 h of incubation with 3-NP, only negligible enzyme activity could be recovered by jump dilution, an observation that was not influenced by the addition of DTT. However, the presence of glyoxylate increased reaction rates, suggesting cooperative binding. ESI-MS and X-ray crystallography (PDB 6C4A) revealed the formation of a thiohydroximate by reaction of the inhibitor with Cys191, an active site residue acting as a general base in ICL catalysis.184 An inverse solvent isotope effect indicated the thiolate form to be the nucleophilic species. The reaction could be rationalized by the CH-acidic nature of 3-NP (pKa = 9.0) generating significant amounts of the conjugate base propionate-3-nitronate (P3N, 67b) at neutral pH. A mechanism was proposed in which Glu285 protonates P3N at an oxygen atom to transform the

Figure 25. X-ray crystal structure of the K-Ras G12C mutant covalently bound to compound 66a (PDB 5V6V). Cys12 forms the covalent bond by opening the aziridine ring at the β-position. The indole NH and quinazoline N1-atom are involved in charge-assisted hydrogen bonds to the side chains of of Asp69 and Arg68, respectively, while the piperidine carboxamide oxygen interacts with the side chains of Tyr96 and Asp92 via water-bridged hydrogen bonds.

Figure 26. 3-Nitropropionate, propionate-3-nitronate, and the suggested reaction mechanism with ICL.

nucleophilic nitronate into an electrophilic nitronic acid. The latter could react with Cys191 to form the thiohydroximate by water elimination, probably via a second protonation step involving Arg228. The reaction mechanism was supported by kinetic analyses showing that preformed P3N (kinact/KI = 2.6 × 104 M−1 s−1) inactivated the enzyme approximately 100 times faster than 3-NP. Accordingly, nitroalkanes should be suitable as cysteine-targeted warheads for proteins hosting a proper combination of a sufficiently reactive cysteine and proximal acidic residues capable of generating the nitronic acid electrophile. Besides the aforementioned concerns on nitro groups in drug discovery, the generalizability of this concept P



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Figure 27. α-Cyanoacrylamide-derived covalent-reversible inhibitors.

Figure 28. Optimization of covalent-reversible FGFR4 inhibitors possessing an aldehyde warhead.

studies assessing target and off-target binding in cells have been published to date. For example, compound 69 (Figure 27), a low micromolar covalent-reversible inhibitor of glycosaminoglycan (GAG) sulfotransferases189 was tested for competition with a known alkynylated iodoacetamide probe.13,156 The compound neither competed with iodoacetamide-labeling in some isolated proteins containing hyper-reactive cysteines156 nor did it change the labeling profile in the cellular proteome of Neu7 astrocytes, indicating good specificity despite the highly reactive warhead.189 The compound further showed good stability in rodent and human liver microsomes and moderate in vivo PK properties after intravenous injection in rats. In another study, prolonged cellular target engagement has been demonstrated for α-cyanoacrylamide-derived covalent-reversible BTK inhibitors (e.g., compound 70, Figure 27) in isolated Ramos B cells and in PBMCs after oral dosing in rodents.11 Notably, further development of this compounds furnished PRN1008,190−192 a covalent-reversible oral BTK inhibitor of undisclosed structure currently being in phase II/ III clinical trials for the treatment of pemphigus ( N C T0 27 04 42 9 /N C T0 37 62 26 5 on https://www . clinicaltrials.gov) and immune thrombocytopenic purpura (NCT03395210). A dually activated Michael acceptor is also present in the approved catecholamine-O-methyltransferase (COMT) inhibitor entacapone (71).193 Although further studies are clearly required to determine the biological fate of α-cyanoacrylamides and analogous Michael acceptors, the currently available data suggest them to be promising warheads for in vivo use. Beyond α-cyanoacrylamides, benzylidene rhodanines have been reported as covalent-reversible Michael-type warheads.136 However, covalent binding does not seem to be a general feature of this compound class.194 In contrast, arylidene dinitriles are very reactive reversible Michael acceptors, but the reverse reaction seems to be competed by side reactions in this

remains to be demonstrated. To this end, chemoproteomic studies with nitroalkyl-derived probes would provide valuable information on the scope of this reactivity with respect to other target proteins. 2.11. Reversible Cysteine Addition to α-Cyanoacrylamides. Covalent reversible targeting of noncatalytic cysteines has recently emerged as a strategy to benefit from the advantages of irreversible TCIs (e.g., long residence times and the (potentially) increased selectivity) while avoiding liabilities like permanent off-target modification and the risk of idiosyncratic toxicity.28 The rational implementation of this concept in TCI design has been pioneered by the group of Jack Taunton, who introduced the β-substituted α-cyanoacrylamide functionality as a promising covalent-reversible warhead for cysteine.25 Although this electrophile can be considered highly reactive toward thiols, the increased α-CH acidity and thermodynamic destabilization of the β-thioether adduct185 favor the reverse reaction and thereby dissociation from offtargets or peptides, which are not capable of stabilizing the covalent complex by noncovalent interactions. Interestingly, a recent crystal structure of the kinase JAK3 in complex with the selective α-cyanoacrylamide-based inhibitor FM-409 (68, Figure 27) shows the coexistence of both the covalently bound and the unreacted form (PDB 5LWN), supporting the notion of covalent-reversible binding.179 By adding steric bulk to the β-position of the α-cyanoacrylamide11 or by replacing the amide group by an electron-withdrawing heteroarene,186 the intrinsic reactivity of this CRG and the dissociation rates of the derived covalent-reversible inhibitors can be modulated.185 More details are provided in a recent Perspective article40 and subsequent publications highlighting the merit of this warhead class in the design of EGFR-187 and highly isoform-specific JAK3 inhibitors.188 Global profiling of the covalent interactions of this chemotype in proteomes, however, is complicated by the reversible nature of protein modification and only a few Q



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significant off-rates (between 3.7 × 10−3 and 9.6 × 10−3 min−1) pinpoint the highly reversible nature of hemithioacetal formation.203 Consistently, no covalent adducts were found in mass-spectrometric experiments. Although no inhibitorbound X-ray crystal structure was determined, SAR and modeling provided insight into the binding interactions. Replacement of the pyridyl side chain by pyrimidine and pyrazine was tolerated with a slight loss in potency, while the use of five-membered N-heterocycles was detrimental to activity. Methylation of the urea NH furnished a completely inactive compound. Constraining and opening the piperidine ring was also tolerated, while increasing the size of this saturated ring system by one methylene group led to a substantial potency loss. The advanced lead compound 73d was further optimized to furnish clinical candidate FGF401 (74), a compound with a good oral bioavailability, PK, and safety profile, which is currently in phase I/II clinical studies (NCT02325739) for FGFR4 and β-klotho positive solid tumors and hepatocellular carcinoma.204 Although the final optimization steps as well as the outcome of clinical evaluation remain yet to be reported, the presented preclinical investigations emphasize that aldehydes, despite potential metabolic liabilities, can indeed be valuable warheads for the covalent-reversible targeting of cysteines beyond the active sites of hydrolases. 2.13. Reversible Cysteine Addition to Activated Nitriles. Besides aldehydes, nitriles have a long history as covalent-reversible warheads for protease inhibitors196,205 and are employed in approved drugs such as saxagliptin.206 As indicated by their prevalence in noncovalent inhibitors,207 nitriles behave relatively inert, and covalent adduct formation generally requires highly reactive active site nucleophiles along with precise positioning of the electrophilic carbon atom. However, the electrophilicity of the nitrile group can be increased by the attachment to electron-withdrawing moieties, e.g., heteroaryl rings, alkylamines,12,208 or acylated N,N′dialkylhydrazines.209 Pyridine and pyrimidinecarbonitriles show tunable reactivities toward glutathione, which are in a similar range as the ones of acrylamides (Figure 29A,B), suggesting these structural elements as suitable CRGs for targeting noncatalytic cysteines. Nitriles reversibly react with cysteines to form thioimidates via

case.188 Moreover alkynone-derived Michael acceptors (cf. section 2.3)79 and β-thiomethyl α-cyanovinylsulfones195 have recently been shown to react reversibly with thiols. According to the mechanism in Figure 5B, however, the reverse reaction is rather a thiol exchange, which might complicate the implementation in TCI design. Nevertheless, the current combinations of electron-withdrawing groups for generating covalent-reversible Michael acceptors with specifically tuned reactivities are by far not exhaustive, leaving room for further optimization. It will be interesting to see if alternative chemotypes such as vinyl sulfones or sulfonamides equipped with an additional electron-withdrawing group in the αposition can further extend the scope (e.g., toward lysines, vide infra) of this covalent-reversible chemistry. 2.12. Reversible Cysteine Addition to Aldehydes. Aldehydes and ketones are common warheads in proteolytic enzyme inhibitors.7,26,196 They react reversibly by forming tetrahedral hemi(thio)acetals and ketals mimicking the transition state of amide bond cleavage.197 These moieties, however, are far less commonly employed to address noncatalytic cysteine or lysine residues (some recent examples for lysine targeting by Schiff base formation can be found in refs 198−200). In a recent drug discovery program by Fairhurst and co-workers, the utility of aldehydes in covalent-reversible kinase targeting has been demonstrated.132,201 In the screening campaign, which also identified SNAr-based irreversible inhibitor 41 (see section 2.6), a set of closely related 2-formylquinoline amides exemplified by compound 72 (Figure 28, IC50 = 65 nM) was discovered. These compounds were potent FGFR4 inhibitors with good selectivity in the FGFR family but also against the FGFR4 C552A mutant and a panel of over 50 kinases. Replacement of the formyl warhead by a proton, hydroxymethyl, or acid group was detrimental to activity. In accordance with these data, modeling studies suggested covalent-reversible binding to the middle-hinge Cys552 by hemithioacetal formation. The quinoline nitrogen atom and the carboxamide proton were found to be of crucial importance because they stabilize a pseudotricyclic arrangement via intramolecular hydrogen bonding, thereby positioning the aldehyde for covalent interaction. Moreover, the electron-withdrawing ortho-quinoline scaffold further increases the aldehyde’s electrophilicity. Although certain questions concerning toxicity and metabolic stability of the aldehyde moiety remained,202 this compound class was selected for optimization due to its excellent potency and selectivity profile. Covalent-reversible inhibition was considered more promising than irreversible targeting due to the short resynthesis half-life (50% at 1 μM in a panel containing 375 kinases. The increased number of kinases captured in the panel could reflect the expression pattern of kinases in Jurkat cells. On the other hand, it might be an artifact from the nonphysiological conditions of in vitro screening panels or arise from noncovalent inhibition that cannot be captured in the cellular labeling experiment. Although some nonkinase off-targets were also modified, kinases accounted for the bulk of signal in MS experiments. Competition experiments with dasatinib subsequently validated the use of probe 112 for cellular selectivity profiling. As an application example of aryl fluorosulfates, these warheads were used by Jeffery Kelly and co-workers in AA



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Figure 47. Development of activated esters covalently targeting Lys779 in PI3Kδ.

Figure 48. N-Acyl-N-alkyl sulfonamides addressing surface-exposed lysine side chains. (A) Biotin-transferring probes. (B) Covalent ligand design and application example. The transferable residue is highlighted in red.

fluorogenic probes to image transthyretin in living cells and Caenorhabditis elegans.288 Cell permeable inhibitors 113a,b (Figure 46A) were designed to target the pKa-perturbed Lys15 side chain, which had previously been addressed with the analogous sulfonyl fluoride 114.289 Although the probes were well-suited for the envisaged imaging purposes, covalent modification was very slow and only 6% and 29% labeling were observed after 24 h with 113a and 113b, respectively. These findings comply with the known low reactivity of the fluorosulfate group, however, the slow reaction could also be indicative of a suboptimal alignment between the reactants. Despite the slow reaction, MS-based analysis confirmed Lys15 as the site of modification while little general reactivity toward the proteome could be detected. Unexpectedly, the ε-amino group was present as a free sulfamate without the ligand attached (Figure 46B) indicating that the substitution product had been hydrolyzed. Although the authors stress the probable catalytic effect of the target protein on hydrolysis, these results demonstrate the limited stability of such covalent linkages that will have to be evaluated on a case-by-case basis. In proteins where the lysine residue is required for catalytic activity, the εsulfamate would still be detrimental to activity while one equivalent of the unreactive ligand with a free phenolic hydroxy group would be released. Further data on the general stability of protein-linked disubstituted sulfates or sulfamates

under physiological conditions would be appreciated. Examples for the use of fluorosulfates in tyrosine and serine targeting can be found in the sections 5.1 and 6.1. 4.4. Lysine Acylation by Activated Esters. The potential of activated esters for targeting lysine side chains was recently emphasized by a team around Sebastien Campos at GSK.290 With the aim of generating a potent and selective covalent PI3Kδ inhibitor, they modified the clinical candidate GSK2292767 (115, Figure 47), a highly optimized and isoform-selective reversible PI3Kδ inhibitor. The sulfonamide moiety in 115 had been shown to interact with the conserved Lys779 ε-amino group, suggesting that replacement by an activated ester (116a−f) would be a promising strategy to address this residue. A variety of phenolic esters with electronwithdrawing or releasing substituents in the para position was prepared and tested for their ability to covalently inactivate PI3Kδ. Isoform selectivity and activity in human whole blood were assessed in parallel. Activity in the isolated kinase assay correlated roughly with the electronic properties of the substituents. The 4-nitro derivative 116b proved to be most potent (pIC50 = 9.2) but featured a slightly decreased isoform selectivity and limited stability in DMSO. In contrast, slightly less reactive esters 116a,c,d maintained low nanomolar potency, high isoform selectivity (ca. 2−3 orders of magnitude), and excellent activity (≈ 10 nM) in human AB



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approach, an irreversible Hsp90 inhibitor (120, Figure 48B) was generated by using the known ligand PU-H71 (119) as the transferable N-acyl substituent. A spacer was required to bridge the distance between the target lysine and the ligand binding site. Compound 120 featured a KI of 62 nM and modified Lys58 (kinact/KI = 2.9 × 104 M−1 s−1) as determined by MS. The compound showed durable engagement of Hsp90 in SKBR3 cells, as demonstrated by washout and competition experiments. Encouragingly, an inverted probe featuring a transferable fluorescein marker labeled only four additional proteins, all of those to a lesser extent than Hsp90, indicating good cellular selectivity. Although labeling of surface-exposed lysines, which are typically poor nucleophiles, is a special feature of this compound class, this behavior could not be fully rationalized. Despite the reasonable reaction kinetics and the potential of further tuning by suitable N-alkyl substituents, the CRG is relatively bulky and might be usable only for special applications. For targeting surface-exposed lysine side chains, this bulkiness might not be a major issue, in sharp contrast to such lysines located in spatially restricted binding sites. Finally, despite the good cellular selectivity demonstrated for compound 120, the intrinsic reactivity of this warhead class toward other nucleophiles remains yet to be assessed. Further examples of electrophiles that have been used to irreversibly target lysine residues include natural productderived spiro-epoxides addressing Lys100 in phosphoglycerate mutase 1 (PGAM1)293,294 and quinazolin-4(3H)-one hydroxamate esters modifying nonactive site lysines in the bacterial serine endoprotease DegS.295 Chemoselective methods for irreversible lysine modification, such as capture with diazonium terephthalates296 or ortho-phthalaldehyde-amine condensations,297 have recently emerged, but these can primarily be considered useful for bioconjugation applications rather than for specific covalent targeting in complex biological systems. The interested reader is referred to recent reviews.41−43,46 4.6. Condensation of Lysine with Aldehydes Forming Stabilized Schiff Bases. Schiff bases (imines), which are formed by the reversible condensation of an amine with an aldehyde or ketone, are quite common in biological systems. Prominent examples include pyridoxal phosphate (PLP)dependent enzymes or the light sensitive GPCR rhodopsin, both binding their cofactors via Schiff base formation.298 In medicinal chemistry, however, ketones and especially aldehydes are relatively unpopular. One of the main reasons for their avoidance is metabolic liability, as these chemical functionalities are prone to reduction by aldo-keto reductases and short-chain dehydrogenases/reductases or, in the case of aldehydes, oxidation to the corresponding acids by aldehyde dehydrogenases.299 However, as highlighted in section 2.12, aldehydes can also be surprisingly stable. A recent study showed that metabolically labile aldehydes can be replaced by ketones whose metabolic stability, solubility and, to a lesser extent, the ketone−imine equilibrium can be tuned by the attachment of suitable residues.199 This strategy furnished novel inhibitors of the cystine−glutamate antiporter, but covalent target engagement was not proven experimentally. Although Schiff base formation is highly reversible, massspectrometric analysis of the covalent complex is possible after reducing the Schiff base with borohydride to obtain a stable secondary amine. For example, this technique has been used to demonstrate the covalent binding of the umbelliferon-derived ligand 4μ8C with two distinct lysine residues (Lys509 and Lys907) in the inositol-requiring enzyme (IRE) 1.200

whole blood. Kinetic analysis revealed that the rate of covalent inactivation was similar for all compounds (kinact = 5.5−7.5 × 10−3 s−1) and did not correlate with the leaving group properties of the phenolate, while KI spanned a range from 40 nM (compound 116b) to 7.8 μM (compound 116f). Mass spectrometry and X-ray crystallography confirmed the exclusive labeling of Lys779 by 4-fluorophenyl ester 116d, and as expected, the nonactivated methyl ester 116g bound only reversibly to the enzyme. Compound 116d was stable against hydrolysis and reaction with N-α-Boc-lysine at pH 7.4. Interestingly, 116d showed a selectivity window in which PI3Kδ was covalently inhibited while PI3Kα and β engagement was reversible, indicating that covalent bond formation is critically dependent on reversible interactions. The compound showed high selectivity in a panel of 140 protein and 10 lipid kinases and the analogous azide-labeled probe 117 revealed a relatively clean chemoproteomic profile in Ramos cells. Washout experiments in CD4+ T-cells showed sustained suppression of IFNγ release for 48 h upon stimulation with αCD3, indicating prolonged cellular target engagement. However, no data on the metabolic stability of these compounds were reported. It is not unlikely that such activated esters are rapidly inactivated by esterases, which may erode activity in vivo or, following the rationale of Cravatt’s fumarates (section 2.1), improve kinetic selectivity. Finally, it should be noted that activated carbamates that have been frequently employed to target catalytic serines in drug discovery and crop protection291 might be used in a similar manner as these activated esters. 4.5. Acylation of Surface-Exposed Lysines by N-AcylN-alkyl Sulfonamides. Another structure class that has recently been used to target lysines are N-acyl-N-alkyl sulfonamides. In early 2018, Itaru Hamachi and co-workers reported on the optimization of these moieties to achieve rapid ligand-directed labeling of surface-exposed lysine residues.292 Activation of the acyl functionality was achieved by attaching electron-withdrawing groups to the sulfonamide nitrogen atom via a methylene spacer (Figure 48A). In an initial experiment, FKBP12 was used as a model protein. SLF, a known FKBP12 ligand (reported KD = 20 nM), was attached via a spacer to the sulfonamide sulfur atom while biotin was employed as the transferable N-acyl moiety and a cyanomethyl residue was utilized as the activating group (compound 118, KI = 210 nM). Incubation of this probe with FKBP12 predominantly biotinylated a single surface-exposed lysine, Lys44, located near the ligand binding site. Reasonable rates (kinact/KI = 2.9 × 104 M−1 s−1, kinact = 6.1 × 10−3 s−1) were achieved with this compound, while the replacement of the nitrile group by 4nitrophenyl or 2,4-dinitrophenyl slowed down reaction kinetics substantially. Hydrolysis of compound 118 in aqueous buffer at pH 7.2 was slow, with a half-life of 43 h. The probe selectively labeled FKBP12 with biotin in HeLa cell lysates when the probe and the protein were present at an equimolar concentration (both 1 μM). However, increasing the probe concentration or competition with the high-affinity ligand rapamycin suppressed FKBP12 biotinylation and caused unspecific labeling. This underlines again the importance of potent and selective reversible binding for target specificity, especially when highly reactive electrophiles are employed. Remarkably, similar experiments were conducted with an analogous trimethoprim-derived probe (not shown) specifically labeling Lys32 in E. coli dihydrofolate reductase (kinact/KI = 9.3 × 103 M−1s−1, kinact = 1.3 × 10−2 s−1). In an inverse AC



