Emerging and Re-Emerging Warheads for Targeted Covalent

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Perspective

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., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01153 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Journal of Medicinal Chemistry

Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology Matthias Gehringer* and Stefan A. Laufer Department of Pharmaceutical / Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, DE

KEYWORDS covalent reactive groups; electrophilic warheads; chemical probes; targeted covalent inhibitors, irreversible inhibitors; covalent-reversible inhibitors

ABSTRACT

Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting non-catalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant chemical strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable

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efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of non-catalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheads beyond α,β-unsaturated amides that were 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 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 are key elements 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 inhibition has long been used as a design strategy to address highly reactive active site nucleophiles such as activated serine or cysteine residues.7,8 Within the last 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


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reactivity that, following binding to the target protein, is positioned to react rapidly with a specific non-catalytic residue at the target site."5 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 non-equilibrium kinetics potentially enabling full target occupancy, even if 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 re-synthesis.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 approximately 50% of the drug candidates fail due to ADME issues,9 the above features promise significant benefits. In cases where permanent protein modification is undesired, covalent-reversible chemistry can be used to exploit 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. They can be tuned to fit the target's turnover rate.11 Ideally, the unmodified ligand would be released to re-engage with the target after degradation or after non-specific covalent binding to off-targets incapable of stabilizing the covalent complex. TCIs are typically generated by structure-based design from optimized reversible binders, 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


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have recently been employed to identify covalent binders include fragment-based13–16 or tethering approaches17 and DNA-encoded libraries featuring electrophilic ligands.18,19 In TCI design, binding kinetics require special consideration. Since a detailed perspective on the implications of covalent binding kinetics has recently been provided20 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 halfmaximal rate of covalent modification. The second step is characterized by kinact, the 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 since they vary over time and do not reflect the relative contribution of KI and kinact to the observed overall effect. Notably, time-dependent inhibition can also result from a slow step in the reversible binding event. Therefore, 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 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 distinctly differs from the one of protein and peptide labeling reagents. The latter are used for chemo- but not site-selective covalent modification and usually address surface-exposed side chains. In this case, the reaction


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can be described as a one-step mechanism20 without the requirement of specific reversible binding. Here, comparably reactive reagents relying solely on their intrinsic chemical reactivity towards 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 minimum to prevent non-specific off-target labeling or reaction with GSH. Since 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 non-toxic. 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. However, the latter class of compounds 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 demonstrated 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 α-cyanoacrylamide-derived "hyper-reactive" Michael


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acceptors can further be useful for covalent-reversible targeting.25 Finally, the ideal balance of reversible binding affinity and specificity, inherent reactivity and metabolic stability depends on the 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, and the tremendous amount of work published has been compiled in a plethora of recent reviews.5,6,26–39 However, most of these reports focus on non-catalytic 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 on α,β-unsaturated carbonyl compounds has already been provided in a recent Perspective article by Kay Brummond, Daniel Harki and coworkers, extensively discussing the chemistry and reactivity of Michael acceptors as well as their application in drug discovery and beyond.40 Instead, this Perspective focusses on less common warheads, recent innovations and emerging concepts, highlighting their potential in medicinal chemistry and chemical biology by case studies. Reagents for peptide labeling, 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. 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.


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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 tried 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. cysteine-targeted cyanamides as Janus kinase 3 inhibitors,49 histidine-targeted linear alkyl bromides as 17β-hydroxysteroid dehydrogenase inhibitors50 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 chapters. (Pseudo)irreversible CRGs are discussed first while covalent-reversible warheads may be found at the end of each chapter. 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 part. 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


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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 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 cysteine thiols in proteins,56 non-catalytic cysteine residues typically feature pKa values in the range between 7.4 and 9.1. Catalytic active-site 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, as might have been expected, non-catalytic cysteine residues played a key role in recent TCI design efforts. For example, all the six currently approved targeted covalent kinase inhibitors address a poorly conserved cysteine in the solvent-exposed front region of the epidermal growth factor receptor (EGFR) kinase or 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 high 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 non-catalytic cysteines. As suggested by the differing pKa values, cysteine reactivity can vary over a wide range, and not


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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 in deprotonating the thiol group. Moreover, non-essential 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. the oncogenic KRAS G12C or the p53 Y220C mutant) may open up new avenues even 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 "non-canonical" 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. A novel interesting approach to minimize time-dependent offtarget 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 reactivity;67 a finding that might be attributed to the decreased intrinsic reactivity of monomethyl fumarate, but also reflects the presumably lower cell permeability of MMF. Based on this observation, the hypothesis arose that replacement of a


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conventional α,β-unsaturated amide in a cysteine-targeted inhibitor by a methyl fumarate residue could furnish compounds that rapidly react with their desired targets while the slower inactivation by esterases prevents off-target labeling (Figure 1).

Figure 1: Kinetic selectivity of fumarate 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. 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 case study. Ibrutinib features a 3-piperidinyl-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, ibrutinibderived probe 2 and its methyl fumarate analog 5 labeled BTK in Ramos cell lysates. As expected, fumarate 5 reacted faster and possessed the higher reactivity towards the cellular proteome. When


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incubated with HEK293 T cells stably expressing human carboxylesterase (hCES) 1, ibrutinib remained unaffected while the methylfumarate-derived inhibitor 3 was rapidly converted to the inactive acid 4. This effect was not observed when using HEK293 stably expressing methionine aminopeptidase (MetAP) 2 as a control. Accordingly, a significant reduction in time-dependent proteome labeling accompanied by a modest decrease in BTK activity (ca. 10-fold) was observed for probe 6 when incubated over 24 h with a 6:1 co-culture of the Ramos and the HEK293 cell lines, which was supposed to mimic the hCES1 activity in tumor xenografts. In contrast, the labeling profile of the ibrutinib-derived 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 hCES1-insensitive manner while 2 retained its proteomic profile in the presence of the esterase. Subsequent profiling of probe 6 and its isopropyl ester analog 7 in rodents (20 mg/kg i. p.) demonstrated BTK engagement in vivo, although both compounds were poorly stable in mouse plasma (t1/2 = < 2 min). Pretreatment with the covalent CES inhibitor JZL18472, however, increased the plasma stability to 25.5 and 352 min for 6 and 7. In contrast, inhibitor 3 and the free acid 5 did not require CES inhibitor treatment to obtain reasonable plasma stability (t1/2 = 168 min and 129 min). Both, 6 and 7 demonstrated significantly reduced off-target labeling in different tissues while substantial reactivity with BTK was maintained. Although these results are qualitative in nature, they underline the kinetic BTK selectivity of the fumarate 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


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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 treatment 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 impressively demonstrates that kinetic selectivity is achievable with reactive metabolically labile warheads and it will be interesting to see whether related concepts will expand our arsenal of warheads for TCI design.

Figure 2: Ibrutinib-derived fumarate esters and analogous probes equipped with a click handle. 2.2 Cysteine addition to allenamides Recently, allenamides have been suggested as bioisosteres of α,β-unsaturated amides for protein labeling purposes and inhibitor design. Although the reactivity of allenamides (general structure 8, Figure 3) towards thiols has been known for a long time,73,74 Teck-Peng Loh and colleagues were the first to investigate these reagents for cysteine labeling applications.75 They found a high


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intrinsic reactivity for the thiol of isolated cysteine as well as for terminal and internal cysteine residues in peptides and proteins but neither reaction rates were determined nor was a qualitative comparison of reactivity between aniline-derived allenamides and alkylamine-derived analogs provided. No labeling of other nucleophilic amino acids was observed. Upon reaction with cysteine (pH 8.0) 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 counterparts, terminal allenamides have a much lower tendency to undergo [3+2] cycloaddition reactions. Cysteine labeling was shown to be irreversible even in the presence of a 100-fold excess of glutathione or dithiothreitol (DTT).

Figure 3: Mechanism of cysteine addition to allenamides. The prevalence of the mesomeric structure 10a rationalizes the formation of the non-conjugated product. An alternative meachnism involving attack of the thiol to form a zwitterionic species followed by proton-transfer was proposed by Loh and co-workers.


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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 analog 12 was slightly more active in blocking the EGFR T790M/L858R mutant compared to the parent compound (IC 50 = 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 43-fold for compound 13) or decreased (only 6fold 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 NCI-H1975 cells was shown 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 to be between 7–28-fold higher than for the approved acrylamide-derived kinase inhibitors afatinib (k' = 0.042 min-1), osimertinib (k' = 0.010 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 is currently limited by their 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 over the


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more common α,β-unsaturated amide, assuming that the reversibly binding part of the inhibitor confers sufficient kinetic selectivity to suppress off-target labeling.

Figure 4: Osimertinib-derived allenamides as EGFR inhibitors. 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 in plasma and living cells was also demonstrated. In contrast, structurally related aryl alkynones and terminal alkynoic amides undergo thiol exchange via addition of a second thiol and subsequent elimination of one thiolate (Figure 5B).80 Reactivities were in the same range as for terminal alkynoic amides and thus borderline for in vivo applications. However, reactivity could be decreased by substituents with +M-effect in the para-position (and vice versa with substituents featuring a –M-effect) and dropped dramatically when ortho-substituents were introduced. Moreover, the analogous 3cyclohexylpropiolonitrile (16) reacted about 10 times slower with 2-phenylethane-1-thiol compared to unsubstituted 15, however, the obtained reaction product also proved to be slightly


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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 where precise and rigid spatial placement of the warhead is required (provided that the binding cavity can host this elongate functionality). The published data further suggest that this CRG can be readily attenuated to an appropriate intrinsic reactivity. Still, metabolic stability requires further investigation and the general applicability in drug and probe development remains to be shown.

Figure 5: Propiolonitriles as potential covalent warheads: A) 3-aryl and 3-alkyl propiolonitriles. B). Mechanism of cysteine addition and thiol exchange. 2.4 Cysteine addition to vinyl or alkynyl-substituted heteroaryl rings Electron-deficient heteroaryl rings 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 (thiol-Michael addition or thiolene reaction), in which the intermediate negative charge is stabilized by the heterocycle. 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 conjugate additions are not uncommon (e.g. Ref.83–88). Vinylpyridines have also been applied in cysteine-specific protein labeling89,90 and their reactivity can be readily


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modulated.91 In this light, it is surprising that only few and relatively limited systematic studies evaluating this chemistry for cysteine-targeted inhibitors have been published so far (vide infra). An early isolated series of publications from 2008/2009 was reported by David Uehling and coworkers from GSK.92 They serendipitously found a non-catalytic cysteine of ErbB kinases to react covalently with ethynylthienopyrimidine-based inhibitors. Although the ethynyl moieties were attached to thiophene ring, which is comparably 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[3,2d]pyrimidine (17–20, Figure 6A) and 6-ethynylthieno[2,3-d]pyrimidine series (exemplified by compound 21). Consistent with a mechanism in which the thiol(ate) attacks the triple bond generating a transient negative charge, the 6-ethynylthieno[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) strongly increased covalent inactivation leading to 83 % labeling after 3 h and full labeling after 20 h, respectively. This acceleration was neither observed with an analogous propargylamine featuring a (R)-2-aminoethyl substituent (19) nor with a weakly basic 2-piperazine derivative (20). The rate-accelerating effect of methylene-linked amino groups has been described previously for propiolamide- and acrylamide-derived EGFR inhibitors.94,95 In the suggested mechanism, the basic amine increases the nucleophilicity of the thiol group by hydrogen bonding or 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


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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 nM, 13 nM, and 66 nM, respectively) with cellular IC50's in the mid-nanomolar range, featured > 100-fold selectivity against 30 from 31 kinases in a small panel. Notably, it did not inhibit interleukin-2-inducible T-cell kinase (ITK) possessing an equivalently positioned cysteine. Dosed orally at 30 mg/kg and 100 mg/kg b.i.d., compound 18 was an effective inhibitor of BT464 tumor growth in a xenograft mouse model while compound 19 showed slightly lower activity in the same model.

Figure 6: 6-ethynylthienopyrimidine as covalent ErbB kinase inhibitors. A) 6-ethynylthieno[3,2d]pyrimidine and 6-ethynylthieno[2,3-d]pyrimidine-derived inhibitors. B) Suggested mechanism of cysteine addition. In several follow-up studies, the same team investigated the effects of substituents at the 4position of the pyrrolidine ring,96,97 the stereochemistry97 of the pyrrolidine substituent and


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modifications at the aniline head group.98 Although some improvements of ADMET properties were achieved, no major advances in terms of biochemical and cellular potency and covalent labeling efficiency could be made. Moreover, it should be pointed out that neither biochemical nor cellular inhibitory potencies correlated 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 towards a second thioether addition and accompanying thiol exchange reactions (see chapter 2.3) will necessitate further investigation.