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showed an EC50 of 75 nM in the MCL-1-dependent cell line MOLP-8. Experiments with other MCL-1-dependent cell lines confirmed cellular activity, while MCL-1 independent cell lines remained unaffected. Mass spectrometry confirmed ca. 50% covalent labeling with 122a after 1 h, while acetophenone 122c reacted slightly slower. However, 50% conversion was not exceeded with an excess of 122a even after prolonged exposure, suggesting that an equilibrium stage had been reached. Reversibility was shown by SPR experiments and ligand dissociation fitted a two-state binding model (kd1 = 0.14 s−1 and 0.13 s−1; kd2 = 0.018 s−1 and 0.010 s−1 for 122a and 122b, respectively). It is worth mentioning that the warhead alone did not generate covalent complexes. Although no X-ray data was included, the modification of Lys234 was supported by binding data of covalent and noncovalent analogues to the K234A mutant. As demonstrated by this study, 2-carbonylphenylboronic acids can be used as cell-penetrable warheads that enable the reversible engagement of surface-exposed lysine side chains in the proximity of small molecule binding sites. It will be interesting to see whether addressing buried lysines, e.g., in kinases, will substantially decrease off-rates to enable complete labeling. Finally, the stability of this structure class under physiological conditions, especially with respect to the mentioned susceptibility of aldehydes and ketones toward redox biotransformation, will have to be assessed.

The stabilization of Schiff bases by coordination of the nitrogen lone pair to a boronic acid has been pursued by Pedro Gois and co-workers as a strategy for reversible protein modification.300 In drug discovery, boronic acids are wellknown as warheads for catalytic serine or threonine residues, as exemplified by the approved proteasome inhibitor bortezomib.301 In this study, however, a boronic acid was introduced to the ortho-position of a benzaldehyde moiety in order to obtain an iminoboronate upon Schiff base formation. The latter is stabilized by an intramolecular dative bond between the nucleophilic nitrogen lone pair and the electrophilic boron center (Figure 49). In contrast to many of the above-

Figure 49. 2-Formylbenzenboronic acid reversibly forming stabilized Schiff bases with amines.

mentioned methods, iminoboronate formation did not require pKa perturbation and proceeded readily between the model substrate 2-formylbenzeneboronic acid (121) and n-butyl amine at pH 6−9. The reaction was reversed upon the addition of GSH, fructose, and dopamine, suggesting reversibility under physiological conditions, which was later confirmed by Gao and co-workers.302,303 Proteins such as cytochrome c, ribonuclease a, and myoglobin fully converted with excess 121 within 5 min. Applications of this chemistry include the labeling of Grampositive bacteria by targeting amine-presenting lipids302 and more recently the inhibition of the induced myeloid leukemia cell differentiation protein (MCL-1), a protein−protein interaction target which is a key survival factor for various human cancers.304 In the latter study, a group of researchers around Qibin Su and Neil Grimster attached a 2-carbonylphenylboronic acid warhead to a known indole-acid based MCL-1 inhibitor to reversibly target the surface-exposed Lys234 side chain. Formylphenylboronic acids 122a,b (Figure 50) as well as the analogous acetophenone 122c featured low nanomolar IC50 values in a TR-FRET assay, while derivatives lacking either the boronic acid or the ortho-carbonyl group were substantially less active. The improved potency translated into superior cellular activity, and the best compound (122b)

5. TARGETING THE TYROSINE SIDE CHAIN Neutral tyrosine has a relatively low intrinsic nucleophilicity when compared to cysteine or unprotonated lysine. Therefore, the selective modification of nonactivated tyrosine can be challenging. However, its phenolic hydroxy group possesses a slightly higher acidity compared to the protonated lysine side chain (pKa ≈ 9.7 vs 10.5),305 and tyrosine residues in proteins can have a significantly lowered pKa, favoring the formation of the highly nucleophilic phenoxy anion.306 Increased reactivity of tyrosine toward sulfonyl fluorides, for example, correlated with the proximity of a basic amino acid residue.307 The phenoxy anion is a relatively hard nucleophile and favors the reaction with hard Lewis acids. Besides the nucleophilic properties of the phenolic hydroxy group, the electron-rich nature of the phenyl ring has been exploited for tyrosineselective bioconjugation reactions, e.g., with in situ generated Mannich reagents,308 diazonium salts,309 or via ene-like reactions with triazoline diones.310,311 5.1. Tyrosine Targeting with Sulfur (VI) Fluorides. Because the inherent reactivity of tyrosine and lysine toward sulfur (VI) fluorides has already been discussed extensively in section 4.2, only application examples are provided in this section. Sulfonyl fluoride warheads have recently been used by Lyn Jones and colleagues from Pfizer to address the mRNAdecapping scavenger enzyme DcpS.307 In a structure-based design approach, they modified known diaminoquinazolinederived DcpS inhibitors to covalently address two proximal tyrosines, Tyr113 and 143, or the neighboring Lys142. The generated inhibitors featured low picomolar IC50 values in an ELISA-based DcpS activity assay (no binding kinetics were provided) and were approximately 2 orders of magnitude more potent than the reversible parent compound D153249 (123, Figure 51). X-ray and MS experiments showed that the orthoand meta-substituted inhibitors 124a,b selectively modify Tyr113, while compound 124c with the CRG in the paraposition reacts exclusively with Tyr143 (Figure 52). However, all of these inhibitors left Lys142 and His139 untouched,

Figure 50. 2-Formylbenzenboronic acids and analogous acetophenones targeting MCL-1 via Schiff base formation. AD



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Figure 51. Design of tyrosine-targeted sulfonyl fluorides as DcpS inhibitors.

indicating that precise positioning of the warhead in conjunction with the appropriate intrinsic reactivity of the sulfonyl fluoride group for tyrosine confers specificity. It is worth mentioning that an analogous inhibitor with a silent click-tag revealed some off-targets that were not further disclosed. In a very recent study, Nathanael Gray and co-workers made use of a sulfonyl fluoride warhead to generate SRPKIN-1 (126, Figure 53), the first targeted kinase ligand covalently addressing a tyrosine residue.312 While screening an internal compound library in the cellular KiNativ profiling assay,313 the approved anaplastic lymphoma kinase (ALK) inhibitor alectinib (125)314 was found to exhibit strong inhibitory activity toward the SR-protein kinase SRPK1 (IC50 = 11 nM on isolated SRPK1). A cocrystal structure gave insight into the binding mode of alectinib in complex with SRPK1, suggesting that replacement of the 4-morpholinopiperidine moiety by a meta-substituted phenyl ring bearing a sulfonyl fluoride warhead might enable covalent targeting of a unique tyrosine (Tyr227) adjacent to the solvent-exposed front region of the ATP binding site. Installation of this headgroup conserved

Figure 53. Design of sulfonyl fluoride SRPKIN-1, the first tyrosinetargeted covalent kinase inhibitor.

most of the SRPK1 activity (IC50 = 36 nM) while rendering the compound a less potent ALK inhibitor (IC50 = 195 nM). No kinetic data was provided, thus the contribution of the covalent interaction to the observed potency remains unclear. Cellular KiNativ profiling demonstrated selectivity for SRPK1 and SRPK2, and washout experiments supported the anticipated covalent binding mode. Labeling of Tyr227 was subsequently confirmed by MS but no X-ray crystal structure was published. Notably, the compound suppressed neovascularization in a choroidal neovascularization model (CNV) after intravitreal injection in mice. The less reactive aryl fluorosulfates have been suggested as privileged tyrosine-targeted electrophiles as they preferably react with phenolic hydroxy groups when compared to free alkanols, amines, thiols, guanidines, and heterocyclic NH groups.315 Interesting work highlighting the special properties

Figure 52. Distinct binding modes of 124a−c in the respective X-ray crystal structures in complex with DcpS. The ligand is completely embedded in the protein environment, and the 2,4-diaminquinazoline core adopts a similar orientation in all structures. Conserved interactions include two hydrogen bonds of the quinazoline 2-amino group to the carboxylate of Glu185 and the backbone carbonyl of Pro204 as well as a hydrogen bond between the quinazoline 4-amino group and the Asp205 side chain. The second proton of the quinazoline 4-amine forms an intramolecular H-bond to the ether linker. The (protonated) quinazoline N1-atom is also hydrogen-bonded to Glu185. (A) ortho-Substituted derivative 124a covalently bound to Tyr113 (PDB 4QDE). The phenyl ring points to the “bottom” of the binding pocket. Additional hydrogen bonds are formed to the side chains of Lys142 and Tyr273. (B) meta-Substituted derivative 124b covalently bound to Tyr113 (PDB 4QEB). Covalent attachment is enabled by an “upward” orientation of the phenyl ring, giving rise to an additional hydrogen bond between the sulfonyl group and the Tyr143 side chain. (C) para-Substituted derivative 124c covalently bound to Tyr143 (PDB 4QDV). The overall orientation resembles that of 124a, but Tyr143 is labeled instead of Tyr113. No hydrogen bonds with Lys142, Tyr273 and His139 are observed in (B) and (C), and the latter two residues were omitted for clarity. No covalent modification of the proximal nucleophiles Lys142 and His139 was observed in any of the experiments. AE



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Figure 54. Aryl fluorosulfate probes targeting CRABP2 used in chemical proteomics studies.

of aryl fluorosulfate warheads was published in 2016 by Kelly, Sharpless, Wilson, and colleagues. They showed that structurally simple aryl fluorosulfates can react specifically with tyrosine side chains in certain intracellular lipid binding proteins.268 By using a clickable PEGylated phenyl fluorosulfate (127, Figure 54), it was demonstrated that this compound class selectively (and slowly) labels relatively few proteins as contrasted by an analogous sulfonyl fluoride. The cellular retinoic acid binding protein (CRABP) 2 was identified as the key target of 127 in HeLa cell proteomes. Rudimentary optimization furnished biphenyl-derived analogue 128 and fluorescent probe 129, enabling higher (but still moderate) CRABP2 labeling rates (kinact = 0.106 min−1 for 129). Structural comparison of CRABP2 with other lipid-binding proteins targeted by 127 suggested a spatially conserved Arg− Arg−Tyr carboxylic acid binding motif to be the key for covalent modification. Intriguingly, mutational depletion of either of the arginine residues impaired labeling. Studies at variable pH values indicated that the arginine side chain lowers the pKa of the tyrosine phenol moiety, and a simultaneous stabilization of the fluoride leaving group is likely. Cellpermeable probe 128 showed a very low background proteome labeling in HEK293T cells. An X-ray crystal structure in complex with CRABP2 (Figure 55) confirmed covalent bond formation with Tyr134. In contrast to the above-mentioned

Figure 56. Alkyne-tagged aryl fluorosulfate-based probes used in an “inverse drug discovery” approach.

modification of transthyretin−Lys15 (section 4.3), the covalent linkage was stable. Finally, 128 was shown to inhibit CRABP-mediated delivery of retinoic acid to the nuclear retinoic acid receptor α in MCF-7 breast cancer cells. Notably, later studies indicated that aryl fluorosulfates have a low susceptibility to hepatic metabolism suggesting that such probes might also be applied in vivo.316 Subsequently, the same groups reported on a strategy they denominated ″inverse drug discovery″, where the highly specific nature of latent fluorosulfate electrophiles was utilized to identify proteins with hotspots activating the latter for covalent lysine or tyrosine binding.269 By capitalizing on three distinct alkyne-labeled probes of intermediate complexity (130−132, Figure 56) in conjunction with unlabeled competitors, quantitative proteomics were employed to identify proteins that are efficiently labeled. In this approach, only a very low number of proteins was retrieved. This can be attributed to the very low reactivity of aryl fluorosulfates, however, the susceptibility of the reaction products to hydrolysis and elimination (vide infra), or the reaction with thiols to form unstable thiosulfate-S-esters (compare section 4.2) might also hamper product detection. Labeling at a specific site was validated for 11 targets by MS, mutagenesis, and X-ray crystallography using the recombinant proteins. As expected, covalent modification was only observed at tyrosine and lysine residues. Interestingly, probe 131 reacted with a largely different set of proteins compared to probes 130 and 132. Although no further optimization was performed, this study identified interesting targets that might be covalently addressed by sulfur (VI) chemistry. It should not remain unmentioned that the labeling sites could be reliably predicted by covalent docking which might be interesting for future drug discovery efforts.

Figure 55. X-ray crystal structure of the fluorosulfate-based ligand 128 covalently bound to Tyr134 in CRABP2 (PDB 5HZQ). The ligand is deeply buried in the binding site, and the sulfate group is engaged in a direct hydrogen bond to the Arg132 side chain and water-mediated hydrogen bonds to the Arg111 side chain. The PEGlinker is not resolved and a second, slightly deviating conformation of the ligand and the Tyr134 side chain was omitted for clarity. AF



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5.2. Tyrosine Targeting by SNAr Reactions. LAS17 (133, Figure 57), an inhibitor with a 4,6-dichloro-1,3,5-triazine

emphasizing that intrinsic reactivity is not the only determinant for promiscuity and that the specificity and kinetics of reversible binding play a critical role for the selectivity of covalent inhibitors. In this context, further investigation of the specificity in cellular proteomes upon prolonged exposure to higher compound concentrations as well as a systematic evaluation of the minimal reactivity requirements for modifying Tyr108 with SNAr-type warheads would be very informative.