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 vinylthioether adduct. The


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N1-atom of the pyrimidine ring is further anchored to the backbone NH of Met799 in the hinge region via hydrogen bond. The pyrimidine N3-atom is engaged in a water-mediated hydrogen bond to the side chain of Thr860 preceeding the conserved DFG motif. 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, and its non-electrophilic analog VUF14481 (23) bound the hH4 receptor with similar 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. However, timedependence was not determined in either assay system. As expected, 22 reacted with GSH and cysteine ethyl ester to form covalent adducts 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, 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 wild type receptor and the C98S mutant suggesting reversible binding to be the main contributor to the observed affinity, which is consistent with slow covalent modification.


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

2.5 Cysteine addition to non-activated 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 disease100,101 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 non-activated 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 non-activated, and thus poorly electrophilic terminal alkynes are quite remarkable (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 derived peptides intended for click-chemistrybased labeling. The active site cysteine of deubiquitinating isopeptidases (DUBs) and SUMO proteases selectively attacked the triple bond at the 2-position forming the Markovnikov vinylthioether adduct. Interestingly, N-but-3-ynyl amide (24a) and even N-hex-5-ynyl amide homologs (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


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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 for 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 SUMOprotease Senp1, however, mutation of key catalytic residues in the catalytic triad (i. e. H553A and D550A) and the oxyanion hole (Q597A) did not prevent the formation of the covalent adduct108. Further mechanistic investigations will be required to elucidate the structural requirements promoting these reactions and it remains to be seen, if non-catalytic cysteines could covalently trap propargylamides and similar terminal alkynes.

Figure 9: Non-activated terminal alkynes as cysteine traps. A) Reaction of C-terminally propargylated ubiquitin 24 with the active site cysteine in DUBs. B) Reactive and non-reactive analogs.

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 thiol(ate), displacing a


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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 SN1type 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 electron-withdrawing (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 halogenides typically ranks F >> Cl ≈ Br > I roughly correlating with the increasing polarization of the Cδ+−Xδ− bond. However, different rankings might be observed since 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 and can be found elsewhere.113 Yet, 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. Early studies have exploited SNAr warheads in inhibitors like 2-chloroquinoxaline L-764406 (25, Figure 10B),115 the electron-deficient phenyl derivative GW9662 (26)116 or the 2-sulfonylpyridine GDK3787 (27),117 which target peroxisome proliferator-activated receptors (PPARs) by labeling different non-catalytic 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


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reagents such as MSTB (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-s-tetrazine124 have been further been exploited for peptide stapling. Notably, the cysteine reactivity of highly activated SNAr electrophiles has been correlated with their skin sensitization potential.125 In 2011, the team of researchers around Kiplin Guy identified methylsulfonyl nitrobenzoates exemplified by MLS000389544 (30) as low micromolar irreversible inhibitors of the thyroid hormone receptor (TR) β/steroid co-activator (SRC) 2 interaction by high throughput screening (HTS) of a library of 500.000 compounds.126 These compounds selectively labeled Cys298 as shown 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 poorly reactive analog, was detrimental to activity. However, the replacement of the methylsulfonyl group by chloride or fluoride also furnished inactive compounds, which is more surprising since sulfones are typically less reactive than iodides in SNAr reactions.113 This finding might reflect the requirement of the methylsulfonyl residue for reversible binding or point to the complex interplay of different factors on SNAr reactivity.


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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. In 2015, John Kuryan and co-workers reported the sulfonyltetrazole (31, Figure 11), the NBDdye 32 and the dichloropyrimidine carboxamide 33 as low molecular weight inhibitors antagonizing the interaction between the tandem Src homology (SH) 2 domains of the non-receptor 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 time-dependent manner. X-ray and MS experiments confirmed the specific binding to single cysteines when no excess of the reagents


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was used. The X-ray structures of compounds 31 (PDB: 4XZ0) and 32 (PDB: 4XZ1) in complex with the ZAP-tSH2 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 intrinsic reactivity of these electrophiles precludes application in cells or in vivo.

Figure 11: Covalent inhibitors antagonizing the interaction of ZAP-70 and Syk with ITAMs. Leaving groups are highlighted in red. 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 alkynetagged 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) gave very potent and promiscuous labeling reflecting the highly electrondeficient nature of this electrophile. Intriguingly, however, 34a and 35a affected distinct sets of proteins, and peptide mapping showed that 34a preferentially labeled cysteine thiols while 35a


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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 for reactivity within protein binding sites.

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. A recent study from Walter Fast and co-workers investigated 4-halopyridines as quiescent electrophilic warheads.131 They found the reactivity of these compounds to increase drastically when the pyridine nitrogen atom is protonated. To this end, reactivity with GSH at neutral pH was compared between 4-chloropyridine (36, Figure 13A) and its charged N-methylated 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 rate constants in the same range as for Nmethylacrylamide.12 Similarly, 36 showed very low reactivity comparable to that of ampicillin, styrene oxide and acrylamide. In contrast, the N-methylated analogue's reactivity increased by more than three orders of magnitude, thus being in the same range as for iodoacetamide. Arguing


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with the classical SNAr mechanism, this 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 iodo-derivative, 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 dimethyl arginine 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 4.7–0.4), a clear correlation between the pKa of the protonated pyridine and reactivity was observed. Counterintuitively, more electron-deficient compounds reacted slower. 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 E. coli proteomes. Gelbased analysis showed that 39 only modifies few proteins and the number even decreases when cell lysates are denatured prior to exposure. In contrast, N-methylated analog 40 labeled much more proteins and the number increased substantially in denatured lysates. These observations suggest that the neutral 4-halopyridines require activation by their protein environment for


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becoming reactive, while the charged N-methyl-4-halopyridines may promiscuously label surfaceexposed nucleophiles, which are more abundant in the denatured samples.

Figure 13. 4-Halopyridines as SNAr-warheads. A) SNAr-reaction with 4-chloropyridine according to the classical mechanism. An anionic Meisenheimer intermediate is formed. B) Analogous mechanism of the SNAr-reaction with N-methyl-4-chloropyridine. A neutral dihydropyridine species is formed as the intermediate. C) General structures of the investigated compounds. D) Alkyne-tagged probes used for proteomic analysis. In a recently published drug discovery campaign, Fairhurst and co-workers from Novartis aimed to identify covalent inhibitors targeting a rare cysteine (Cys552)35 located in the hinge region at the gatekeeper (GK)+2 position of the fibroblast growth factor receptor (FGFR) 4 tyrosine kinase.132 A high throughput screen yielded compound 41 (Figure 14A) as a nanomolar inhibitor (IC50 = 32 nM) of wild type FGFR4 while 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 affinity of this structurally simple molecule can be attributed to displacement of the 6-pyridyl chlorine atom by the cysteine thiol. Covalent modification at Cys552 was confirmed by MS and X-ray crystallography (Figure 14B). The compound showed a 170-fold slower reaction rate (kinact/KI = 3.0 x 104 M-1s-1) than an acrylamide-based inhibitor from the same study targeting the


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more reactive 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 seemed to leave kinase activity unaffected, highlighting the fact, that enzymatic or competition-based kinase assays are typically not suited for the identification of non-active 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 reversibly binding aldehyde-derived compound (vide infra) owing to the high FGFR4 re-synthesis rate (< 2 h).

Figure 14: A) Structure of the SNAr-based FGFR4 inhibitor 41. B) X-ray cystal structure of 41 in complex with the FGFR4 kinase domain (PDB: 5NUD). Both pyridine rings form a hydrogen bond with the backbone NH group of Ala553 while the nitro group stabilizes the active conformation via a weak intramolecular hydrogen bond with the diarylamino NH. The covalent bond with Cys552 is formed by SNAr displacement of the 6-chloro group from the 2-amino-3-nitropyridine moiety.


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Weijie Hou and co-workers reported in early 2018 on putatively covalent EGFR inhibitors featuring SNAr warheads to address Cys797.133 Their rationale was the replacement of the α,βunsaturated amide found in afatinib and analogous compounds by an electron-deficient aryl ring equipped with a halide leaving group (general structure 42, Figure 15). Several potent inhibitors with low nanomolar affinities were identified but activity differences between electrophilic and non-electrophilic compounds were only moderate. Covalent modification was not directly assessed but washout experiments with key compound 42a indicated only reversible binding although the inhibitor reacted with GSH confirming its inherent reactivity. As the EGFR is known to react with various acrylamide-derived inhibitors,134 it can be assumed that inappropriate positioning of the electrophile prevents covalent trapping in this case.

Figure 15: EGFR inhibitors with SNAr warhead which do not form the predicted covalent bond with Cys797. Although the above studies show that targeted covalent inhibition with SNAr warheads is feasible, no substantial efforts on the (successful) compound optimization were reported nor were the compounds profiled in a broader array of assays. One of the few comprehensive medicinal chemistry studies on verifiable SNAr-based irreversible inhibitors was published by Kevin Chen and colleagues at Merck Sharp & Dohme.135 The researchers optimized a screening hit based on


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an indole core (43, Figure 16) to inhibit the hepatitis C virus (HCV) NS5B polymerase. While optimizing the indole N1-substituent, the introduction of a nitrile group ortho to the fluoride at a 2-fluoro-5-methylsulfonyl benzyl residue (45) was serendipitously found to promote 100-fold improvement in activity in a cellular replicon assay. As the parent compound 44 was known to bind to the "palm" site of the NS5B protein, covalent bond formation with the non-catalytic Cys366, a residue that had previously been targeted with benzylidene rhodanine-derived covalentreversible inhibitors,136 was assumed to be responsible for this unexpected leap in activity. A variety of analogs with electron-withdrawing substituents and leaving groups at the phenyl ring was prepared but no electron-deficient heterocycles had been explored at that stage. While most modifications gave only weak to moderately active compounds, a strongly electron-withdrawing nitro group in the para-position of the halogen leaving group (46 and 47) was able to promote excellent biochemical and cellular activity. Notably, binding kinetics were not investigated. Further optimization yielded key compound 48, which exhibited an excellent PK profile in monkeys and dogs. The inhibitor featured an oral bioavailability of 35% and 95%, an AUC (p.o., 3 mg/kg) of 3.4 μM·h and 11 μM·h, and a half-life of 1.3 h and 1.2 h, respectively, in the aforementioned species. The X-ray crystal structure of 48 in complex with the HCV NS5B polymerase (PDB: 3TYQ) as well as MS experiments unambiguously confirmed the expected covalent binding mode. An NMR-based method (ALARM-NMR)137 exploiting the high nucleophilicity of cysteines in human La antigen showed no promiscuous thiol reactivity. Moreover, the compound was found to be clean in kinase and protease panels. Although the nitro group was regarded with skepticism due to known potential safety issues caused by partial reduction products,138 no toxicity could be observed in a Salmonella/mammalian microsome miniAMES assay, an in vitro micronucleus induction assay and in single-dose animal PK studies.


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Figure 16: Development of covalent HCV NS5B polymerase inhibitors with SNAr warheads. In a follow-up study, the same team evaluated heterocyclic substitutes for the 2-fluoro-5nitrobenzyl residue.139 Although the nitro group did not promote any obvious toxicity in the above study, it was considered to be safer to avoid this functionality. An initial meta-linked 2chloropyridine series exemplified by 49 (Figure 17A) gave acceptable potencies in the biochemical assay, however cellular EC50 values increased to the low micromolar range. As it was shown later, these moieties were not capable of covalent target engagement. Replacement of the pyridine ring by a 2-chloroquinoline moiety rescued cellular activity. Although small substituents in the C6- and C7-position of the quinoline ring were tolerated, the best results were obtained with an undecorated 2-chloroquinoline substituent. Key compound 50 featured low nanomolar activities in both assays. Covalent binding to Cys366 was proven by MS and X-ray crystallography (Figure 17B) and no labeling of any of the enzyme's other 21 cysteines was detected. Intriguingly, the compound was stable towards harsh chemical conditions such as refluxing in aqueous lithium hydroxide or 0.5 M ammonia/dioxane pointing out the low intrinsic reactivity of the warhead necessitating proximitydriven covalent binding. The compound possessed a similar clean profile as 48, showed no significant CYP or hERG interactions, limited toxicity up to 2000mg/kg in rats and proved


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favorable in vivo drug metabolism and pharmacokinetic (DMPK) parameters in rat, dog and monkey. Compound 50 was thus suggested as a candidate for further development.