6. TARGETING NONCATALYTIC SERINE AND THREONINE RESIDUES Serine and threonine are abundant as the key catalytic residues in the active sites of proteases and other hydrolase enzymes. At these locations, the side chain hydroxy groups are activated by neighboring residues, e.g., in catalytic triads, dramatically increasing their nucleophilicity.320 A plethora of covalent inhibitors has been developed for these enzymes, and as mentioned already, many of the CRGs described in this Perspective had initially been employed to target the active sites of hydrolases.7,321 A comprehensive discussion of covalent inhibitors of this enzyme class, however, is far beyond the scope of this article. In contrast, noncatalytic serine and threonine residues are usually poorly acidic and therefore hardly reactive. Targeting these amino acids is still a significant challenge in bioconjugation chemistry. There is a very prominent example for the covalent modification of a noncatalytic serine by a drug, namely the acetylation of Ser530 in cyclooxygenases by aspirin.322 Nevertheless, reports on compounds targeting noncatalytic serine and threonine residues in a proximitydriven manner remain very rare. In general, hard Lewis acids, such as sulfur (VI) fluorides or oxophilic phosphorus (V) compounds but also boron-based reagents, may be suited best for addressing such hydroxy groups. 6.1. Targeting Noncatalytic Serine by Fluorosulfates. In a follow-up of the study on diaminoquinazolines targeting tyrosine residues in DcpS (see section 5.1, Figure 51), Lyn Jones and co-workers sought to reduce off-target labeling by replacing the sulfonyl fluoride warheads by less reactive fluorosulfates.316 Key compound FS-p1 (134a, Figure 58) featured increased stability toward hydrolysis and adequate membrane permeability. The turnover of this inhibitor in human liver microsomes was even lower as for the unsubstituted parent compound DAQ1 (134b), albeit at the expense of potency (IC50 = 3.2 nM) compared to the corresponding sulfonyl fluoride 124c (IC50 = 95% in less than 10 min) and only marginal cleavage was observed within 24 h. Finally, reintroduction of the piperidine nitrogen atom furnished 141, another promising compound from this series. Interestingly, removing the (piperidin-4-yl)methyl substituent from the sulfonamide nitrogen atom yielded a compound which did not effectuate any labeling pinpointing the critical role of reversible binding and accurate warhead positioning. Compound 140d possessed low reactivity toward the side chains of protected lysine and serine but cross-reacted with cysteine. However, no labeling of the PDE6δ E88A mutant was observed. As indicated by MS and fluorescence quenching studies, ARL proteins were unable to release the covalent ligands from the protein. Covalent attachment to Glu88 was further demonstrated by an X-ray crystal structure (Figure 60). It is worth noting that the ligand

8. TARGETING THE HISTIDINE SIDE CHAIN Although the histidine imidazole group is frequently mentioned as a potential nucleophile for covalent targeting, only limited rational efforts have been described to address this amino acid. Histidine residues react with sulfonyl fluorides to form readily hydrolyzable sulfonylimidazoles as shown by the covalent modification of His130 in the active site of Salmonella typhimurium ribose-phosphate diphosphokinase by 5′-FSBA (102, Figure 42).336 Moreover, histidine residues can undergo aza-Michael addition reactions with α,β-unsaturated ketones or aldehydes. For example, histidine residues were shown to react with 4-hydroxynonenal337 or prostaglandin J2,338 and more recently, a histidine of the vitamin D receptor has been covalently addressed with vitamin D-derived enones.339 8.1. Alkylation of Histidine by Spiro-epoxides. One of the prominent compounds addressing the histidine side chain is the natural product fumagillin (143, Figure 61) and the derived clinical candidate beloranib (142), which bind to one of the active site histidines in methionine aminopeptidase (MetAP) 2 via opening of a spiro-epoxide (Figure 61B).340 To improve the poor pharmacokinetics of this substance class, Aubry Miller and colleagues designed spiro-epoxytriazoles as drug-like fumagillin analogues (Figure 61C, general structures 144 and 145).341 Several potent inhibitors of human MetAP2 were generated with key compound 145a (Figure 61D), featuring an IC50 value of 220 nM while its stereoisomers were inactive. As expected, enzyme inhibition was time-dependent and correlated well with cellular activities in human umbilical vein endothelial (HUVE) and HT1080 cells. Although the keto and benzoyl derivatives possessed poor stability in plasma and mouse liver microsomes, carbamate analogues were significantly more stable, even when compared with the former drug candidate beloranib. The labeling of His231 was confirmed by X-ray crystallography (Figure 62), while solvent-exposed nucleophiles (such as Cys290 and Lys427) remained untouched. These results indicate that histidine residues can be addressed specifically with relatively weak

Figure 60. X-ray crystal structure of compound 140d covalently bound to Glu88 of PDE6δ (PDB 5NAL). The ligand is predominantly bound in the less stable O-acylated form and deeply buried in the binding site. Hydrogen bonds are formed by both oxygen atoms of the first sulfonyl group to the side chains of Arg61 and Gln78. An additional hydrogen bond is established between the second sulfonyl group and the side chain of Tyr149 (omitted for clarity).

appears to be predominantly bound in the less stable form (compare structure 138). However, the electron density of the covalent tether is slightly less pronounced as for the rest of the ligand which might be attributed to partial conversion to the more stable β-enol ester product. Investigation of selectivity in cell lysates using a cellular thermal shift assay (CETSA)332,333 revealed only three off-targets (GDPGP1, PSMG3, PTGES2) along with PDE6δ. AI



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Figure 61. Spiro-epoxides as histidine-targeted covalent inhibitors of hMetAP2. (A) Former drug candidate beloranib. (B) Reaction of the natural product fumagillin with His231 in hMetAP2. (C) Simplified fumagillin-derived structures. (D) hMetAP2 inhibitor 145a with improved PK properties.

addition to the α-cyanoenone moiety was confirmed by Xray crystallography (Figure 63B), while no concomitant modification of exposed cysteines was observed. In accordance with the X-ray crystal structure showing a hydrogen bond between the backbone of Ser326 and the nitrile group, the removal of the α-cyano substituent in series 146 was detrimental to activity, while saturation of the double bond precluding covalent modification led to a moderate 16-fold loss in inhibitory potency. The latter finding suggests that the hydrogen bond to the nitrile group not only increases the inhibitors electrophilicity but is also crucial as a noncovalent key recognition element. Reversibility was demonstrated by wash out and jump dilution experiments. Consistent with transient covalent binding, no adducts were observed in mass spectrometric experiments. Figure 62. X-ray crystal structure of the covalent complex between hMetAP2 and compound 145a (PDB 5CLS). His231 is covalently attached to the methylene group formed from the terminal carbon atom of the epoxide ring. The ensuing hydroxy group is linked to Asp251 and His382 by water-mediated hydrogen bonds. A direct hydrogen bond between the carbamate’s carbonyl group and the Asn329 backbone NH, and further water-mediated hydrogen bonds additionally anchor the ligand in the binding site.

9. TARGETING METHIONINE SIDE CHAINS Methionine is among the rarest amino acids in vertebrates and due to its high lipophilicity, typically buried within proteins. As mentioned before, the nucleophilicity of the methionine side chain is only moderate, however, its sulfur atom can be readily oxidized. Protocols for methionine labeling typically rely on highly reactive electrophiles at low pH.343 9.1. Redox-Activated Labeling of Methionine by Oxaziridines. An interesting new method for the selective labeling of methionine residues in proteins and cell lysates has recently been identified by the groups of Dean Toste and Christopher Chang.343 They aimed to exploit the distinct redox activity of methionine for redox-activated chemical tagging (ReACT) under physiological conditions. A screening identified oxaziridines as suitable strain-driven sulfur imidation reagents.344 Incorporating the oxaziridine nitrogen atom into a weakly electron-withdrawing urea moiety (exemplified by compound 147a, Figure 64A) avoided problems with concomitant sulfoxide formation observed for analogous carbamates (e.g., compound 147b). Sulfur imidation proceeds via a nucleophilic attack of the sulfur lone pair at the oxaziridine nitrogen atom followed by ring opening and release of benzylic aldehydes or ketones to furnish the S-imidation product (Figure 64B/C). The S-oxidation byproduct is formed

electrophiles, making them suitable targets for covalent inhibitor design. However, as for other moderate nucleophiles, the ligandability may largely depend on the precise positioning of the CRG and the target residues distinct nucleophilicity and thus on the surrounding protein environment. 8.2. Reversible Addition of Histidine to α-Cyanoenones. A covalent-reversible approach was recently pursued by Clarissa Jakob and co-workers from AbbVie to address a histidine in wild-type isocitrate dehydrogenase (IDH)1.342 A high-throughput screen identified inhibitor 146a (Figure 63A) with an IC50 value of 410 nM. Optimization furnished the most potent carboxy-substituted analogue 146b (IC50 = 41 nM) and the slightly less active but cell permeable key compound 146c (IC50 = 110 nM). The latter inhibitor decreased reductive glutaminolysis in A498 cells in a dose-dependent manner, suggesting cellular target occupancy. Covalent modification of His315 in the NADPH binding pocket via aza-Michael AJ



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Figure 63. α-Cyanoenones as histidine-targeted covalent-reversible IDH1 inhibitors. (A) Hit compound 146a and optimized derivatives. (B) X-ray crystal structure of 146c bound to IDH1 (PDB 6BL1). His315 is covalently attached to the β-position of the enone precursor. A key hydrogen bond is formed between the α-cyano moiety and the backbone NH of Se326. The enone keto group is hydrogen-bonded to the Lys374 side chain, while the diarylamino group forms a charge-assisted hydrogen bond to the carboxylate of Asp375.

Figure 64. Methionine-targeted oxaziridines. (A) Urea and carbamate-derived analogues. (B) Reaction with methionine via S-imidation or concomitant S-oxidation. Conditions: (1) D2O/CD3OD = 1:1, 2.5 min or (2) D2O/CD3OD = 95:5, 20 min. (C) Reaction mechanism of covalent methionine modification.

in an analogous manner by attack at the oxygen atom. Labeling was selective for methionine over other amino acids (free cysteine and selenocysteine were oxidized to their cystine forms), and the products were relatively stable even in the presence of acid, base, and reducing agents. Alkynylated probe 147c at a 1 mM concentration incubated for 10 min with HeLa cell lysates labeled 235 methionine residues, and only a single lysine residue. Different concentrations of 147c showed a dosedependent increase in the number of labeled residues allowing the identification of hyper-reactive methionines. The suitability of the approach for selective protein labeling and the construction of antibody−drug conjugates was also demonstrated. Despite their chemoselectivity, the presented oxaziridine-based probes are still highly reactive and convert methionine at rates similar to CuAAC reactions. It remains to be seen if steric shielding and/or tuning of electronic properties could furnish more specific warheads for chemical probe or drug design and if the bulkiness of such CRGs will limit their use with respect to the spatial constrains of many binding pockets. The underlying principle, however, is very

promising and might stimulate research on similar approaches for specifically addressing methionine side chains.

10. TARGETING OTHER AMINO ACIDS It is not surprising that relatively few methods have been described to selectively address weak nucleophiles such as arginine, asparagine, glutamine, or tryptophan. For example, the bidentate nature of arginine’s guanidinium group has been exploited for reactions with glyoxal-derived reagents,345 forming comparably stable cyclic products while the reaction with lysine or cysteine is highly reversible. However, the latter compound class is unlikely to find broad application in drug discovery due to metabolic liability and potential toxicity issues. A recent method has been described for metal-free tryptophan-selective bioconjugation in proteins.346 Although being conceptually interesting, this method requires an organoradical reagent and sodium nitrite as an additive precluding applications in TCI design. Addressing phenylalanine or amino acids with nonactivated aliphatic side chains is even more challenging. Although catalysis-based approaches AK



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Table 2. Overview of the Warhead Classes Discussed in This Article

AL



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Table 2. continued

a

Excluding catalytic nucleophiles. Data based on the studies discussed in the respective chapters. bL: Protein/peptide/amino acid (labeling or reactivity assay);. P: Protein (activity, binding affinity, kinact/KI). Y: Cell lysate (chemical proteomics study). C: Intact cell (functional assay or chemical proteomics study). I: In vivo (mammals or human). Data based on the reports discussed in the respective sections and additional searches in PubMed and the DrugBank.146 cApplication restricted to the catalytic cysteine of DUBs and related cysteine proteases so far. dModified in solution but aziridines were unable to address Asp12 in the K-Ras G12D mutant. eSuggested by experiments with n-butylamine as a model nucleophile. fForms unstable S-ester reaction products with cysteine. gHemithioacetal formation with Cys is likely but rapidly reversible. AM



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targeting needs to be combined with good metabolic stability and a highly tunable reactivity, especially if some additional steric bulk is not a problem. In the best case scenario, electrondeficient (hetero)arenes, which are already present in many biologically active compounds may simply be equipped with suitable leaving groups for covalently addressing proximal nucleophilic amino acids. Carbocyclic strain release warheads, which have a very low literature precedence, may be valuable alternatives especially if IP claims are an issue. Despite their promising chemical properties, however, the latter moieties have only been evaluated against isolated peptides so far. Systematic implementation of metabolically labile CRGs, such as fumaric acid esters or epoxides, is an option where short time exposure could offer a kinetic selectivity advantage. If reversible cysteine targeting is desired, α-cyanoacrylamides and analogous dually activated Michael acceptors are an up-to-date option with clinically approved representatives. Activated nitriles such as cyanamides or 2-cyanopyri(mi)dines represent suitable alternatives that have shown promising in vivo properties and aldehydes may also be employed in some cases. Although the above-mentioned CRGs are typically used for cysteine targeting, many of them can also react with weaker nucleophiles such as lysine, tyrosine, or histidine. As discussed before, selective engagement of these less reactive residues is more challenging. Harder electrophiles, e.g., sulfonyl fluorides or activated esters, are more suitable to react with amino or hydroxy bases. Vinyl sulfones and the corresponding sulfonamides have further been shown to react readily with the lysine ε-amino group. Although the latter CRGs may be used for targeting lysines and tyrosines, most of these moieties have certain liabilities such as high intrinsic reactivity (vinyl sulfones and sulfonyl fluorides), limited stability toward hydrolysis (sulfonyl fluorides), and susceptibility to metabolic inactivation (activated esters). In the case of sulfur (VI) fluorides, the weakly reactive aryl fluorosulfates and the highly tunable sulfonimidoyl fluorides may address these issues and clearly merit further investigations. For reversible lysine targeting, 2-carbonylarylboronic acids forming stabilized Schiff bases may open up future avenues and further proof of concept, especially in vivo, would be highly desirable. However, despite these advances, the scope of lysine and tyrosine-targeted warheads is still limited and studies addressing the histidine side chain are even less abundant in the recent literature. Consequently, there is an urgent need for the development of novel CRGs which reliably and specifically address these residues without cross-reacting with cysteines. Many weakly nucleophilic (e.g., glutamine, asparagine, arginine as well as nonactivated serine or threonine) or electrophilic (e.g., tryptophan) amino acids cannot be targeted reliably with the current warhead chemistry, necessitating further research to expand our scope of ligandable residues. Encouragingly, recent studies on glutamate/aspartate or methionine targeted CRGs hold the promise that at least some of the challenges associated with weaker nucleophiles could be addressed in the near future. Furthermore, the recognition of the role of cysteine oxidation in redox regulation has stimulated promising research on nucleophilic CRGs targeting electrophilic sulfenic acids and sulfenamides, which represent transient key oxidation products of cysteines’ thiols. Nevertheless, these concepts still have to prove their applicability in drug discovery and further refinement or alternative chemical approaches will be necessary when aiming for clinical applications.

might be practicable for bioconjugation in vitro, such chemistries are difficult to realize in cells or even in vivo. Therefore, it is unlikely that these amino acids will be amenable to covalent targeting in a medicinal chemistry setting in the near future.

11. SUMMARY AND PERSPECTIVE Covalent targeting has become an extremely powerful tool in drug discovery and chemical biology and substantial effort has recently been put into developing or repurposing warheads for TCI design. An overview of the warhead classes discussed in this article and their key characteristics is provided in Table 2. The reader is referred to the respective sections and the literature cited therein for details. Despite the plethora of CRGs described in the current literature, selecting the right warhead for a specific application remains a nontrivial task. Especially in vivo, factors to be considered reach far beyond the structure of the protein and the nature of the target amino acid and include properties like target turnover, tissue distribution, and (sub)-cellular location, among others. Metabolic stability and chemical reactivity of the ligand also need to be well balanced, and the ideal window largely depends on the projected application. In this context, it is important to note that extrahepatic clearance is a major determinant of the pharmacokinetics of common acrylamidederived TCIs.347 Oxidative metabolism via reactive epoxide intermediates is another well-known biotransformation of α,βunsaturated amides.348 However, little is known about the metabolic fate of most CRGs discussed here. Reactivity toward glutathione, but also other thiol-containing reagents, has frequently been employed as a surrogate parameter to describe the nonspecific reactivity toward biological nucleophiles. In cells, however, much of the GSH-conjugation is mediated by glutathione S-transferases. Consequently, GSH addition under real physiological conditions might be much faster than estimated by the common (enzyme-free) GSH binding assays. An additional level of complexity is added by the presence of hyper-reactive cysteine residues in various proteins that could potentially engage even weakly reactive electrophiles. Similarly, certain features of protein binding sites might activate individual CRGs to become more electrophilic. In the light of this complexity, it should not remain unmentioned that some of the electrophiles discussed in this Perspective have only been evaluated in isolated proteins or peptides so far. Although these chemotypes may become useful in TCI design, the potential of such CRGs remains unclear until extensive profiling of reactivity, specificity, and stability in cells or in vivo has been performed. Warhead selection typically starts with the estimation of reactivity required with respect to the desired target amino acid. Although computational reactivity prediction for certain electrophiles has significantly advanced within the last years,12,158,349 precise and comparable rating over different warhead classed and nucleophiles remains a major challenge. When sufficiently exposed and reactive cysteines are to be addressed and slow depletion by GSH or other thiols is not an issue, α,β-unsaturated amides generally represent a safe bet. However, problems may arise when the target residue is poorly reactive, difficult to access, or incompatible with the spatial and geometric requirements of this electrophilic headgroup. Here, alternative acceptors, such as alkenylated or alkynylated heteroarenes or the rigid propiolonitriles, might come into play. SNAr warheads may offer advantages if spatially defined AN



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ligands. Many other issues are known and a further discussion may be found elsewhere.351 Combination with emerging techniques, e.g., cellular thermal shift assays (CETSA)332 or thermal proteome profiling,333 and the ever increasing number of biochemical and cellular screening platforms, provides an opportunity to obtain a more complete picture. In this context, it is worth mentioning that little effort has been made so far to investigate the binding of reactive ligands to nonprotein offtargets, for example, the nucleobases in RNA and DNA. Such investigations would become even more important when employing more versatile warhead chemistries because each functional group features a distinct reactivity toward these nucleophiles. Altogether, understanding the fate of reactive ligands in complex living organisms still represents a fascinating challenge and a continuously developing area of research. Ultimately, it seems unlikely that alternative warheads will replace cysteine-targeted α,β-unsaturated amides as the dominant chemical species in TCI design in the near future. Nevertheless, α,β-unsaturated amides are still far from being perfect warheads and do often not provide the general and predictable solutions they promise. Therefore, the CRGs presented here offer a valuable toolbox for the manifold applications where cysteine targeting with α,β-unsaturated amides cannot satisfy all needs or cases where simply no cysteine residues are available for covalent modification. Finally, we can expect a broadening of the scope of CRGs employed in drug discovery as challenging targets require the deliberate use of tailor-made warheads for success.

Likewise, many issues with the more established warheads remain to be resolved. For example, none of the presented studies on sulfonyl fluorides, SNAr electrophiles, or activated esters investigated the toxic potential of the leaving group. Depending on the dose and the abundance/distribution of the target protein, it is conceivable that high local fluoride concentrations may result from fluoride-containing warheads in vivo. Similarly, phenols and other alcohols eliminated from activated esters could possess a distinct biological activity or toxicity themselves. Although these factors are not major issues for many chemical biology applications, they necessitate a detailed assessment prior to utilization in humans. As it can be noticed throughout this article, IC50 values still dominate the literature on covalent ligands although the more laborious determination of binding kinetics is becoming more common. This reliance on IC50 data has implications on covalent ligand design because the observed differences in potency can be driven by reversible binding (KI) but also by the efficiency of covalent bond formation (kinact). In this context, it is interesting to note that the diffusion-based limit for the rate of reversible ligand association (often referred to as kon) is in the range of 108−109 M−1 s−1. Lower kon values are typically observed because various factors, e.g. desolvation or induced-fit mechanisms, can retard ligand association. The same upper limit also applies to the specificity constants (kcat/ KM) of catalytically perfect enzymes.34,350 Naturally, kinact/KI cannot exceed this rate as well. A well-designed TCI should be a potent and specific reversible binder to allow for selective target modification at low inhibitor concentrations. It should place the reactive moieties in favorable positions in terms of distance and angle. Similarly, the orientation toward activating residues in the binding cleft can play a role. The latter factors are crucial to enable a rapid bond formation between the reaction partners. The proper alignment of the reactive residues becomes especially important when low reactivity warheads are employed because the key properties of covalent binders (see the Introduction) would be lost with inactivation kinetics becoming too slow. In such a case, increased reversible potency could easily be mistaken as a result of covalent binding. Remarkably, recent data suggest that the efficacy of approved covalent EGFR inhibitors relies predominantly on reversible interactions.219 Consequently, understanding binding kinetics is key to generate a properly balanced TCI which benefits from both the reversible and the covalent binding event. To determine the specificity of novel CRGs and the derived TCIs in cells or even tissues, robust and convenient assays are required. Fortunately, considerable advances in chemical proteomics have made the assessment of off-targets in cellular proteomes becoming more and more common. Proteomic approaches provide very powerful tools to determine the fate of reactive ligands. Nevertheless, they also have their limitations. Methods employing tagged ligands to enrich covalently bound proteins, for example, may ignore the influence of the tag on ligand binding. On the other hand, approaches exploiting the differential labeling pattern of promiscuous reactive probes in the presence of a competitor ligand capture only a fraction of all potentially reactive amino acids present (e.g., the entire “cysteinome” or the “lysinome”) suggesting that relevant reaction sites could be missed. Even more of an issue are the intrinsic limitations of reactivity-based methods in identifying noncovalent interactions, which can significantly contribute to the biological profiles of reactive

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 7071 29-72466. E-mail: [email protected] ORCID

Matthias Gehringer: 0000-0003-0163-3419 Stefan A. Laufer: 0000-0001-6952-1486 Notes

The authors declare no competing financial interest. Biographies Matthias Gehringer studied chemistry at the Karlsruhe Institute of Technology, the Ecole Nationale Supérieure de Chimie de Montpellier, and the University of Heidelberg, and obtained his Ph.D. from Tü bingen University working on reversible and irreversible kinase inhibitors. As a postdoctoral researcher at the Swiss Federal Institute of Technology (ETH) Zürich, he focused on the total synthesis of mycolactones and on targeted antibiotic−protein conjugates. He recently returned to Tübingen, where he is currently establishing an independent research group. His research interests include medicinal chemistry, chemical biology, natural-product synthesis, and innovative drug targeting approaches. Stefan A. Laufer studied Pharmacy and completed his Ph.D. from Regensburg University. After postdoctoral research in Frankfurt, he took a position in the pharmaceutical industry but maintained lectureships at Frankfurt and later at Mainz University, where he finished his habilitation in 1997. Since 1999, he has been full professor (chair) for pharmaceutical and medicinal chemistry at Tübingen University. He is cofounder/spokesman of ICEPHA (Interfaculty Center for Pharmacogenomics and Pharma Research), TüCADD (Tübingen Center for Academic Drug Discovery), and cofounder of the two startups CAIR Biosciences and Heparegenix. Three AO



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compounds from his lab made it first into man. He is currently (2016−2019) president of the German Pharmaceutical Society (DPhG). His research interests are protein kinase inhibitors and eicosanoid modulators.

bank; PDIA1, protein disulfide isomerase A1; PEG, polyethylene glycol; PEITC, phenethyl isothiocyanate; PGAM1, phosphoglycerate mutase 1; PLK, polo-like kinase; PLP, pyridoxal phosphate; PMBCs, peripheral blood mononuclear cells; PMSF, phenylmethane sulfonyl fluoride; PPARs, peroxisome proliferator-activated receptors; ReACT, redoxactivated chemical tagging; ROS, reactive oxygen species; SAR, structure−activity relationship; SH, Scr homology; SNAr, nucleophilic aromatic substitution; SPR, surface plasmon resonance; SRC, steroid receptor coactivator; SRPK1, SRprotein kinase 1; SuFEx, sulfur (VI) fluoride exchange; SUMO, small ubiquitin-related modifier; TCEP, tris(2-carboxyethyl)phosphine; TCI, targeted covalent inhibitor; TM, transmembrane domain; TR, thyroid hormone receptor; TRFRET, time-resolved Fö rster resonance energy transfer; TRPA1, transient receptor potential cation channel A1; Ub, ubiquitin; WDK, Woodward’s reagent K

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ACKNOWLEDGMENTS We thank Dr. Michael Forster and Dr. Marcel Günther for fruitful discussions and Dr. Apirat Chaikuad for scientific advice. Kristine Schmidt, Dr. Michael Forster, Dr. Marcel Günther, and Bent Präfke are gratefully acknowledged for proof-reading. We thank Valentin Wydra and Nathanael Disch for assistance in the preparation of the manuscript and the TOC graphic. M.G. gratefully acknowledges financial support by the Institutional Strategy of the University of Tübingen (Deutsche Forschungsgemeinschaft, ZUK 63) and the Postdoctoral Fellowship Programme of the Baden-Württemberg Stiftung.

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ABBREVIATIONS USED ABPP, activity-based protein profiling; AChE, acetylcholinesterase; ADME, absorption, distribution, metabolism and excretion; ALDH2, aldehyde dehydrogenase 2; ALK, anaplastic lymphoma kinase; AR, androgen receptor; ARL, allosteric release factor; BTK, Bruton’s tyrosine kinase; CDK, cyclin-dependent kinase; CETSA, cellular thermal shift assay; CNV, choroidal neovascularization model; CRABP, cellular retinoic acid binding protein; CRG, covalent reactive group; CuAAC, copper(I)-catalyzed alkyne−azide cycloaddition; CYP, cytochrome P; DcpS, scavenger mRNA-decapping enzyme; DDAH, dimethylarginine dimethylaminohydrolase; DFT, density functional theory; Dha, dehydroalanine; DMF, dimethyl fumarate; DMPK, drug metabolism and pharmacokinetics; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; DUBs, deubiquitinating isopeptidases; EC50, half-maximum effective concentration; EGFR, epidermal growth factor receptor; ErbB, protein family of four receptor tyrosine kinases (ErbB/Her 1−4); ESI-MS, electrospray ionization-mass spectrometry; FDA, Food and Drug Administration; FSBA, 5′-(4-fluorosulfonylbenzoyl)adenosine; FGFR, fibroblast growth factor receptor; FMK, fluoromethylketone; GAG, glycosaminoglycan; GK, gatekeeper; GPCR, G-protein coupled receptor; GPX, glutathione peroxidase; GSH, glutathione; GSTP1, glutathione S-transferase π or P1; H1975, human lung-cell line; hCES, human carboxylesterase; HCV, hepatitis C virus; HDAC8, histone deacetylase 8; HEK293, human embryonic kidney 293 cells; hERG, human ether-à-go-gorelated gene; HSAB, hard and soft acids and bases; HTS, high throughput screening; HUVE, human umbilical vein endothelial; IC50, half-maximum inhibitory concentration; ICL, isocitrate lyase; IDH, isocitrate dehydrogenase; IRE, inositolrequiring enzyme; ITAM, immunoreceptor tyrosine-based activation motif; ITK, interleukin-2-inducible T-cell kinase; JAK, Janus kinase; K-Ras, p21 GTPase (oncogen first found in Kirsten rat sarcoma virus); MAO, monoamine oxidase; MetAP, methionine aminopeptidase; MGMT, (O 6 -)methylguanine-DNA-methyltransferase; MMF, monomethyl fumarate; MSF, methanesulfonyl fluoride; MSBT, 2(methanesulfonyl)benzothiazole; MurA, UDP-N-acetylglucosamine enolpyruvyl transferase; NAC, N-acetylcysteine; NBDdye, nitrobenzoxadiazole-dye; NCI, National Cancer Institute; NHS, N-hydroxysuccinimid; NMR, nuclear magnetic resonance; NS5B, nonstructural protein 5B; PDB, protein data

REFERENCES

(1) Mann, M.; Jensen, O. N. Proteomic Analysis of PostTranslational Modifications. Nat. Biotechnol. 2003, 21 (3), 255−261. (2) Schopfer, F. J.; Cipollina, C.; Freeman, B. A. Formation and Signaling Actions of Electrophilic Lipids. Chem. Rev. 2011, 111 (10), 5997−6021. (3) Allis, C. D.; Jenuwein, T. The Molecular Hallmarks of Epigenetic Control. Nat. Rev. Genet. 2016, 17 (8), 487−500. (4) Uetrecht, J. Idiosyncratic Drug Reactions: Current Understanding. Annu. Rev. Pharmacol. Toxicol. 2007, 47 (1), 513−539. (5) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307−317. (6) Bauer, R. A. Covalent Inhibitors in Drug Discovery: From Accidental Discoveries to Avoided Liabilities and Designed Therapies. Drug Discovery Today 2015, 20 (9), 1061−1073. (7) Powers, J. C.; Asgian, J. L.; Ekici, Ö . D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. Chem. Rev. 2002, 102 (12), 4639−4750. (8) Bachovchin, D. A.; Cravatt, B. F. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases. Nat. Rev. Drug Discovery 2012, 11 (1), 52−68. (9) Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. How to Improve R&D Productivity: The Pharmaceutical Industry’s Grand Challenge. Nat. Rev. Drug Discovery 2010, 9 (3), 203−214. (10) Bandyopadhyay, A.; Gao, J. Targeting Biomolecules with Reversible Covalent Chemistry. Curr. Opin. Chem. Biol. 2016, 34, 110−116. (11) Bradshaw, J. M.; McFarland, J. M.; Paavilainen, V. O.; Bisconte, A.; Tam, D.; Phan, V. T.; Romanov, S.; Finkle, D.; Shu, J.; Patel, V.; Ton, T.; Li, X.; Loughhead, D. G.; Nunn, P. A.; Karr, D. E.; Gerritsen, M. E.; Funk, J. O.; Owens, T. D.; Verner, E.; Brameld, K. A.; Hill, R. J.; Goldstein, D. M.; Taunton, J. Prolonged and Tunable Residence Time Using Reversible Covalent Kinase Inhibitors. Nat. Chem. Biol. 2015, 11 (7), 525−531. (12) Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57 (23), 10072−10079. (13) Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning, B. D.; González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F. Proteome-Wide Covalent Ligand Discovery in Native Biological Systems. Nature 2016, 534 (7608), 570−574. AP



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(14) Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I. M.; Taunton, J. Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors. J. Am. Chem. Soc. 2013, 135 (14), 5298− 5301. (15) Jöst, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: A Systematic Study of Electrophilic Fragments. J. Med. Chem. 2014, 57 (18), 7590−7599. (16) Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 2014, 57 (11), 4969−4974. (17) Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M. K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions. Nature 2013, 503 (7477), 548−551. (18) Zimmermann, G.; Rieder, U.; Bajic, D.; Vanetti, S.; Chaikuad, A.; Knapp, S.; Scheuermann, J.; Mattarella, M.; Neri, D. A Specific and Covalent JNK-1 Ligand Selected from an Encoded SelfAssembling Chemical Library. Chem. - Eur. J. 2017, 23 (34), 8152− 8155. (19) Zambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N. Screening for Covalent Inhibitors Using DNA-Display of Small Molecule Libraries Functionalized with Cysteine Reactive Moieties. MedChemComm 2016, 7 (7), 1340−1351. (20) Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discov. 2017, 22 (1), 3−20. (21) Miyahisa, I.; Sameshima, T.; Hixon, M. S. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (Kinact/Ki) by Means of an Endpoint Competition Assay. Angew. Chem., Int. Ed. 2015, 54 (47), 14099−14102. (22) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry. Annu. Rev. Biochem. 2008, 77 (1), 383−414. (23) Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase Inhibitors. Nat. Chem. Biol. 2014, 10 (9), 760−767. (24) Zaro, B. W.; Whitby, L. R.; Lum, K. M.; Cravatt, B. F. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors. J. Am. Chem. Soc. 2016, 138 (49), 15841− 15844. (25) Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen, M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Frödin, M.; Taunton, J. Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles. Nat. Chem. Biol. 2012, 8 (5), 471− 476. (26) Noe, M. C.; Gilbert, A. M. Targeted Covalent Enzyme Inhibitors. In Annual Reports in Medicinal Chemistry; Desai, M. C., Ed.; Academic Press, 2012; Vol. 47, pp. 413−439. DOI: 10.1016/ B978-0-12-396492-2.00027-8. (27) Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20 (2), 146−159. (28) Miller, R. M.; Taunton, J. Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors. In Methods in Enzymology; Shokat, K. M., Ed.; Academic Press, 2014; Vol. 548, pp 93−116. DOI: 10.1016/B978-0-12-397918-6.00004-5. (29) Gilbert, A. M. Recent Advances in Irreversible Kinase Inhibitors. Pharm. Pat. Anal. 2014, 3 (4), 375−386. (30) Adeniyi, A. A.; Muthusamy, R.; Soliman, M. E. New Drug Design with Covalent Modifiers. Expert Opin. Drug Discovery 2016, 11 (1), 79−90. (31) Baillie, T. A. Targeted Covalent Inhibitors for Drug Design. Angew. Chem., Int. Ed. 2016, 55 (43), 13408−13421. (32) Hallenbeck, K. K.; Turner, D. M.; Renslo, A. R.; Arkin, M. R. Targeting Non-Catalytic Cysteine Residues Through StructureGuided Drug Discovery. Curr. Top. Med. Chem. 2016, 17, 4−15.

(33) Lagoutte, R.; Patouret, R.; Winssinger, N. Covalent Inhibitors: An Opportunity for Rational Target Selectivity. Curr. Opin. Chem. Biol. 2017, 39, 54−63. (34) De Cesco, S.; Kurian, J.; Dufresne, C.; Mittermaier, A. K.; Moitessier, N. Covalent Inhibitors Design and Discovery. Eur. J. Med. Chem. 2017, 138, 96−114. (35) Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The Cysteinome of Protein Kinases as a Target in Drug Development. Angew. Chem., Int. Ed. 2018, 57 (16), 4372−4385. (36) Zhao, Z.; Bourne, P. E. Progress with Covalent Small-Molecule Kinase Inhibitors. Drug Discovery Today 2018, 23 (3), 727−735. (37) Lonsdale, R.; Ward, R. A. Structure-Based Design of Targeted Covalent Inhibitors. Chem. Soc. Rev. 2018, 47 (11), 3816−3830. (38) Ferguson, F. M.; Gray, N. S. Kinase Inhibitors: The Road Ahead. Nat. Rev. Drug Discovery 2018, 17 (5), 353−377. (39) Pettinger, J.; Jones, K.; Cheeseman, M. D. Lysine-Targeting Covalent Inhibitors. Angew. Chem., Int. Ed. 2017, 56 (48), 15200− 15209. (40) Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,βUnsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60 (3), 839−885. (41) Baslé, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17 (3), 213−227. (42) Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 2174−2195. (43) Shannon, D. A.; Weerapana, E. Covalent Protein Modification: The Current Landscape of Residue-Specific Electrophiles. Curr. Opin. Chem. Biol. 2015, 24, 18−26. (44) Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529−553. (45) Dondoni, A.; Marra, A. SuFEx: A Metal-Free Click Ligation for Multivalent Biomolecules. Org. Biomol. Chem. 2017, 15 (7), 1549− 1553. (46) deGruyter, J. N.; Malins, L. R.; Baran, P. S. Residue-Specific Peptide Modification: A Chemist’s Guide. Biochemistry 2017, 56 (30), 3863−3873. (47) Hoch, D. G.; Abegg, D.; Adibekian, A. Cysteine-Reactive Probes and Their Use in Chemical Proteomics. Chem. Commun. 2018, 54 (36), 4501−4512. (48) Cromm, P. M.; Crews, C. M. The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug Target. ACS Cent. Sci. 2017, 3 (8), 830−838. (49) Casimiro-Garcia, A.; Trujillo, J. I.; Vajdos, F.; Juba, B.; Banker, M. E.; Aulabaugh, A.; Balbo, P.; Bauman, J.; Chrencik, J.; Coe, J. W.; Czerwinski, R.; Dowty, M.; Knafels, J. D.; Kwon, S.; Leung, L.; Liang, S.; Robinson, R. P.; Telliez, J.-B.; Unwalla, R.; Yang, X.; Thorarensen, A. Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors. J. Med. Chem. 2018, 61, 10665−10699. (50) Li, T.; Maltais, R.; Poirier, D.; Lin, S.-X. Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide. J. Phys. Chem. Lett. 2018, 9 (18), 5275−5280. (51) Kharenko, O. A.; Patel, R. G.; Brown, S. D.; Calosing, C.; White, A.; Lakshminarasimhan, D.; Suto, R. K.; Duffy, B. C.; Kitchen, D. B.; McLure, K. G.; Hansen, H. C.; van der Horst, E. H.; Young, P. R. Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine. J. Med. Chem. 2018, 61 (18), 8202−8211. (52) Pearson, R. G.; Sobel, H. R.; Songstad, J. Nucleophilic Reactivity Constants toward Methyl Iodide and Trans-Dichlorodi(Pyridine)Platinum(II). J. Am. Chem. Soc. 1968, 90 (2), 319−326. (53) Sardi, F.; Manta, B.; Portillo-Ledesma, S.; Knoops, B.; Comini, M. A.; Ferrer-Sueta, G. Determination of Acidity and Nucleophilicity in Thiols by Reaction with Monobromobimane and Fluorescence Detection. Anal. Biochem. 2013, 435 (1), 74−82. (54) Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc. 1963, 85 (22), 3533−3539. AQ