Figure 17: Second generation of NS5B polymerase inhibitors with SNAr warheads. A) Reversibly binding 2-chloro pyridine 46 and the irreversibly binding quinazoline analog 47. B) X-ray crystal structure of key compound 47 covalently bound to Cys366 of HCV NS5B polymerase (PDB: 4MZ4). The compound is further anchored by hydrogen bonds between the 2-pyridone moiety and the backbone carbonyl atom of Gln446 and the NH group of Tyr448. The carboxylate and the quinoline N1-atom are linked to different residues via water-mediated hydrogen bond networks (the second sphere of water molecules and beyond was omitted for clarity). A second conformation of the Cys366 was omitted as well. In summary, the highlighted reports point out the enormous potential of SNAr chemistry for targeted covalent inhibition. Although only medicinal chemistry programs have been published and little systematic investigation has been performed, the tunability, synthetic accessibility and structural rigidity of SNAr warheads will probably stimulate further exploration of this structure


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class. Moreover, electron-deficient heterocycles are already present in many known ligands and approved drugs with excellent ADME properties. If a properly positioned nucleophile is present in the binding site, such a ligand might be readily turned into an irreversible modifier by the simple addition of suitable leaving groups. Besides the efforts discussed above, it is worth mentioning that an elegant approach to target DNA cytosine-5-methyltransferases (DNMTs) covalently via an SNAr mechanism has very recently been reported in two studies by Akira Matsuda, Satoshi Ichikawa and co-workers.140,141 However, their approach addressing an active site cysteine via a suicide mechanism does not fully fit the scope of this manuscript. The interested reader is therefore referred to the original articles.

2.7 Cysteine alkylation by strain release reagents A very elegant method for selective cysteine labeling by using strain release reagents was described in 2016 by the group of Phil Baran.142,143 Although the strain-promoted addition of thiols to [1,1,1]-propellane derivatives has been known since the 1980s,144 this reaction has not been applied to cysteine conjugation until recently. While [1,1,1]-propellane was found to be a useful building block the for the derivatization ("propellerization") of alkylamines by means of "turboGrignard" reagents,145 phenylsulfonyl bicyclobutanes (e.g. compound 48, Figure 18) proved to be suitable for cysteine labeling applications. These bench-stable reagents selectively cyclobutanated cysteine in glutathione and in highly functionalized peptides under basic conditions while leaving other nucleophilic amino acid side chains untouched (Figure 18).142 Analogous cyclopentylation reactions were carried out even in a stereospecific manner by using chiral 1-(phenylsulfonyl)bicyclo[2.1.0]pentanes (housanes, e.g. compound (+)-49) as the strain-release reagent.143 The reactivity of the strained cycles could be readily tuned by modulating the electronic properties of


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the aryl system linked to the sulfonyl group. As expected, the attachment of electron-withdrawing substituents increased reaction rates while electron-donating substituents had the opposite effect. The adducts were chemically stable even in the presence of TCEP, but stability studies in a more physiologically relevant setting remain elusive. In a GSH reactivity assay previously reported by researchers from Pfizer (vide infra),12 strain-release warheads were benchmarked against common α,β-unsaturated amides and vinyl sulfonamides (see Table 1 for a representative selection). Halflives spanned from 4 h for the 3,5-difluoro derivative (no analogs with stronger electronwithdrawing groups have been evaluated) to 19 h for the 4-methoxy analog. The compounds thus proved to be significantly less reactive than the common N-phenylacrylamide motif (t1/2 = 0.9 h) or N-(vinylsulfonyl)pyrrolidine (t1/2 = 0.53 h) but in the same range as N-benzyl acrylamide (t1/2 = 15 h). Given the well-balanced, selective and tunable reactivity towards cysteines along with the limited spatial requirements and the defined geometry, these electrophiles might be well-suited as warheads in drug discovery. Moreover, the replacement of the aryl sulfone moiety by sulfonamides, carboxamides, esters or ketones or the attachment of substituents to the strained ring system may enable the modulation of reactivity over a wider range. Unfortunately, no studies demonstrating the applicability of strain-release warheads in a complex biological setting have been published so far. One potential issue with this CRG is the underlying synthetic chemistry. Although Baran and co-workers have developed an operationally simple protocol for the preparations of the strain-release reagents shown above, the flexible implementation of the latter into chemically more complex setting may require optimization and profound chemical expertise.


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Figure 18: Strain-release warheads and their reaction with a cysteine-containing peptide.

R

t1/2 (h)

R

t1/2 (h)

3,5-di-F

4

4-OMe

19

4-CF3

10

0.53

4-Cl

13

0.9

4-Me

15

15

Table 1: Influence of the aryl substituent on the reactivity of phenylsulfonyl bicyclobutanes in a GSH stability assay and comparison with common acrylamides.


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2.8 Cysteine alkylation by nucleophilic displacement of alkyl halides Although aryl halides are abundant in approved and investigational drugs, their alkyl counterparts are rare with the notable exception of DNA alkylating agents146 and unreactive alkyl fluorides motifs, that have been frequently applied in drug discovery, e.g. as bioisosteres or for modulating physicochemical properies.147,148 The most representative sp3-halides used to covalently modify biological targets are α-halomethylketones and the analogous esters and amides. α-Halomethylketones have a long history as protease probes where the keto group is attacked by the active site nucleophile while the proximal alkyl halide irreversibly labels the enzyme, for example by reacting with a histidine of the catalytic triad.7 Mometasone, an approved corticosteroid, contains an α-chloromethylketone moiety. Reactive fluoroalkanes can be found in mechanism-based inhibitors such as eflornithine149 or trifluoromethyl deoxyuridine derivatives.150 Moreover, fluoromethylketone (FMK)-tagged adenine analogs were amongst the first rationally designed covalent kinase inhibitors.151,152 The nucleophilic substitution reactions discussed here proceed via a SN2-mechanism (Figure 19A), in which the reaction rate depends on the nucleophile, the stability of the sp2-hybridized transition state and the leaving group. SN1 reactions, where the rate depends solely on the loss of the nucleofuge to generate a highly reactive carbocation, are not utile in the context of TCI design. Nucleophilic substitutions of non-activated linear haloalkanes are relatively slow and increasing steric bulk around the electrophilic carbon center further decreases reactivity. Benzyl or allyl halides are more reactive since the transition state of the SN2-reaction (but also the carbocation in the case of an SN1-mechanism) is stabilized via conjugation with the π-system. A tremendous increase in reactivity is typically observed for α-halomethylketones, which may be rationalized by a further increase in stabilization of the SN2-transition state via conjugation or by the dual attraction