Perspective

(55) Awoonor-Williams, E.; Rowley, C. N. Evaluation of Methods for the Calculation of the PKa of Cysteine Residues in Proteins. J. Chem. Theory Comput. 2016, 12 (9), 4662−4673. (56) Grimsley, G. R.; Scholtz, J. M.; Pace, C. N. A Summary of the Measured PK Values of the Ionizable Groups in Folded Proteins. Protein Sci. 2008, 18, 247−251. (57) Alcock, L. J.; Perkins, M. V.; Chalker, J. M. Chemical Methods for Mapping Cysteine Oxidation. Chem. Soc. Rev. 2018, 47 (1), 231− 268. (58) Pace, N. J.; Weerapana, E. Diverse Functional Roles of Reactive Cysteines. ACS Chem. Biol. 2013, 8 (2), 283−296. (59) Jones, A.; Zhang, X.; Lei, X. Covalent Probe Finds Carboxylic Acid. Cell Chem. Biol. 2017, 24 (5), 537−539. (60) Shirley, M. Dacomitinib: First Global Approval. Drugs 2018, 78, 1947. (61) Markham, A.; Dhillon, S. Acalabrutinib: First Global Approval. Drugs 2018, 78 (1), 139−145. (62) Forster, M.; Gehringer, M.; Laufer, S. A. Recent Advances in JAK3 Inhibition: Isoform Selectivity by Covalent Cysteine Targeting. Bioorg. Med. Chem. Lett. 2017, 27 (18), 4229−4237. (63) Garzón, B.; Oeste, C. L.; Díez-Dacal, B.; Pérez-Sala, D. Proteomic Studies on Protein Modification by Cyclopentenone Prostaglandins: Expanding Our View on Electrophile Actions. J. Proteomics 2011, 74 (11), 2243−2263. (64) Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60 (7), 2879−2889. (65) Günther, M.; Juchum, M.; Kelter, G.; Fiebig, H.; Laufer, S. Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant. Angew. Chem., Int. Ed. 2016, 55 (36), 10890−10894. (66) Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.; Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFRDirected Covalent Inhibitors. Cell Chem. Biol. 2017, 24, 1388−1400. (67) Blewett, M. M.; Xie, J.; Zaro, B. W.; Backus, K. M.; Altman, A.; Teijaro, J. R.; Cravatt, B. F. Chemical Proteomic Map of Dimethyl Fumarate−Sensitive Cysteines in Primary Human T Cells. Sci. Signaling 2016, 9 (445), rs10. (68) Deeks, E. D. Ibrutinib: A Review in Chronic Lymphocytic Leukaemia. Drugs 2017, 77 (2), 225−236. (69) Bender, A. T.; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.; Grenningloh, R.; Head, J.; Morandi, F.; Haselmayer, P.; Liu-Bujalski, L. Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but Not B-Cell Receptor Signaling. Mol. Pharmacol. 2017, 91 (3), 208−219. (70) Pan, Z.; Scheerens, H.; Li, S.-J.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase. ChemMedChem 2007, 2 (1), 58−61. (71) Mann, M. Innovations: Functional and Quantitative Proteomics Using SILAC. Nat. Rev. Mol. Cell Biol. 2006, 7 (12), 952−958. (72) Crow, J. A.; Bittles, V.; Borazjani, A.; Potter, P. M.; Ross, M. K. Covalent Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by the Carbamates JZL184 and URB597. Biochem. Pharmacol. 2012, 84 (9), 1215−1222. (73) Buynak, J. D.; Mathew, J.; Rao, M. N.; Haley, E.; George, C.; Siriwardane, U. The Preparation of the First α-Vinylidene-β-Lactams. J. Chem. Soc., Chem. Commun. 1987, 0 (10), 735−737. (74) Roedig, A.; Ritschel, W. Reaktionen von 3,4,4-Trichlor-3butenamiden mit Nucleophilen, II. Thiol- und Aminaddukte von 3,3Dichlorallencarboxamiden. Chem. Ber. 1983, 116 (4), 1595−1602.

(75) Abbas, A.; Xing, B.; Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem., Int. Ed. 2014, 53 (29), 7491−7494. (76) Pedzisa, L.; Li, X.; Rader, C.; Roush, W. R. Assessment of Reagents for Selenocysteine Conjugation and the Stability of Selenocysteine Adducts. Org. Biomol. Chem. 2016, 14 (22), 5141− 5147. (77) Chen, D.; Guo, D.; Yan, Z.; Zhao, Y. Allenamide as a Bioisostere of Acrylamide in the Design and Synthesis of Targeted Covalent Inhibitors. MedChemComm 2018, 9 (2), 244−253. (78) Awoonor-Williams, E.; Rowley, C. N. How Reactive Are Druggable Cysteines in Protein Kinases? J. Chem. Inf. Model. 2018, 58 (9), 1935−1946. (79) Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J.-S.; Strub, J.-M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles. Bioconjugate Chem. 2014, 25 (2), 202−206. (80) Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.; Liu, Y.; Wong, M.-K.; Che, C.-M. Electron-Deficient Alkynes as Cleavable Reagents for the Modification of Cysteine-Containing Peptides in Aqueous Medium. Chem. - Eur. J. 2009, 15 (15), 3839−3850. (81) Friedman, M.; Wall, J. S. Additive Linear Free-Energy Relationships in Reaction Kinetics of Amino Groups with α,βUnsaturated Compounds. J. Org. Chem. 1966, 31 (9), 2888−2894. (82) Cavins, J. F.; Friedman, M. An Internal Standard for Amino Acid Analyses: S-β-(4-Pyridylethyl)-l-Cysteine. Anal. Biochem. 1970, 35 (2), 489−493. (83) Gill, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr, M. G.; Woodhead, A. J.; Walker, M. T.; Congreve, M. S.; Devine, L. A.; Tisi, D.; O’Reilly, M.; Seavers, L. C. A.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A. E.; Jhoti, H. Identification of Novel P38α MAP Kinase Inhibitors Using Fragment-Based Lead Generation. J. Med. Chem. 2005, 48 (2), 414− 426. (84) Raux, E.; Klenc, J.; Blake, A.; Sączewski, J.; Strekowski, L. Conjugate Addition of Nucleophiles to the Vinyl Function of 2Chloro-4-Vinylpyrimidine Derivatives. Molecules 2010, 15 (3), 1973− 1984. (85) Burns, A. R.; Kerr, J. H.; Kerr, W. J.; Passmore, J.; Paterson, L. C.; Watson, A. J. B. Tuned Methods for Conjugate Addition to a Vinyl Oxadiazole; Synthesis of Pharmaceutically Important Motifs. Org. Biomol. Chem. 2010, 8 (12), 2777−2783. (86) Kuchař, M.; Hocek, M.; Pohl, R.; Votruba, I.; Shih, I.; Mabery, E.; Mackman, R. Synthesis, Cytostatic, and Antiviral Activity of Novel 6-[2-(Dialkylamino)Ethyl]-, 6-(2-Alkoxyethyl)-, 6-[2-(Alkylsulfanyl)Ethyl]-, and 6-[2-(Dialkylamino)Vinyl]Purine Nucleosides. Bioorg. Med. Chem. 2008, 16 (3), 1400−1424. (87) Il’yasov, E. A.; Galust’yan, G. G. Homolytic Addition of 1Alkanethiols to 5-Ethynyl-2-Methylpyridine. Chem. Heterocycl. Compd. 1999, 35 (10), 1187−1189. (88) Wipf, P.; Graham, T. H. Synthesis and Hetero-Michael Addition Reactions of 2-Alkynyl Oxazoles and Oxazolines. Org. Biomol. Chem. 2005, 3 (1), 31−35. (89) Li, Q.-F.; Yang, Y.; Maleckis, A.; Otting, G.; Su, X.-C. Thiol− Ene Reaction: A Versatile Tool in Site-Specific Labelling of Proteins with Chemically Inert Tags for Paramagnetic NMR. Chem. Commun. 2012, 48 (21), 2704−2706. (90) Yang, Y.; Li, Q.-F.; Cao, C.; Huang, F.; Su, X.-C. Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study. Chem. - Eur. J. 2013, 19 (3), 1097−1103. (91) Ma, F.-H.; Chen, J.-L.; Li, Q.-F.; Zuo, H.-H.; Huang, F.; Su, X.C. Kinetic Assay of the Michael Addition-Like Thiol−Ene Reaction and Insight into Protein Bioconjugation. Chem. - Asian J. 2014, 9 (7), 1808−1816. (92) Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.; Dickerson, S. H.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell, A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S. M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J. AR



Perspective

Promoted Azide−Alkyne Cycloaddition. Bioconjugate Chem. 2012, 23 (3), 392−398. (105) Tian, H.; Sakmar, T. P.; Huber, T. A Simple Method for Enhancing the Bioorthogonality of Cyclooctyne Reagent. Chem. Commun. 2016, 52 (31), 5451−5454. (106) Haldón, E.; Nicasio, M. C.; Pérez, P. J. Copper-Catalysed Azide−Alkyne Cycloadditions (CuAAC): An Update. Org. Biomol. Chem. 2015, 13 (37), 9528−9550. (107) Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J. R.; Komander, D.; Ovaa, H. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases. J. Am. Chem. Soc. 2013, 135 (8), 2867−2870. (108) Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Covalent Inhibition of SUMO and Ubiquitin-Specific Cysteine Proteases by an in Situ Thiol−Alkyne Addition. Bioorg. Med. Chem. 2013, 21 (9), 2511−2517. (109) Swatek, K. N.; Aumayr, M.; Pruneda, J. N.; Visser, L. J.; Berryman, S.; Kueck, A. F.; Geurink, P. P.; Ovaa, H.; van Kuppeveld, F. J. M.; Tuthill, T. J.; Skern, T.; Komander, D. Irreversible Inactivation of ISG15 by a Viral Leader Protease Enables Alternative Infection Detection Strategies. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), 2371−2376. (110) Arkona, C.; Rademann, J. Propargyl Amides as Irreversible Inhibitors of Cysteine ProteasesA Lesson on the Biological Reactivity of Alkynes. Angew. Chem., Int. Ed. 2013, 52 (32), 8210− 8212. (111) Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507−515. (112) Terrier, F. Rate and Equilibrium Studies in JacksonMeisenheimer Complexes. Chem. Rev. 1982, 82 (2), 77−152. (113) Terrier, F. Modern Nucleophilic Aromatic Substitution, 1st ed.; Wiley-VCH: Weinheim, 2013. (114) Kwan, E. E.; Zeng, Y.; Besser, H. A.; Jacobsen, E. N. Concerted Nucleophilic Aromatic Substitutions. Nat. Chem. 2018, 10 (9), 917−923. (115) Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.; Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moller, D. E. L764406 Is a Partial Agonist of Human Peroxisome ProliferatorActivated Receptor γ. The Role of Cys13 in Ligand Binding. J. Biol. Chem. 1999, 274 (12), 7913−7922. (116) Leesnitzer, L. M.; Parks, D. J.; Bledsoe, R. K.; Cobb, J. E.; Collins, J. L.; Consler, T. G.; Davis, R. G.; Hull-Ryde, E. A.; Lenhard, J. M.; Patel, L.; Plunket, K. D.; Shenk, J. L.; Stimmel, J. B.; Therapontos, C.; Willson, T. M.; Blanchard, S. G. Functional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662. Biochemistry 2002, 41 (21), 6640−6650. (117) Shearer, B. G.; Wiethe, R. W.; Ashe, A.; Billin, A. N.; Way, J. M.; Stanley, T. B.; Wagner, C. D.; Xu, R. X.; Leesnitzer, L. M.; Merrihew, R. V.; Shearer, T. W.; Jeune, M. R.; Ulrich, J. C.; Willson, T. M. Identification and Characterization of 4-Chloro-N-(2-{[5Trifluoromethyl)-2-Pyridyl]Sulfonyl}ethyl)Benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist. J. Med. Chem. 2010, 53 (4), 1857−1861. (118) Babaoglu, K.; Simeonov, A.; Irwin, J. J.; Nelson, M. E.; Feng, B.; Thomas, C. J.; Cancian, L.; Costi, M. P.; Maltby, D. A.; Jadhav, A.; Inglese, J.; Austin, C. P.; Shoichet, B. K. Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase. J. Med. Chem. 2008, 51 (8), 2502−2511. (119) Patterson, J. T.; Asano, S.; Li, X.; Rader, C.; Barbas, C. F. Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone Linkers. Bioconjugate Chem. 2014, 25 (8), 1402−1407. (120) Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R.; Xian, M. Methylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking Reagent. Org. Lett. 2012, 14 (13), 3396−3399. (121) Toda, N.; Asano, S.; Barbas, C. F. Rapid, Stable, Chemoselective Labeling of Thiols with Julia−Kocieński-like Reagents: A

D.; White, W. L.; Uehling, D. E. 6-Ethynylthieno[3,2-d]- and 6Ethynylthieno[2,3-d]Pyrimidin-4-Anilines as Tunable Covalent Modifiers of ErbB Kinases. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (8), 2773−2778. (93) Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A. Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth Factor Receptor: 4(Phenylamino)Quinazoline- and 4-(Phenylamino)Pyrido[3,2-d]Pyrimidine-6-Acrylamides Bearing Additional Solubilizing Functions. J. Med. Chem. 2000, 43 (7), 1380−1397. (94) Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.; Wissner, A. 6-Substituted-4-(3-Bromophenylamino)Quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor Activity. J. Med. Chem. 2001, 44 (17), 2719−2734. (95) Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, H.-R. Synthesis and Structure−Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-Carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2). J. Med. Chem. 2003, 46 (1), 49−63. (96) Hubbard, R. D.; Dickerson, S. H.; Emerson, H. K.; Griffin, R. J.; Reno, M. J.; Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G. Dual EGFR/ErbB-2 Inhibitors from Novel Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines. Bioorg. Med. Chem. Lett. 2008, 18 (21), 5738−5740. (97) Stevens, K. L.; Alligood, K. J.; Alberti, J. G. B.; Caferro, T. R.; Chamberlain, S. D.; Dickerson, S. H.; Dickson, H. D.; Emerson, H. K.; Griffin, R. J.; Hubbard, R. D.; Keith, B. R.; Mullin, R. J.; Petrov, K. G.; Gerding, R. M.; Reno, M. J.; Rheault, T. R.; Rusnak, D. W.; Sammond, D. M.; Smith, S. C.; Uehling, D. E.; Waterson, A. G.; Wood, E. R. Synthesis and Stereochemical Effects of PyrrolidinylAcetylenic Thieno[3,2-d]Pyrimidines as EGFR and ErbB-2 Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (1), 21−26. (98) Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D. M.; Smith, S. C.; Dickson, H. D.; Caferro, T. R.; Hinkle, K. W.; Stevens, K. L.; Dickerson, S. H.; Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J.; Uehling, D. E. Synthesis and Evaluation of Aniline Headgroups for Alkynyl Thienopyrimidine Dual EGFR/ErbB-2 Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (5), 1332−1336. (99) Nijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R. Design and Pharmacological Characterization of VUF14480, a Covalent Partial Agonist That Interacts with Cysteine 983.36 of the Human Histamine H4 Receptor. Br. J. Pharmacol. 2013, 170, 89−100. (100) Schapira, A.; Bate, G.; Kirkpatrick, P. Rasagiline. Nat. Rev. Drug Discovery 2005, 4 (8), 625−626. (101) Youdim, M. B. H.; Gross, A.; Finberg, J. P. M. Rasagiline [NPropargyl-1R(+)-Aminoindan], a Selective and Potent Inhibitor of Mitochondrial Monoamine Oxidase B. Br. J. Pharmacol. 2001, 132 (2), 500−506. (102) Wright, A. T.; Song, J. D.; Cravatt, B. F. A Suite of ActivityBased Probes for Human Cytochrome P450 Enzymes. J. Am. Chem. Soc. 2009, 131 (30), 10692−10700. (103) Wright, A. T.; Cravatt, B. F. Chemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In Vivo. Chem. Biol. 2007, 14 (9), 1043−1051. (104) van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C. Preventing Thiol-Yne Addition Improves the Specificity of StrainAS



Perspective

Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA Polymerase. J. Med. Chem. 2006, 49 (3), 1034−1046. (137) Huth, J. R.; Mendoza, R.; Olejniczak, E. T.; Johnson, R. W.; Cothron, D. A.; Liu, Y.; Lerner, C. G.; Chen, J.; Hajduk, P. J. ALARM NMR: A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens. J. Am. Chem. Soc. 2005, 127 (1), 217−224. (138) Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Bioactivation and Hepatotoxicity of Nitroaromatic Drugs. Curr. Drug Metab. 2006, 7, 715−727. (139) Zeng, Q.; Nair, A. G.; Rosenblum, S. B.; Huang, H.-C.; Lesburg, C. A.; Jiang, Y.; Selyutin, O.; Chan, T.-Y.; Bennett, F.; Chen, K. X.; Venkatraman, S.; Sannigrahi, M.; Velazquez, F.; Duca, J. S.; Gavalas, S.; Huang, Y.; Pu, H.; Wang, L.; Pinto, P.; Vibulbhan, B.; Agrawal, S.; Ferrari, E.; Jiang, C.; Li, C.; Hesk, D.; Gesell, J.; Sorota, S.; Shih, N.-Y.; Njoroge, F. G.; Kozlowski, J. A. Discovery of an Irreversible HCV NS5B Polymerase Inhibitor. Bioorg. Med. Chem. Lett. 2013, 23 (24), 6585−6587. (140) Sato, K.; Kunitomo, Y.; Kasai, Y.; Utsumi, S.; Suetake, I.; Tajima, S.; Ichikawa, S.; Matsuda, A. Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-CNucleoside. ChemBioChem 2018, 19 (8), 865−872. (141) Kasai, Y.; Sato, K.; Utsumi, S.; Ichikawa, S. Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor. ChemBioChem 2018, 19 (17), 1866−1872. (142) Gianatassio, R.; Lopchuk, J. M.; Wang, J.; Pan, C.-M.; Malins, L. R.; Prieto, L.; Brandt, T. A.; Collins, M. R.; Gallego, G. M.; Sach, N. W.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Amination. Science 2016, 351 (6270), 241−246. (143) Lopchuk, J. M.; Fjelbye, K.; Kawamata, Y.; Malins, L. R.; Pan, C.-M.; Gianatassio, R.; Wang, J.; Prieto, L.; Bradow, J.; Brandt, T. A.; Collins, M. R.; Elleraas, J.; Ewanicki, J.; Farrell, W.; Fadeyi, O. O.; Gallego, G. M.; Mousseau, J. J.; Oliver, R.; Sach, N. W.; Smith, J. K.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Heteroatom Functionalization: Development, Scope, and Stereospecificity. J. Am. Chem. Soc. 2017, 139 (8), 3209−3226. (144) Semmler, K.; Szeimies, G.; Belzner, J. Tetracyclo[5.1.0.01,6.02,7]Octane, a [1.1.1]Propellane Derivative, and a New Route to the Parent Hydrocarbon. J. Am. Chem. Soc. 1985, 107 (22), 6410−6411. (145) Haag, B.; Mosrin, M.; Ila, H.; Malakhov, V.; Knochel, P. Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide Bases. Angew. Chem., Int. Ed. 2011, 50 (42), 9794−9824. (146) Wishart, D. S.; Feunang, Y. D.; Guo, A. C.; Lo, E. J.; Marcu, A.; Grant, J. R.; Sajed, T.; Johnson, D.; Li, C.; Sayeeda, Z.; Assempour, N.; Iynkkaran, I.; Liu, Y.; Maciejewski, A.; Gale, N.; Wilson, A.; Chin, L.; Cummings, R.; Le, D.; Pon, A.; Knox, C.; Wilson, M. DrugBank 5.0: A Major Update to the DrugBank Database for 2018. Nucleic Acids Res. 2018, 46 (D1), D1074−D1082. (147) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of Fluorine in Medicinal Chemistry. J. Med. Chem. 2015, 58 (21), 8315−8359. (148) Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018, 61 (14), 5822−5880. (149) Grishin, N. V.; Osterman, A. L.; Brooks, H. B.; Phillips, M. A.; Goldsmith, E. J. X-Ray Structure of Ornithine Decarboxylase from Trypanosoma Brucei: The Native Structure and the Structure in Complex with α-Difluoromethylornithine. Biochemistry 1999, 38 (46), 15174−15184. (150) Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2’-Deoxyuridine 5′-Monophosphate. Biochemistry 1994, 33 (50), 15086−15094.