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model (Figure 19B).153 However, the relative increase in reaction rates depends on the nucleophile.154 Nucleophilic substitution of α-halomethylcarbonyl compounds exclusively proceed via SN2-mechanism even if good leaving groups are employed, which can be attributed to the electron-withdrawing carbonyl group destabilizing the intermediate carbocation in SN1 reactions. Reactivity of α-halomethylcarbonyl compounds decreases if +M-substituents are added to the carbonyl group. Consequently, α-halogenated esters are less reactive than the corresponding ketones and the respective amides possess an even lower reactivity.153 On the other hand, it rises with increasing leaving group properties of the halogen (I > Br > Cl > F). α-Haloacetamides are quite versatile 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 reactivity12 are by far less common. α-Chloroacetamides, which are the most frequently employed haloacetamides in covalent ligand design, possess even further reduced reactivities. Their stability against GSH at pH 7.4 is in the same range as the one of α,βunsaturated amides12 and a linear chloroacetamide-derived alkyne probe show only moderate levels of labeling in soluble mouse liver proteoms.157 In the case of aniline-derived αhaloacetamides, GSH reaction rates correlate with the Hammet parameter of the aryl substituent, i.e. electron-donating substituents decrease reactivity an vice versa.158 Reactivity of αhaloacetamides can be further decreases 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 N-benzyl acrylamide (Figure 19).12 In support of the low and specific reactivity of α-chloropropionamides,


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a recent ABPP-based study identified (S)-CW3554 (53, Figure 19B) as irreversible inhibitor of the 57 kDa protein disulfide isomerase (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 TCIs design remain to be demonstrated.160,161

Figure 19: Alkyl halides as cystein-targeted warheads A) General mechanism of the SN2 reaction. B) Dual attraction model rationalizing the enhanced reactivity of α-halocarbonyl compounds. C) Reactivities of α-halopropion- and acetamides compared in a GSH assay. Half-lives were determined in the presence of 100 mM GSH at pH 7.4 and a 37 °C or b 60 °C. N-Phenylacrylamide is shown for comparison. D) 2-chloropropionamide (S)-53, a covalent PDIA1 inhibitor. As an example of cysteine targeted alkyl halides devoid of an α-carbonyl group, 1,4disubstituted 5-chloromethyl-1,2,3-triazoles have recently been identified by Alexander Adibekian and co-workers as inhibitors of the O6-methylguanine-DNA-methyltransferase (MGMT), a non-


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catalytic 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 5-chloromethyl 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 amongst 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, analog AA-CW236 (55) was identified as a highly potent MGMT inhibitor (KI = 24 nM), albeit with slow inactivation kinetics (kinact of 0.03 min-1). The compound did neither cross-react with any of 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 temozolimide, significantly increased the O6-alkylguanine levels in MCF7 cells compared to temozolimide alone indicating a sensitization to the chemotherapeutic drug. Due to the low inactivation rates, however, it is not unlikely that the bulk of the observed effects are promoted by the potent reversible interactions.

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


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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 time as warheads to target different types of proteases7,164 and glycosidases165,166. In contrast, other three-membered 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 reactions and their reactivity is influenced by similar factors as discussed in the previous section. In the absence of an acid catalyst, nucleophilic attack occurs at the sterically less hindered position. Epoxides are used as CRGs in approved drugs, e.g. the epoxy-ketone 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 non-essential cysteine (Cys115 in E. Coli).172,173 Despite being structurally simple, this orally available drug can be safely administered in multi-gram doses illustrating the tolerability of epoxide-derived compounds.171 Moreover, epoxides can behave unreactive even if a proximal cysteine is present in the target 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


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reactivity as acrylamide or ampicillin towards 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 wellknown toxic effects of epoxy-metabolites such as the CYP oxidation products of benzene.6 Unfortunately, systematic studies on the factors determining the reactivity of epoxides and other three-membered heterocycles in a biological environment are still elusive. Another reason for the low abundance of epoxides in drug discovery campaigns is their facile hydrolysis by epoxide hydrolases, and factors determining metabolic stability of this compound class are not fully elucidated. On the other hand, this liability might be used to confer kinetic selectivity in a similar manner as the fumarate esters discussed in chapter 2.2. 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 non-catalytic cysteine residues are highlighted below.

Figure 21: Examples of epoxide-containing drugs. 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 ethers, carbamates, nitriles and heterocycles containing an electrophilic sulfur atom were also evaluated in this study. Starting from the covalent inhibitor PD168393 (59), replacement of the acrylamide


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moiety by three different epoxides furnished highly potent compounds (IC50 = 0.5 nM, 0.5 nM 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-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 co-workers observed only very weak activity of the analogous epoxide 63 on the cSRC S345C mutant sharing an equivalently positioned cysteine.177

Figure 22: α-acyl epoxides as warheads for putatively covalent EGFR inhibitors. In a similar approach from our own group, an epoxide moiety was used to invert the 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


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address JAK3 Cys909, which is unique within the JAK family. We prepared a series of ruxolitinibderived triazoles and epoxy analog 65 appeared to be a potent JAK3 inhibitor (IC50 = 35 nM) with a high selectivity (70−160 fold) in the JAK family. Since 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 interaction was not unambiguously proven as we discontinued this series in favor of tricyclic covalent-reversible JAK3 inhibitors.179

Figure 23: Ruxolitinib-derived triazoles with a propylene oxide warhead as selective JAK3 inhibitors. Aziridines haven been used for targeting active site carboxylates in glucosidases,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 well-known 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 E. Burke.182 With the aim of covalently addressing the oncogenic K-Ras G12D mutant, different electrophilic head groups, known to react


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with aspartates, were attached to an optimized reversible ligand in a structure-guided manner (general structure 66, Figure 24). Despite the reactivity of the representative aziridine 66a towards 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 LC-MS (HDX-MS). Xray-crystallography and MS finally revealed the binding mode (Figure 25). Interestingly, the attack of the thiol group occurred at the sterically more hindered β-carbon atom.

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


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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 piperdine carboxamide oxygen interacts with the side chains of Tyr96 and Asp92 via a water-bridged hydrogen bond.

2.10 Cysteine targeting by nitroalkyl groups as masked electrophiles The covalent complex formation of between 3-nitropropionate (3-NP, 67a, Figure 26) and M. 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 co-factors 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


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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 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 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 pre-formed P3N (kinact/KI = 2.6 x 104 M-1s-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 nucleophile. Besides the aforementioned concerns on nitro groups in drug discovery, the generalizability of this reactivity 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.