Serum-Stable Alternative to Maleimide-Based Protein Conjugation. Angew. Chem., Int. Ed. 2013, 52 (48), 12592−12596. (122) Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.; Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135 (16), 5946−5949. (123) Alapour, S.; de la Torre, B. G.; Ramjugernath, D.; Koorbanally, N. A.; Albericio, F. Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in Bioconjugation. Bioconjugate Chem. 2018, 29 (2), 225−233. (124) Brown, S. P.; Smith, A. B. Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/Protein. J. Am. Chem. Soc. 2015, 137 (12), 4034−4037. (125) Roberts, D. W.; Aptula, A. O. Electrophilic Reactivity and Skin Sensitization Potency of SNAr Electrophiles. Chem. Res. Toxicol. 2014, 27 (2), 240−246. (126) Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.; Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.; Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone. J. Biol. Chem. 2011, 286 (14), 11895−11908. (127) Arnold, L. A.; Kosinski, A.; Estébanez-Perpiña,́ E.; Guy, R. K. Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary Structure−Activity Relationships. J. Med. Chem. 2007, 50 (22), 5269−5280. (128) Visperas, P. R.; Winger, J. A.; Horton, T. M.; Shah, N. H.; Aum, D. J.; Tao, A.; Barros, T.; Yan, Q.; Wilson, C. G.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Modification by Covalent Reaction or Oxidation of Cysteine Residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding. Biochem. J. 2015, 465 (1), 149−161. (129) Visperas, P. R.; Wilson, C. G.; Winger, J. A.; Yan, Q.; Lin, K.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen. SLAS Discov. 2017, 22 (3), 324−331. (130) Shannon, D. A.; Banerjee, R.; Webster, E. R.; Bak, D. W.; Wang, C.; Weerapana, E. Investigating the Proteome Reactivity and Selectivity of Aryl Halides. J. Am. Chem. Soc. 2014, 136 (9), 3330− 3333. (131) Schardon, C. L.; Tuley, A.; Er, J. A. V.; Swartzel, J. C.; Fast, W. Selective Covalent Protein Modification by 4-Halopyridines through Catalysis. ChemBioChem 2017, 18 (15), 1551−1556. (132) Fairhurst, R. A.; Knoepfel, T.; Leblanc, C.; Buschmann, N.; Gaul, C.; Blank, J.; Galuba, I.; Trappe, J.; Zou, C.; Voshol, J.; Genick, C.; Brunet-Lefeuvre, P.; Bitsch, F.; Graus-Porta, D.; Furet, P. Approaches to Selective Fibroblast Growth Factor Receptor 4 Inhibition through Targeting the ATP-Pocket Middle-Hinge Region. MedChemComm 2017, 8, 1604−1613. (133) Hou, W.; Ren, Y.; Zhang, Z.; Sun, H.; Ma, Y.; Yan, B. Novel Quinazoline Derivatives Bearing Various 6-Benzamide Moieties as Highly Selective and Potent EGFR Inhibitors. Bioorg. Med. Chem. 2018, 26 (8), 1740−1750. (134) Juchum, M.; Günther, M.; Laufer, S. A. Fighting Cancer Drug Resistance: Opportunities and Challenges for Mutation-Specific EGFR Inhibitors. Drug Resist. Updates 2015, 20, 12−28. (135) Chen, K. X.; Lesburg, C. A.; Vibulbhan, B.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennett, F.; Anilkumar, G. N.; Duca, J.; Jiang, Y.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, F. G. A Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase Inhibitors. J. Med. Chem. 2012, 55 (5), 2089−2101. (136) Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P. C.; Wang, Z. SAR and Mode of Action of Novel AT



Perspective

(171) Falagas, M. E.; Vouloumanou, E. K.; Samonis, G.; Vardakas, K. Z. Fosfomycin. Clin. Microbiol. Rev. 2016, 29 (2), 321−347. (172) Kim, D. H.; Lees, W. J.; Kempsell, K. E.; Lane, W. S.; Duncan, K.; Walsh, C. T. Characterization of a Cys115 to Asp Substitution in the Escherichia Coli Cell Wall Biosynthetic Enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) That Confers Resistance to Inactivation by the Antibiotic Fosfomycin. Biochemistry 1996, 35 (15), 4923−4928. (173) Eschenburg, S.; Priestman, M.; Schönbrunn, E. Evidence That the Fosfomycin Target Cys115 in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Is Essential for Product Release. J. Biol. Chem. 2005, 280 (5), 3757−3763. (174) Porter, N. J.; Christianson, D. W. Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8. ACS Chem. Biol. 2017, 12 (9), 2281−2286. (175) Lopus, M.; Smiyun, G.; Miller, H.; Oroudjev, E.; Wilson, L.; Jordan, M. A. Mechanism of Action of Ixabepilone and Its Interactions with the ΒIII-Tubulin Isotype. Cancer Chemother. Pharmacol. 2015, 76 (5), 1013−1024. (176) Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap Portion. J. Med. Chem. 2010, 53 (5), 2038−2050. (177) Klüter, S.; Simard, J. R.; Rode, H. B.; Grütter, C.; Pawar, V.; Raaijmakers, H. C. A.; Barf, T. A.; Rabiller, M.; van Otterlo, W. A. L.; Rauh, D. Characterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug Resistance. ChemBioChem 2010, 11 (18), 2557−2566. (178) Gehringer, M.; Forster, M.; Laufer, S. A. Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide−Alkyne Cycloaddition. ACS Comb. Sci. 2015, 17 (1), 5−10. (179) Forster, M.; Chaikuad, A.; Bauer, S. M.; Holstein, J.; Robers, M. B.; Corona, C. R.; Gehringer, M.; Pfaffenrot, E.; Ghoreschi, K.; Knapp, S.; Laufer, S. A. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem. Biol. 2016, 23 (11), 1335−1340. (180) Rempel, B. P.; Withers, S. G. Covalent Inhibitors of Glycosidases and Their Applications in Biochemistry and Biology. Glycobiology 2008, 18 (8), 570−586. (181) Povirk, L. F.; Shuker, D. E. DNA Damage and Mutagenesis Induced by Nitrogen Mustards. Mutat. Res., Rev. Genet. Toxicol. 1994, 318 (3), 205−226. (182) McGregor, L. M.; Jenkins, M. L.; Kerwin, C.; Burke, J. E.; Shokat, K. M. Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes. Biochemistry 2017, 56 (25), 3178−3183. (183) Ray, S.; Kreitler, D. F.; Gulick, A. M.; Murkin, A. S. The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition. ACS Chem. Biol. 2018, 13 (6), 1470−1473. (184) Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178−187. (185) Krenske, E. H.; Petter, R. C.; Houk, K. N. Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to Cysteines. J. Org. Chem. 2016, 81 (23), 11726−11733. (186) Krishnan, S.; Miller, R. M.; Tian, B.; Mullins, R. D.; Jacobson, M. P.; Taunton, J. Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis. J. Am. Chem. Soc. 2014, 136 (36), 12624−12630. (187) Smith, S.; Keul, M.; Engel, J.; Basu, D.; Eppmann, S.; Rauh, D. Characterization of Covalent-Reversible EGFR Inhibitors. ACS Omega 2017, 2 (4), 1563−1575. (188) Forster, M.; Chaikuad, A.; Dimitrov, T.; Döring, E.; Holstein, J.; Berger, B.-T.; Gehringer, M.; Ghoreschi, K.; Müller, S.; Knapp, S.; Laufer, S. A. Development, Optimization, and Structure−Activity

(151) Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors. Science 2005, 308 (5726), 1318−1321. (152) Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. A Clickable Inhibitor Reveals Context-Dependent Autoactivation of P90 RSK. Nat. Chem. Biol. 2007, 3 (3), 156−160. (153) Carroll, F. A. Perspectives on Structure and Mechanism in Organic Chemistry, 2nd ed.; Wiley-VCH: Weinheim, 2014. (154) Shaik, S. S. The .Alpha.- and .Beta.-Carbon Substituent Effect on SN2 Reactivity. A Valence-Bond Approach. J. Am. Chem. Soc. 1983, 105 (13), 4359−4367. (155) Lundell, N.; Schreitmüller, T. Sample Preparation for Peptide Mapping A Pharmaceutical Quality-Control Perspective. Anal. Biochem. 1999, 266 (1), 31−47. (156) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes. Nature 2010, 468 (7325), 790−795. (157) Weerapana, E.; Simon, G. M.; Cravatt, B. F. Disparate Proteome Reactivity Profiles of Carbon Electrophiles. Nat. Chem. Biol. 2008, 4 (7), 405−407. (158) Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57 (12), 3124−3137. (159) Allimuthu, D.; Adams, D. J. 2-Chloropropionamide As a LowReactivity Electrophile for Irreversible Small-Molecule Probe Identification. ACS Chem. Biol. 2017, 12 (8), 2124−2131. (160) Steinmetz, C. G.; Xie, P.; Weiner, H.; Hurley, T. D. Structure of Mitochondrial Aldehyde Dehydrogenase: The Genetic Component of Ethanol Aversion. Structure 1997, 5 (5), 701−711. (161) Karala, A.-R.; Lappi, A.-K.; Ruddock, L. W. Modulation of an Active-Site Cysteine PKa Allows PDI to Act as a Catalyst of Both Disulfide Bond Formation and Isomerization. J. Mol. Biol. 2010, 396 (4), 883−892. (162) Wang, C.; Abegg, D.; Hoch, D. G.; Adibekian, A. Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT. Angew. Chem., Int. Ed. 2016, 55 (8), 2911−2915. (163) Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. Ruthenium-Catalyzed Azide−Alkyne Cycloaddition: Scope and Mechanism. J. Am. Chem. Soc. 2008, 130 (28), 8923−8930. (164) Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide Electrophiles as Activity-Dependent Cysteine Protease Profiling and Discovery Tools. Chem. Biol. 2000, 7 (8), 569−581. (165) Willems, L. I.; Jiang, J.; Li, K.-Y.; Witte, M. D.; Kallemeijn, W. W.; Beenakker, T. J. N.; Schröder, S. P.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase Probes. Chem. Eur. J. 2014, 20 (35), 10864−10872. (166) Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P. N -Alkylated Aziridines Are Easily-Prepared, Potent, Specific and Cell-Permeable Covalent Inhibitors of Human β-Glucocerebrosidase. Chem. Commun. 2015, 51 (57), 11390−11393. (167) Singh, G. S. Synthetic Aziridines in Medicinal Chemistry: A Mini-Review. Mini-Rev. Med. Chem. 2016, 16, 892−904. (168) Pitscheider, M.; Mäusbacher, N.; Sieber, S. A. Antibiotic Activity and Target Discovery of Three-Membered Natural ProductDerived Heterocycles in Pathogenic Bacteria. Chem. Sci. 2012, 3, 2035−2041. (169) Lee, M.; Ikejiri, M.; Klimpel, D.; Toth, M.; Espahbodi, M.; Hesek, D.; Forbes, C.; Kumarasiri, M.; Noll, B. C.; Chang, M.; Mobashery, S. Structure−Activity Relationship for Thiirane-Based Gelatinase Inhibitors. ACS Med. Chem. Lett. 2012, 3 (6), 490−495. (170) Harshbarger, W.; Miller, C.; Diedrich, C.; Sacchettini, J. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure 2015, 23 (2), 418−424. AU



Perspective

Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]Pyrrolo[2,3-b]Pyridine Scaffold. J. Med. Chem. 2018, 61 (12), 5350−5366. (189) Cheung, S. T.; Miller, M. S.; Pacoma, R.; Roland, J.; Liu, J.; Schumacher, A. M.; Hsieh-Wilson, L. C. Discovery of a SmallMolecule Modulator of Glycosaminoglycan Sulfation. ACS Chem. Biol. 2017, 12 (12), 3126−3133. (190) Cully, M. Rational Drug Design: Tuning Kinase Inhibitor Residence Time. Nat. Rev. Drug Discovery 2015, 14, 457. (191) Langrish, C. L.; Bradshaw, J. M.; Owens, T. D.; Campbell, R. L.; Francesco, M. R.; Karr, D. E.; Murray, S. K.; Quesenberry, R. C.; Smith, P. F.; Taylor, M. D.; Zhu, J.; Nunn, P. A.; Gourlay, S. G. PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura. Blood 2017, 130, 1052. (192) Masjedizadeh, M. R.; Gourlay, S. Salts and Solid Form of a Btk Inhibitor. WO2015127310, August 28, 2015. (193) Holm, K. J.; Spencer, C. M. Entacapone. Drugs 1999, 58 (1), 159−177. (194) Mendgen, T.; Steuer, C.; Klein, C. D. Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal Chemistry. J. Med. Chem. 2012, 55 (2), 743−753. (195) Schneider, T. H.; Rieger, M.; Ansorg, K.; Sobolev, A. N.; Schirmeister, T.; Engels, B.; Grabowsky, S. Vinyl Sulfone Building Blocks in Covalently Reversible Reactions with Thiols. New J. Chem. 2015, 39 (7), 5841−5853. (196) Siklos, M.; BenAissa, M.; Thatcher, G. R. J. Cysteine Proteases as Therapeutic Targets: Does Selectivity Matter? A Systematic Review of Calpain and Cathepsin Inhibitors. Acta Pharm. Sin. B 2015, 5 (6), 506−519. (197) Cleary, J. A.; Doherty, W.; Evans, P.; Malthouse, J. P. G. Quantifying Tetrahedral Adduct Formation and Stabilization in the Cysteine and the Serine Proteases. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 1382−1391. (198) Sanches, M.; Duffy, N. M.; Talukdar, M.; Thevakumaran, N.; Chiovitti, D.; Canny, M. D.; Lee, K.; Kurinov, I.; Uehling, D.; Al-awar, R.; Poda, G.; Prakesch, M.; Wilson, B.; Tam, V.; Schweitzer, C.; Toro, A.; Lucas, J. L.; Vuga, D.; Lehmann, L.; Durocher, D.; Zeng, Q.; Patterson, J. B.; Sicheri, F. Structure and Mechanism of Action of the Hydroxy−Aryl−Aldehyde Class of IRE1 Endoribonuclease Inhibitors. Nat. Commun. 2014, 5, 4202. (199) Larraufie, M.-H.; Yang, W. S.; Jiang, E.; Thomas, A. G.; Slusher, B. S.; Stockwell, B. R. Incorporation of Metabolically Stable Ketones into a Small Molecule Probe to Increase Potency and Water Solubility. Bioorg. Med. Chem. Lett. 2015, 25 (21), 4787−4792. (200) Cross, B. C. S.; Bond, P. J.; Sadowski, P. G.; Jha, B. K.; Zak, J.; Goodman, J. M.; Silverman, R. H.; Neubert, T. A.; Baxendale, I. R.; Ron, D.; Harding, H. P. The Molecular Basis for Selective Inhibition of Unconventional MRNA Splicing by an IRE1-Binding Small Molecule. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (15), E869−E878. (201) Knoepfel, T.; Furet, P.; Mah, R.; Buschmann, N.; Leblanc, C.; Ripoche, S.; Graus-Porta, D.; Wartmann, M.; Galuba, I.; Fairhurst, R. A. 2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4. ACS Med. Chem. Lett. 2018, 9 (3), 215−220. (202) LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 1081−1091. (203) Caraballo, R.; Dong, H.; Ribeiro, J. P.; Jiménez-Barbero, J.; Ramström, O. Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System. Angew. Chem., Int. Ed. 2010, 49 (3), 589−593. (204) Buschmann, N.; Fairhurst, R. A.; Knoepfel, T.; Furet, P.; Leblanc, C.; Mah, R.; Kiffe, M.; Graus-Porta, D.; Weiss, A.; KinyamuAkunda, J.; Wartmann, M.; Trappe, J.; Gabriel, T. R.; Hofmann, F.; Sellers, W. R. A Reversible Covalent Approach to Selective FGFR4 Inhibition; Book of AbstractsFrontiers in Medicinal Chemistry Symposium, Jena, 2018; p 26.

(205) Otto, H.-H.; Schirmeister, T. Cysteine Proteases and Their Inhibitors. Chem. Rev. 1997, 97 (1), 133−172. (206) Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. Discovery and Preclinical Profile of Saxagliptin (BMS-477118): A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes. J. Med. Chem. 2005, 48 (15), 5025−5037. (207) Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. Nitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile Pharmacophore. J. Med. Chem. 2010, 53 (22), 7902−7917. (208) Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A Generally Applicable Method for Assessing the Electrophilicity and Reactivity of Diverse NitrileContaining Compounds. Bioorg. Med. Chem. Lett. 2007, 17 (4), 998− 1002. (209) Schmitz, J.; Beckmann, A.-M.; Dudic, A.; Li, T.; Sellier, R.; Bartz, U.; Gütschow, M. 3-Cyano-3-Aza-β-Amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins. ACS Med. Chem. Lett. 2014, 5 (10), 1076−1081. (210) Cai, J.; Fradera, X.; van Zeeland, M.; Dempster, M.; Cameron, K. S.; Bennett, D. J.; Robinson, J.; Popplestone, L.; Baugh, M.; Westwood, P.; Bruin, J.; Hamilton, W.; Kinghorn, E.; Long, C.; Uitdehaag, J. C. M. 4-(3-Trifluoromethylphenyl)-Pyrimidine-2Carbonitrile as Cathepsin S Inhibitors: N3, Not N1 Is Critically Important. Bioorg. Med. Chem. Lett. 2010, 20 (15), 4507−4510. (211) Mac Sweeney, A.; Grosche, P.; Ellis, D.; Combrink, K.; Erbel, P.; Hughes, N.; Sirockin, F.; Melkko, S.; Bernardi, A.; Ramage, P.; Jarousse, N.; Altmann, E. Discovery and Structure-Based Optimization of Adenain Inhibitors. ACS Med. Chem. Lett. 2014, 5 (8), 937− 941. (212) de Jesus Cortez, F.; Nguyen, P.; Truillet, C.; Tian, B.; Kuchenbecker, K. M.; Evans, M. J.; Webb, P.; Jacobson, M. P.; Fletterick, R. J.; England, P. M. Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen. ACS Chem. Biol. 2017, 12 (12), 2934−2939. (213) Deaton, D. N.; Hassell, A. M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.; Shewchuk, L. M.; Tavares, F. X.; Willard, D. H.; Wright, L. L. Novel and Potent Cyclic Cyanamide-Based Cathepsin K Inhibitors. Bioorg. Med. Chem. Lett. 2005, 15 (7), 1815−1819. (214) Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S. B.; Percival, M. D. Novel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and L. J. Med. Chem. 2001, 44 (1), 94−104. (215) Rydzewski, R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S. B.; Bass, K. E.; Wong, D. H. Peptidic 1-Cyanopyrrolidines: Synthesis and SAR of a Series of Potent, Selective Cathepsin Inhibitors. Bioorg. Med. Chem. 2002, 10 (10), 3277−3284. (216) Lainé, D.; Palovich, M.; McCleland, B.; Petitjean, E.; Delhom, I.; Xie, H.; Deng, J.; Lin, G.; Davis, R.; Jolit, A.; Nevins, N.; Zhao, B.; Villa, J.; Schneck, J.; McDevitt, P.; Midgett, R.; Kmett, C.; Umbrecht, S.; Peck, B.; Davis, A. B.; Bettoun, D. Discovery of Novel CyanamideBased Inhibitors of Cathepsin C. ACS Med. Chem. Lett. 2011, 2 (2), 142−147. (217) Hill, S. V.; Williams, A.; Longridge, J. L. Acid-Catalysed Hydrolysis of Cyanamides: Estimates of Carbodi-Imide Basicity and Tautomeric Equilibrium Constant between Carbodi-Imide and Cyanamide. J. Chem. Soc., Perkin Trans. 2 1984, 0 (6), 1009−1013. (218) Benson, M. J.; Rodriguez, V.; von Schack, D.; Keegan, S.; Cook, T. A.; Edmonds, J.; Benoit, S.; Seth, N.; Du, S.; Messing, D.; Nickerson-Nutter, C. L.; Dunussi-Joannopoulos, K.; Rankin, A. L.; Ruzek, M.; Schnute, M. E.; Douhan, J. Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune System. J. Immunol. 2014, 193 (1), 185. AV



Perspective

(219) Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (1), 173−178. (220) Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay. J. Med. Chem. 2012, 55 (22), 10047−10063. (221) Gupta, P.; Wright, S. E.; Kim, S.-H.; Srivastava, S. K. Phenethyl Isothiocyanate: A Comprehensive Review of Anti-Cancer Mechanisms. Biochim. Biophys. Acta, Rev. Cancer 2014, 1846 (2), 405−424. (222) Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. TRP Channel Activation by Reversible Covalent Modification. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (51), 19564−19568. (223) van Bladeren, P. J. Glutathione Conjugation as a Bioactivation Reaction. Chem.-Biol. Interact. 2000, 129 (1), 61−76. (224) Drobnica, L.; Kristián, P.; Augustín, J. The Chemistry of the NCS Group. Cyanates Their Thio Derivatives; Patai, P., Ed.; Wiley: New York, 1977; Part 2, pp 1003−1221. (225) Shibata, T.; Kimura, Y.; Mukai, A.; Mori, H.; Ito, S.; Asaka, Y.; Oe, S.; Tanaka, H.; Takahashi, T.; Uchida, K. Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates. J. Biol. Chem. 2011, 286, 42150. (226) Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y. Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine. Chem. Res. Toxicol. 2009, 22 (3), 536−542. (227) Kumari, V.; Dyba, M. A.; Holland, R. J.; Liang, Y.-H.; Singh, S. V.; Ji, X. Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 2016, 11 (9), e0163821. (228) Wilson, A. J.; Kerns, J. K.; Callahan, J. F.; Moody, C. J. Keap Calm, and Carry on Covalently. J. Med. Chem. 2013, 56 (19), 7463− 7476. (229) Lewis, S. M.; Li, Y.; Catalano, M. J.; Laciak, A. R.; Singh, H.; Seiner, D. R.; Reilly, T. J.; Tanner, J. J.; Gates, K. S. Inactivation of Protein Tyrosine Phosphatases by Dietary Isothiocyanates. Bioorg. Med. Chem. Lett. 2015, 25 (20), 4549−4552. (230) Cross, J. V.; Foss, F. W.; Rady, J. M.; Macdonald, T. L.; Templeton, D. J. The Isothiocyanate Class of Bioactive Nutrients Covalently Inhibit the MEKK1 Protein Kinase. BMC Cancer 2007, 7 (1), 183. (231) Mi, L.; Xiao, Z.; Hood, B. L.; Dakshanamurthy, S.; Wang, X.; Govind, S.; Conrads, T. P.; Veenstra, T. D.; Chung, F.-L. Covalent Binding to Tubulin by Isothiocyanates: A Mechanism of Cell Growth Arrest and Apoptosis. J. Biol. Chem. 2008, 283 (32), 22136−22146. (232) Ouertatani-Sakouhi, H.; El-Turk, F.; Fauvet, B.; Roger, T.; Le Roy, D.; Karpinar, D. P.; Leng, L.; Bucala, R.; Zweckstetter, M.; Calandra, T.; Lashuel, H. A. A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor. Biochemistry 2009, 48 (41), 9858−9870. (233) Brown, K. K.; Hampton, M. B. Biological Targets of Isothiocyanates. Biochim. Biophys. Acta, Gen. Subj. 2011, 1810 (9), 888−894. (234) Pearson, R. J.; Blake, D. G.; Mezna, M.; Fischer, P. M.; Westwood, N. J.; McInnes, C. The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1. Cell Chem. Biol. 2018, 25, 1107−1116. (235) Federici, L.; Lo Sterzo, C.; Pezzola, S.; Di Matteo, A. D.; Scaloni, F.; Federici, G.; Caccuri, A. M. Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases. Cancer Res. 2009, 69 (20), 8025−8034.

(236) Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Site-Directed Ligand Discovery. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (17), 9367−9372. (237) Zong, L.; Bartolami, E.; Abegg, D.; Adibekian, A.; Sakai, N.; Matile, S. Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension. ACS Cent. Sci. 2017, 3 (5), 449−453. (238) Tjin, C. C.; Otley, K. D.; Baguley, T. D.; Kurup, P.; Xu, J.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases. ACS Cent. Sci. 2017, 3 (12), 1322−1328. (239) Weichert, D.; Gmeiner, P. Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications. ACS Chem. Biol. 2015, 10 (6), 1376−1386. (240) Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G. F.; Choi, H.-J.; DeVree, B. T.; Sunahara, R. K.; Chae, P. S.; Gellman, S. H.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Caffrey, M.; Gmeiner, P.; Kobilka, B. K. Structure and Function of an Irreversible Agonist-Β2 Adrenoceptor Complex. Nature 2011, 469 (7329), 236−240. (241) Schwalbe, T.; Kaindl, J.; Hübner, H.; Gmeiner, P. Potent Haloperidol Derivatives Covalently Binding to the Dopamine D2 Receptor. Bioorg. Med. Chem. 2017, 25 (19), 5084−5094. (242) Liu, Y.; Xie, Z.; Zhao, D.; Zhu, J.; Mao, F.; Tang, S.; Xu, H.; Luo, C.; Geng, M.; Huang, M.; Li, J. Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors. J. Med. Chem. 2017, 60 (6), 2227−2244. (243) Napolitano, L.; Scalise, M.; Koyioni, M.; Koutentis, P.; Catto, M.; Eberini, I.; Parravicini, C.; Palazzolo, L.; Pisani, L.; Galluccio, M.; Console, L.; Carotti, A.; Indiveri, C. Potent Inhibitors of Human LAT1 (SLC7A5) Transporter Based on Dithiazole and Dithiazine Compounds for Development of Anticancer Drugs. Biochem. Pharmacol. 2017, 143, 39−52. (244) Nagy, P. Kinetics and Mechanisms of Thiol−Disulfide Exchange Covering Direct Substitution and Thiol OxidationMediated Pathways. Antioxid. Redox Signaling 2013, 18 (13), 1623−1641. (245) Go, Y.-M.; Jones, D. P. Thiol/Disulfide Redox States in Signaling and Sensing. Crit. Rev. Biochem. Mol. Biol. 2013, 48 (2), 173−181. (246) García-Santamarina, S.; Boronat, S.; Hidalgo, E. Reversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal Transduction. Biochemistry 2014, 53 (16), 2560−2580. (247) Gupta, V.; Carroll, K. S. Profiling the Reactivity of Cyclic CNucleophiles towards Electrophilic Sulfur in Cysteine Sulfenic Acid. Chem. Sci. 2016, 7 (1), 400−415. (248) Truong, T. H.; Carroll, K. S. Redox Regulation of Epidermal Growth Factor Receptor Signaling through Cysteine Oxidation. Biochemistry 2012, 51 (50), 9954−9965. (249) Garcia, F. J.; Carroll, K. S. Redox-Based Probes as Tools to Monitor Oxidized Protein Tyrosine Phosphatases in Living Cells. Eur. J. Med. Chem. 2014, 88, 28−33. (250) Alcock, L. J.; Farrell, K. D.; Akol, M. T.; Jones, G. H.; Tierney, M. M.; Kramer, H. B.; Pukala, T. L.; Bernardes, G. J. L.; Perkins, M. V.; Chalker, J. M. Norbornene Probes for the Study of Cysteine Oxidation. Tetrahedron 2018, 74 (12), 1220−1228. (251) Poole, T. H.; Reisz, J. A.; Zhao, W.; Poole, L. B.; Furdui, C. M.; King, S. B. Strained Cycloalkynes as New Protein Sulfenic Acid Traps. J. Am. Chem. Soc. 2014, 136 (17), 6167−6170. (252) Gupta, V.; Carroll, K. S. Rational Design of Reversible and Irreversible Cysteine Sulfenic Acid-Targeted Linear C-Nucleophiles. Chem. Commun. 2016, 52 (16), 3414−3417. (253) Forman, H. J.; Davies, M. J.; Krämer, A. C.; Miotto, G.; Zaccarin, M.; Zhang, H.; Ursini, F. Protein Cysteine Oxidation in Redox Signaling: Caveats on Sulfenic Acid Detection and Quantification. Arch. Biochem. Biophys. 2017, 617, 26−37. AW



Perspective

(254) Gupta, V.; Yang, J.; Liebler, D. C.; Carroll, K. S. Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles. J. Am. Chem. Soc. 2017, 139 (15), 5588−5595. (255) Holliday, G. L.; Mitchell, J. B. O.; Thornton, J. M. Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis. J. Mol. Biol. 2009, 390 (3), 560−577. (256) Platzer, G.; Okon, M.; McIntosh, L. P. PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements. J. Biomol. NMR 2014, 60 (2−3), 109−129. (257) Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; Garcia-Moreno, B. E. Large Shifts in PKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (13), 5260−5265. (258) Hacker, S. M.; Backus, K. M.; Lazear, M. R.; Forli, S.; Correia, B. E.; Cravatt, B. F. Global Profiling of Lysine Reactivity and Ligandability in the Human Proteome. Nat. Chem. 2017, 9 (12), 1181−1190. (259) Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine. Mol. Cell 2000, 6 (4), 909−919. (260) Wymann, M. P.; Bulgarelli-Leva, G.; Zvelebil, M. J.; Pirola, L.; Vanhaesebroeck, B.; Waterfield, M. D.; Panayotou, G. Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction. Mol. Cell. Biol. 1996, 16 (4), 1722−1733. (261) Pettinger, J.; Le Bihan, Y.-V.; Widya, M.; van Montfort, R. L. M.; Jones, K.; Cheeseman, M. D. An Irreversible Inhibitor of HSP72 That Unexpectedly Targets Lysine-56. Angew. Chem., Int. Ed. 2017, 56 (13), 3536−3540. (262) Altmeyer, M.; Amtmann, E.; Heyl, C.; Marschner, A.; Scheidig, A. J.; Klein, C. D. Beta-Aminoketones as Prodrugs for Selective Irreversible Inhibitors of Type-1 Methionine Aminopeptidases. Bioorg. Med. Chem. Lett. 2014, 24 (22), 5310−5314. (263) Dahal, U. P.; Gilbert, A. M.; Obach, R. S.; Flanagan, M. E.; Chen, J. M.; Garcia-Irizarry, C.; Starr, J. T.; Schuff, B.; Uccello, D. P.; Young, J. A. Intrinsic Reactivity Profile of Electrophilic Moieties to Guide Covalent Drug Design: N-α-Acetyl-L-Lysine as an Amine Nucleophile. MedChemComm 2016, 7 (5), 864−872. (264) Anscombe, E.; Meschini, E.; Mora-Vidal, R.; Martin, M. P.; Staunton, D.; Geitmann, M.; Danielson, U. H.; Stanley, W. A.; Wang, L. Z.; Reuillon, T.; Golding, B. T.; Cano, C.; Newell, D. R.; Noble, M. E. M.; Wedge, S. R.; Endicott, J. A.; Griffin, R. J. Identification and Characterization of an Irreversible Inhibitor of CDK2. Chem. Biol. 2015, 22 (9), 1159−1164. (265) Narayanan, A.; Jones, L. H. Sulfonyl Fluorides as Privileged Warheads in Chemical Biology. Chem. Sci. 2015, 6 (5), 2650−2659. (266) Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry. Angew. Chem., Int. Ed. 2014, 53 (36), 9430−9448. (267) Baker, B. R. Irreversible Enzyme Inhibitors. CXLIX. TissueSpecific Irreversible Inhibitors of Dihydrofolic Reductase. Acc. Chem. Res. 1969, 2 (5), 129−136. (268) Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016, 138 (23), 7353−7364. (269) Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I. A.; Kelly, J. W. “Inverse Drug Discovery” Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1), 200−210. (270) James, G. T. Inactivation of the Protease Inhibitor Phenylmethylsulfonyl Fluoride in Buffers. Anal. Biochem. 1978, 86 (2), 574−579.