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


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2.11 Reversible cysteine addition to α-cyanoacrylamides Covalent reversible targeting of non-catalytic 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 "hyper-reactive" towards thiols, the increased α-CH-acidity and thermodynamic destabilization of the β-thioether adduct185 favor the reverse reaction and thereby dissociation from off-targets or peptides, which are not capable of stabilizing the covalent complex by non-covalent 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 nitrile group by an electron-withdrawing heterocycle,186 the intrinsic reactivity of this CRG and the dissociation rates of the derived covalent-reversible inhibitors can be modified.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 covalent interactions of this chemotype, however, is complicated by the reversible nature of protein modification and only few study 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)


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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 cysteines,156 nor did it change the labeling profile in the cellular proteome of Neu7 astrocytes indicating good specificity despite the highly reactive electrophile.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 PMBCs 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 (NCT02704429/NCT03762265) and immune thrombocytopenic purpura (NCT03395210). A similar 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 αcyanoacrylamides and analogous Michael acceptors, the currently available data suggest them to be promising warheads for in vivo use.

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


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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, arylidenedinitriles are very reactive reversible Michael acceptors but the reverse reaction seems to be competed by side reactions in this case.188 Moreover alkynonederived Michael acceptors (cf. chapter 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 proceeds via 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 vinylsulfones or sulfonamides equipped with an additional electron-withdrawing group in the α-position can further vary reactivity and extend the scope (e.g. towards 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 non-catalytic cysteines or lysines (some recent examples for Lys targeting by Schiff base formation can be found in Ref. 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 chapter 2.6), a set of closely related 2-formylquinoline amides exemplified by compound 72 (Figure 28, IC50 =


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65 nM) was discovered. These compounds were potent FGFR4 inhibitors with good selectivity in the FGFR family, 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 middlehinge Cys552 by hemiacetal formation. The quinoline nitrogen atom and the carboxamide proton were found to be of crucial importance since they stabilize a pseudo-tricyclic arrangement via intramolecular hydrogen bonding thereby positioning the aldehyde for covalent interaction. The electron-withdrawing ortho-quinoline scaffold further increases the aldehydes’ electrophilicity. Although certain questions concerning toxicity and metabolic stability of aldehyde moiety remained,202 this compound class was selected for development due to its excellent potency and selectivity profile. Covalent-reversible inhibition was considered more promising than irreversible targeting due to the rapid re-synthesis half live (< 2 h in HUH7 and Hep3B cells) of FGFR4. The 2-formylquinoline amide scaffold, however, was unsuited for further development due to very low solubility. A scaffold morphing approach increasing the sp3 portion of the molecule led to bioisosteric 2-formylpyridine ureas (exemplified by 73a–d) with improved solubility. Replacement of the trifluoromethyl (R1) group by a nitrile (73b) decreased lipophilicity, and the introduction of an oxygen atom in the saturated ring system (73c) or polar R2 substituents (73d) further increased both, solubility and potency. The advanced lead compound 73d featured low nanomolar potency in an enzymatic and a Ba/F3 cell assay (IC50 = 1.3 nM and 18 nM, respectively). Residence times (pH 7.0) for the slightly less potent compounds 73a–c were in the range between 105 min (73c) and 270 min (73a) and thus much increased compared to an equipotent non-covalent inhibitor (< 1.4 min). The significant off-rates (between 3.7 x 10-3 min-1 and 9.6 x 10-3 min-1) pinpoint the highly reversible nature arising from the relatively weak


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hemithioacetal C−S bond.203 Consistently, no covalent adducts were found in mass-spectrometric experiments. Although no inhibitor-bound X-ray crystal structure was determined, SAR and modeling provided insight into the binding interactions. Replacement of the pyridine moiety by pyrimidine and piperazine was tolerated with a slight loss in potency while the use of fivemembered N-heterocycles or a phenyl ring was detrimental to activity. Methylation of the urea NH furnished a completely inactive compound. Constraining and opening the piperazine ring was also tolerated while increasing the size of the saturated ring by one methylene group lead to a substantial potency loss.

Figure 28: Optimization of covalent-reversible cysteine-targeted FGFR4 inhibitors possessing an aldehyde warhead. The advanced lead compound 73d was further optimized to furnish clinical candidate FGF401 (74), a compound with good oral bioavailability, PK and safety profile, which is currently in phase I/II clinical studies (NCT02325739 on https://www.clinicaltrials.gov) 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 studies emphasize that aldehydes, despite potential metabolic liabilities, can indeed be valuable


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warheads for the covalent-reversible targeting of cysteines beyond the active sites of hydrolase enzymes.

2.13 Reversible cysteine addition to activated nitriles Besides aldehydes, nitriles have a long history as covalent-reversible warheads for cysteine proteases196,205 and are employed in approved drugs such as saxagliptin.206 As indicated by their prevalence in non-covalent inhibitors,207 nitriles are relatively inert and covalent adduct formation via a Pinner-type reaction generally requires highly nucleophilic active site cysteines along with precise positioning of the electrophilic carbon atom. However, their electrophilicity can be increased by the attachment to electron-withdrawing groups, e.g. heteroaryl rings, alkylamines12,208 or acylated N,N'-dialkylhydrazines.209

Figure 29: Reactivities of activated nitriles in a GSH-based assay. Half-lives (increasing from left to right) were determined in the presence of 100 mM GSH at pH 7.4 and 37 °C. Data for common acrylamides are provided for comparison.


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Pyridine and pyrimidinecarbonitriles show tunable reactivities towards glutathione, which are in a similar range as acrylamides (Figure 29, A and B) suggesting these structural elements as suitable CRGs for targeting non-catalytic cysteines. Nitriles reversibly react with cysteines to form thioimidates, as it has been shown by X-ray crystallography for the cysteine proteases cathepsin S (PDB: 3N3G)210 and the adenovirus protease adenain (PDB: 4PIQ).211 In the case of 2pyridinylcarbonitrile analogs, a double activation mechanism involving the pyridine nitrogen atom as a general base to deprotonate the cysteine while concurrently activating the nitrile group has been proposed.210 A recent study aiming to capitalize on the increased reactivity of 2pyridinecarbonitriles compared to their phenyl counterparts was published by Pamela England and co-workers.212 They hypothesized that a nitrile group present in the approved nonsteroidal antiandrogen bicalutamide (compound 75, Figure 30A) could be activated to react with the proximal Cys784 in the androgen receptor (AR), a residue that is not among the over 160 point mutations associated with prostate cancer. DFT calculations predicted a 10-fold increase in electrophilicity by introduction of a pyridine nitrogen atom ortho to the bicalutamide nitrile group (compound 76a). Reactivity studies with 76a and free cysteine in pH 7.4 buffer showed a 99% conversion to the corresponding thiazoline within 4 h, while no S-adduct formation was observed for 75 after 24 h. The additional nitrogen atom increased binding affinity approximately 150-fold (KI = 0.15 nM) compared to the parent compound bicalutamide. In a cellular luciferase reporter assay detecting AR-mediated transcription, a substantial increase in activity (IC50 = 0.015 µM vs. 0.31 µM for 76a and 75) was confirmed while the two analogs 76b and 76c, devoid of the nitrile group, were much weaker inhibitors (IC50 = 1.41 µM and 1.33 µM, respectively). A mechanism was proposed in which Arg752 activates the nitrile group via hydrogen bonding (Figure 30B). Although not explicitly mentioned, further activation could result from a hydrogen bond between