(271) Lively, M. O.; Powers, J. C. Specificity and Reactivity of Human Granulocyte Elastase and Cathepsin G, Porcine Pancreatic Elastase, Bovine Chymotrypsin and Trypsin toward Inhibition with Sulfonyl Flourides. Biochim. Biophys. Acta BBA - Enzymol. 1978, 525 (1), 171−179. (272) Genov, N. C.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G. Intramolecular Distances between Tryptophan Residues and the Active-Site Serine Residue in Alkaline Bacterial Proteinases as Measured by Fluorescence Energy-Transfer Studies. Biochem. J. 1983, 215 (2), 413−416. (273) Esch, F. S.; Allison, W. S. Identification of a Tyrosine Residue at a Nucleotide Binding Site in the Beta Subunit of the Mitochondrial ATPase with P-Fluorosulfonyl[14C]-Benzoyl-5′-Adenosine. J. Biol. Chem. 1978, 253, 6100−6106. (274) Jörg, M.; Glukhova, A.; Abdul-Ridha, A.; Vecchio, E. A.; Nguyen, A. T. N.; Sexton, P. M.; White, P. J.; May, L. T.; Christopoulos, A.; Scammells, P. J. Novel Irreversible Agonists Acting at the A1 Adenosine Receptor. J. Med. Chem. 2016, 59 (24), 11182− 11194. (275) Yang, X.; Dong, G.; Michiels, T. J. M.; Lenselink, E. B.; Heitman, L.; Louvel, J.; IJzerman, A. P. A Covalent Antagonist for the Human Adenosine A2A Receptor. Purinergic Signalling 2017, 13 (2), 191−201. (276) Beauglehole, A. R.; Baker, S. P.; Scammells, P. J. Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine Receptor. J. Med. Chem. 2000, 43 (26), 4973−4980. (277) Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867−877. (278) Kitz, R.; Wilson, I. B. Esters of Methanesulfonic Acid as Irreversible Inhibitors of Acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245−3249. (279) Moss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C. Methanesulfonyl Fluoride (MSF): A Double-Blind, PlaceboControlled Study of Safety and Efficacy in the Treatment of Senile Dementia of the Alzheimer Type. Alzheimer Dis. Assoc. Disord. 1999, 13 (1), 20. (280) Moss, D. E.; Fariello, R. G.; Sahlmann, J.; Sumaya, I.; Pericle, F.; Braglia, E. A Randomized Phase I Study of Methanesulfonyl Fluoride, an Irreversible Cholinesterase Inhibitor, for the Treatment of Alzheimer’s Disease. Br. J. Clin. Pharmacol. 2013, 75 (5), 1231− 1239. (281) Jones, L. H. Reactive Chemical Probes: Beyond the Kinase Cysteinome. Angew. Chem., Int. Ed. 2018, 57 (30), 9220−9223. (282) Mukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P. A Study of the Reactivity of S(VI)−F Containing Warheads with Nucleophilic Amino-Acid Side Chains under Physiological Conditions. Org. Biomol. Chem. 2017, 15 (45), 9685−9695. (283) Lundblad, R. L. Chemical Reagents for Protein Modification, 4th ed.; CRC Press: Boca Raton, 2017. (284) Chinthakindi, P. K.; Arvidsson, P. I. Sulfonyl Fluorides (SFs): More Than Click Reagents? Eur. J. Org. Chem. 2018, 2018 (27−28), 3648−3666. (285) Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.; Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding Fluorosulfate-l-Tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140 (15), 4995−4999. (286) Zhao, Q.; Ouyang, X.; Wan, X.; Gajiwala, K. S.; Kath, J. C.; Jones, L. H.; Burlingame, A. L.; Taunton, J. Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes. J. Am. Chem. Soc. 2017, 139 (2), 680−685. (287) Becher, I.; Savitski, M. M.; Savitski, M. F.; Hopf, C.; Bantscheff, M.; Drewes, G. Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP. ACS Chem. Biol. 2013, 8 (3), 599−607. AX



Perspective

(288) Baranczak, A.; Liu, Y.; Connelly, S.; Du, W.-G. H.; Greiner, E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encalada, S. E.; Kelly, J. W. A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis Elegans. J. Am. Chem. Soc. 2015, 137 (23), 7404−7414. (289) Grimster, N. P.; Connelly, S.; Baranczak, A.; Dong, J.; Krasnova, L. B.; Sharpless, K. B.; Powers, E. T.; Wilson, I. A.; Kelly, J. W. Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis While Affording a Fluorescent Conjugate. J. Am. Chem. Soc. 2013, 135 (15), 5656− 5668. (290) Dalton, S. E.; Dittus, L.; Thomas, D. A.; Convery, M. A.; Nunes, J.; Bush, J. T.; Evans, J. P.; Werner, T.; Bantscheff, M.; Murphy, J. A.; Campos, S. Selectively Targeting the KinomeConserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition. J. Am. Chem. Soc. 2018, 140 (3), 932−939. (291) Gupta, R. C.; Sachana, M.; Mukherjee, I. M.; Doss, R. B.; Malik, J. K.; Milatovic, D. Organophosphates and Carbamates. In Veterinary Toxicology; Gupta, R. C., Ed.; Academic Press, 2018; pp. 495−508, DOI: 10.1016/B978-0-12-811410-0.00037-4. (292) Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Rapid Labelling and Covalent Inhibition of Intracellular Native Proteins Using Ligand-Directed N -Acyl- N -Alkyl Sulfonamide. Nat. Commun. 2018, 9 (1), 1870. (293) Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F. Target Discovery in Small-Molecule Cell-Based Screens by in Situ Proteome Reactivity Profiling. Nat. Biotechnol. 2005, 23, 1303. (294) Evans, M. J.; Morris, G. M.; Wu, J.; Olson, A. J.; Sorensen, E. J.; Cravatt, B. F. Mechanistic and Structural Requirements for Active Site Labeling of Phosphoglycerate Mutase by Spiroepoxides. Mol. BioSyst. 2007, 3, 495−506. (295) Bongard, J.; Lorenz, M.; Vetter, I. R.; Stege, P.; Porfetye, A. T.; Schmitz, A. L.; Kaschani, F.; Wolf, A.; Koch, U.; Nussbaumer, P.; Klebl, B.; Kaiser, M.; Ehrmann, M. Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent Probes. ACS Chem. Biol. 2018, 13 (5), 1307−1312. (296) Diethelm, S.; Schafroth, M. A.; Carreira, E. M. AmineSelective Bioconjugation Using Arene Diazonium Salts. Org. Lett. 2014, 16 (15), 3908−3911. (297) Tung, C. L.; Wong, C. T. T.; Fung, E. Y. M.; Li, X. Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification. Org. Lett. 2016, 18 (11), 2600−2603. (298) Ritter, E.; Przybylski, P.; Brzezinski, B.; Bartl, F. Schiff Bases in Biological Systems. Curr. Org. Chem. 2009, 13 (3), 241−249. (299) Malátková, P.; Wsól, V. Carbonyl Reduction Pathways in Drug Metabolism. Drug Metab. Rev. 2014, 46 (1), 96−123. (300) Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A. V.; Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. Iminoboronates: A New Strategy for Reversible Protein Modification. J. Am. Chem. Soc. 2012, 134 (24), 10299−10305. (301) Adams, J.; Kauffman, M. Development of the Proteasome Inhibitor VelcadeTM (Bortezomib). Cancer Invest. 2004, 22 (2), 304− 311. (302) Bandyopadhyay, A.; McCarthy, K. A.; Kelly, M. A.; Gao, J. Targeting Bacteria via Iminoboronate Chemistry of Amine-Presenting Lipids. Nat. Commun. 2015, 6, 6561. (303) Bandyopadhyay, A.; Gao, J. Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral PH. Chem. - Eur. J. 2015, 21 (42), 14748−14752. (304) Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through Covalent Modification of a Noncatalytic Lysine Side Chain. Nat. Chem. Biol. 2016, 12 (11), 931− 936. (305) Harris, T. K.; Turner, G. J. Structural Basis of Perturbed PKa Values of Catalytic Groups in Enzyme Active Sites. IUBMB Life 2002, 53 (2), 85−98.

(306) Schwans, J. P.; Sunden, F.; Gonzalez, A.; Tsai, Y.; Herschlag, D. Uncovering the Determinants of a Highly Perturbed Tyrosine PKa in the Active Site of Ketosteroid Isomerase. Biochemistry 2013, 52 (44), 7840−7855. (307) Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H. Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes. ACS Chem. Biol. 2015, 10 (4), 1094−1098. (308) Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A ThreeComponent Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 15942−15943. (309) Jones, M. W.; Mantovani, G.; Blindauer, C. A.; Ryan, S. M.; Wang, X.; Brayden, D. J.; Haddleton, D. M. Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts. J. Am. Chem. Soc. 2012, 134 (17), 7406− 7413. (310) Ban, H.; Gavrilyuk, J.; Barbas, C. F. Tyrosine Bioconjugation through Aqueous Ene-Type Reactions: A Click-Like Reaction for Tyrosine. J. Am. Chem. Soc. 2010, 132 (5), 1523−1525. (311) Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjugate Chem. 2013, 24 (4), 520−532. (312) Hatcher, J. M.; Wu, G.; Zeng, C.; Zhu, J.; Meng, F.; Patel, S.; Wang, W.; Ficarro, S. B.; Leggett, A. L.; Powell, C. E.; Marto, J. A.; Zhang, K.; Ngo, J. C. K.; Fu, X.-D.; Zhang, T.; Gray, N. S. SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform. Cell Chem. Biol. 2018, 25, 460−470. (313) Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou, D.; Zhang, J.; Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.; Nordin, B.; Weissig, H.; Yang, Q.; Lee, J.-D.; Gray, N. S.; Kozarich, J. W. In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases. Chem. Biol. 2011, 18 (6), 699−710. (314) Sakamoto, H.; Tsukaguchi, T.; Hiroshima, S.; Kodama, T.; Kobayashi, T.; Fukami, T. A.; Oikawa, N.; Tsukuda, T.; Ishii, N.; Aoki, Y. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell 2011, 19 (5), 679−690. (315) Choi, E. J.; Jung, D.; Kim, J.-S.; Lee, Y.; Kim, B. M. Chemoselective Tyrosine Bioconjugation through Sulfate Click Reaction. Chem. - Eur. J. 2018, 24 (43), 10948−10952. (316) Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.; Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue. ACS Chem. Biol. 2017, 12 (8), 2015−2020. (317) Crawford, L. A.; Weerapana, E. A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1). Mol. BioSyst. 2016, 12 (6), 1768−1771. (318) Gehringer, M.; Forster, M.; Pfaffenrot, E.; Bauer, S. M.; Laufer, S. A. Novel Hinge-Binding Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure−Activity Relationship Study on Tofacitinib Bioisosteres. ChemMedChem 2014, 9 (11), 2516− 2527. (319) Gu, C.; Shannon, D. A.; Colby, T.; Wang, Z.; Shabab, M.; Kumari, S.; Villamor, J. G.; McLaughlin, C. J.; Weerapana, E.; Kaiser, M.; Cravatt, B. F.; van der Hoorn, R. A. L. Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases. Chem. Biol. 2013, 20 (4), 541−548. (320) Ekici, Ö . D.; Paetzel, M.; Dalbey, R. E. Unconventional Serine Proteases: Variations on the Catalytic Ser/His/Asp Triad Configuration. Protein Sci. 2008, 17 (12), 2023−2037. (321) Long, J. Z.; Cravatt, B. F. The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease. Chem. Rev. 2011, 111 (10), 6022−6063. (322) Lei, J.; Zhou, Y.; Xie, D.; Zhang, Y. Mechanistic Insights into a Classic Wonder DrugAspirin. J. Am. Chem. Soc. 2015, 137 (1), 70− 73. AY



Perspective

(341) Morgen, M.; Jöst, C.; Malz, M.; Janowski, R.; Niessing, D.; Klein, C. D.; Gunkel, N.; Miller, A. K. Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity. ACS Chem. Biol. 2016, 11 (4), 1001−1011. (342) Jakob, C. G.; Upadhyay, A. K.; Donner, P. L.; Nicholl, E.; Addo, S. N.; Qiu, W.; Ling, C.; Gopalakrishnan, S. M.; Torrent, M.; Cepa, S. P.; Shanley, J.; Shoemaker, A. R.; Sun, C. C.; Vasudevan, A.; Woller, K. R.; Shotwell, J. B.; Shaw, B.; Bian, Z.; Hutti, J. E. Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315. J. Med. Chem. 2018, 61 (15), 6647−6657. (343) Lin, S.; Yang, X.; Jia, S.; Weeks, A. M.; Hornsby, M.; Lee, P. S.; Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.; Chang, C. J. Redox-Based Reagents for Chemoselective Methionine Bioconjugation. Science 2017, 355 (6325), 597−602. (344) Bizet, V.; Hendriks, C. M. M.; Bolm, C. Sulfur Imidations: Access to Sulfimides and Sulfoximines. Chem. Soc. Rev. 2015, 44 (11), 3378−3390. (345) Gong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A. Releasable and Traceless PEGylation of Arginine-Rich Antimicrobial Peptides. Chem. Sci. 2017, 8 (5), 4082−4086. (346) Seki, Y.; Ishiyama, T.; Sasaki, D.; Abe, J.; Sohma, Y.; Oisaki, K.; Kanai, M. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 2016, 138 (34), 10798−10801. (347) Shibata, Y.; Chiba, M. The Role of Extrahepatic Metabolism in the Pharmacokinetics of the Targeted Covalent Inhibitors Afatinib, Ibrutinib, and Neratinib. Drug Metab. Dispos. 2015, 43 (3), 375−384. (348) Scheers, E.; Leclercq, L.; de Jong, J.; Bode, N.; Bockx, M.; Laenen, A.; Cuyckens, F.; Skee, D.; Murphy, J.; Sukbuntherng, J.; Mannens, G. Absorption, Metabolism, and Excretion of Oral 14C Radiolabeled Ibrutinib: An Open-Label, Phase I, Single-Dose Study in Healthy Men. Drug Metab. Dispos. 2015, 43, 289−297. (349) Chatterjee, P.; Botello-Smith, W. M.; Zhang, H.; Qian, L.; Alsamarah, A.; Kent, D.; Lacroix, J. J.; Baudry, M.; Luo, Y. Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors? J. Am. Chem. Soc. 2017, 139 (49), 17945−17952. (350) Alberty, R. A.; Hammes, G. G. Application of the Theory of Diffusion-Controlled Reactions to Enzyme Kinetics. J. Phys. Chem. 1958, 62 (2), 154−159. (351) Wright, M. H.; Sieber, S. A. Chemical Proteomics Approaches for Identifying the Cellular Targets of Natural Products. Nat. Prod. Rep. 2016, 33 (5), 681−708.

(323) Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What Is in the Name? J. Am. Soc. Mass Spectrom. 2017, 28 (1), 5−13. (324) Li, Z.; Qian, L.; Li, L.; Bernhammer, J. C.; Huynh, H. V.; Lee, J.-S.; Yao, S. Q. Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications. Angew. Chem., Int. Ed. 2016, 55 (6), 2002−2006. (325) Zhao, S.; Dai, J.; Hu, M.; Liu, C.; Meng, R.; Liu, X.; Wang, C.; Luo, T. Photo-Induced Coupling Reactions of Tetrazoles with Carboxylic Acids in Aqueous Solution: Application in Protein Labelling. Chem. Commun. 2016, 52 (25), 4702−4705. (326) Mix, K. A.; Raines, R. T. Optimized Diazo Scaffold for Protein Esterification. Org. Lett. 2015, 17 (10), 2358−2361. (327) Ban, H. S.; Usui, T.; Nabeyama, W.; Morita, H.; Fukuzawa, K.; Nakamura, H. Discovery of Boron-Conjugated 4-Anilinoquinazoline as a Prolonged Inhibitor of EGFR Tyrosine Kinase. Org. Biomol. Chem. 2009, 7 (21), 4415−4427. (328) Woodward, R. B.; Olofson, R. A.; Mayer, H. A New Synthesis of Peptides. J. Am. Chem. Soc. 1961, 83 (4), 1010−1012. (329) Martín-Gago, P.; Fansa, E. K.; Winzker, M.; Murarka, S.; Janning, P.; Schultz-Fademrecht, C.; Baumann, M.; Wittinghofer, A.; Waldmann, H. Covalent Protein Labeling at Glutamic Acids. Cell Chem. Biol. 2017, 24, 589−597. (330) Martín-Gago, P.; Fansa, E. K.; Klein, C. H.; Murarka, S.; Janning, P.; Schürmann, M.; Metz, M.; Ismail, S.; Schultz-Fademrecht, C.; Baumann, M.; Bastiaens, P. I. H.; Wittinghofer, A.; Waldmann, H. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity That Prevents Efficient Inhibitor Release by Arl2. Angew. Chem., Int. Ed. 2017, 56 (9), 2423−2428. (331) Tsukiji, S.; Hamachi, I. Ligand-Directed Tosyl Chemistry for in Situ Native Protein Labeling and Engineering in Living Systems: From Basic Properties to Applications. Curr. Opin. Chem. Biol. 2014, 21, 136−143. (332) Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Molina, D. M. The Cellular Thermal Shift Assay for Evaluating Drug Target Interactions in Cells. Nat. Protoc. 2014, 9 (9), 2100−2122. (333) Franken, H.; Mathieson, T.; Childs, D.; Sweetman, G. M. A.; Werner, T.; Tögel, I.; Doce, C.; Gade, S.; Bantscheff, M.; Drewes, G.; Reinhard, F. B. M.; Huber, W.; Savitski, M. M. Thermal Proteome Profiling for Unbiased Identification of Direct and Indirect Drug Targets Using Multiplexed Quantitative Mass Spectrometry. Nat. Protoc. 2015, 10 (10), 1567−1593. (334) Komissarov, A. A.; Romanova, D. V.; Debabov, V. G. Complete Inactivation of Escherichia Coli Uridine Phosphorylase by Modification of Asp5 with Woodward’s Reagent K. J. Biol. Chem. 1995, 270 (17), 10050−10055. (335) Qian, Y.; Schü rmann, M.; Janning, P.; Hedberg, C.; Waldmann, H. Activity-Based Proteome Profiling Probes Based on Woodward’s Reagent K with Distinct Target Selectivity. Angew. Chem., Int. Ed. 2016, 55 (27), 7766−7771. (336) Harlow, K. W.; Switzer, R. L. Chemical Modification of Salmonella Typhimurium Phosphoribosylpyrophosphate Synthetase with 5′-(p-Fluorosulfonylbenzoyl)Adenosine. Identification of an Active Site Histidine. J. Biol. Chem. 1990, 265, 5487−5493. (337) Uchida, K.; Stadtman, E. R. Modification of Histidine Residues in Proteins by Reaction with 4-Hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 4544−4548. (338) Yamaguchi, S.; Aldini, G.; Ito, S.; Morishita, N.; Shibata, T.; Vistoli, G.; Carini, M.; Uchida, K. Δ12-Prostaglandin J2 as a Product and Ligand of Human Serum Albumin: Formation of an Unusual Covalent Adduct at His146. J. Am. Chem. Soc. 2010, 132 (2), 824− 832. (339) Yoshizawa, M.; Itoh, T.; Hori, T.; Kato, A.; Anami, Y.; Yoshimoto, N.; Yamamoto, K. Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands. J. Med. Chem. 2018, 61 (14), 6339−6349. (340) Liu, S.; Widom, J.; Kemp, C. W.; Crews, C. M.; Clardy, J. Structure of Human Methionine Aminopeptidase-2 Complexed with Fumagillin. Science 1998, 282 (5392), 1324−1327. AZ


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