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the pyridine nitrogen atom and the cysteine thiol, as mentioned above,210 to facilitate the nucleophilic attack. NMR studies to confirm the covalent-reversible binding where hampered by protein precipitation, and mutation of Cys784 prevented proper folding. Furthermore, no structural data was reported and an ultimate proof of the suggested covalent interaction remains elusive.

Figure 30: Bicalutamide-derived antiandrogens with a putative covalent-reversible binding mode. A) Chemical structures of selected examples. B) Suggested mechanism of covalent binding to Cys784 of the androgen receptor. Cyanamides show similar reaction kinetics with GSH as acrylamides (see Figure 29) and should therefore be applicable in non-active site cysteine targeting. The reversible thiol addition furnishes isothioureas as it has been confirmed for cathepsin K by X-ray crystallography (PDB: 1YK7).213 While cyanamides are not uncommon in cysteine protease inhibitors213–216, reports on targeting non-catalytic cysteines using this functionality are rare. When designing cyanamide-based warheads, it should also be kept in mind that free protons at the amino group could, in principle, enable the tautomerization to carbodiimides,217 which have distinct reactivities. It remains to be tested if this equilibrium is relevant under physiological conditions.


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Micah Benson and colleagues from Pfizer reported the cyanamide-based inhibitor PF-303 (77, Figure 31), which was used as a chemical in vivo probe to investigate the clinical phenotype of BTK inhibition in mice.218 The underlying medicinal chemistry program was not published but PF-303 was claimed to be highly potent (IC50 = 0.64 nM) and orally bioavailable, pointing out the applicability of the cyanamide group in vivo. The compound, which can be considered as a pseudorigid cyanamide analog of the covalent BTK inhibitor ibrutinib possessed a target inactivation rate (kinact/Ki) of 1.44 x 105 M-1s-1 which is in the same range as for many acrylamide-derived kinase inhibitors.21,219 Binding was reversible with a dissociation half-life of 5 h. The compound also inhibited the kinases BMX and TEC featuring an equivalently positioned cysteine. In contrast, an over 10.000-fold selectivity against JAK3 and ITK also sharing this cysteine was observed. The latter finding is in line with SAR reported for a series of ibrutinib-derived ITK inhibitors, where replacement of the acrylamide moiety by cyanamide completely eroded the inhibitors’ activity.220 PF-303 showed high selectivity within the kinome and potently inhibited anti IgM F(ab')2promoted T-cell proliferation with an IC50 of 2 nM.

Figure 31: PF-303, a covalent-reversible BTK inhibitor featuring a cyanamide warhead.


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2.14 Reversible cysteine addition to isothiocyanates Isothiocyanates have long been known as natural electrophilic compounds in foods and their presence has been associated with health benefits. They mainly occur in cruciferous vegetables as metabolic breakdown products of glucosinolates.221 As a representative of this compound class, allyl isothiocyanate (78, Figure 32A) causes the pungent taste of mustard and wasabi, which has been attributed to TRPA1 channel activation by covalent cysteine (and lysine) binding.222 Various clinical trials in all phases have been conducted with phenethyl isothiocyanate (PEITC, 79)221 and sulforaphane (80) e. g. for cancer chemoprevention, the treatment of schizophrenia and autism spectrum disorders (see https://clinicaltrials.gov). Evaluation in a clinical setting and the natural abundance in foods suggest that these electrophiles are well tolerated, however, their biological action has been attributed to multi-target effects. Isothiocyanates rapidly undergo GSH addition to form dithiocarbamates but this process is reversible223,224 and trans-thiocarbamoylation to proteins occurs.225 Reactivity towards amino groups to form stable thioureas is much lower. Nevertheless, lysine residues can be labeled as a consequence of the dithiocarbamate-isothiocyanate equilibrium,226 but also direct reaction pathways seem possible (Figure 32B). Reactivity of isothiocyanates can be increased by the attachment of electron-withdrawing groups, while electron-donating (+I) substituents decrease the electrophilicity of the sp-hybridized carbon center.224 Many proteins, such as glutathione S-transferase pi (GSTP) 1,

227

the cysteine-rich

protein Keap1,228 protein tyrosine phosphatases229 or the protein kinase MEKK1,230 are covalently labeled by isothiocyanates at cysteine residues. Binding to Cys347 of tubulin has been suggested as a key mechanism of action of natural isothiocyanates231 and the N-terminal catalytic proline in macrophage migration inhibitory (MIF) factor is also modified by these compounds.232 Due to the reversible nature of cysteine binding, investigation of targets in living systems is challenging. Despite of the wealth of data on isothiocyanates that has accumulated during several decades,


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systematic studies of SAR and applications in TCI design remain sparse. This might, at least in part be attributed to the very complex equilibria driving isothiocyanate distribution and availability in vivo.233

Figure 32: Isothiocyanates as covalent-reversible warheads for cysteine and irreversible CRGs for lysine. A) Common isothiocyanates found in cruciferous vegetables. B) Reversible reaction of isothiocyanates with GSH and cysteines and slow thiourea formation, e.g. with lysine. Possible direct reaction pathways are depicted as dashed arrows.

2.15 Reversible cysteine addition to electron-deficient heteroarenes forming stable Meisenheimer complexes In a recent publication, Campbell McInnes and co-workers described fragment-like covalentreversible Polo-like kinase (PLK) 1 inhibitors based on a benzothiazole N-oxide scaffold.234 This compound class was identified by a virtual screening campaign and the initial hit 81a (Figure 33) possessed a moderate potency in the low micromolar range (IC50 = 2.5 µM). Subsequent SAR


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studies identified the nitro group in the C7-position and N-oxide as crucial features for activity while replacement C2-carboxamide moiety by a nitrile or a hydroxamate group was tolerated with a moderate loss in potency. Lipophilic –I-substituents were favorable at the C5 position with the most potent compound 81b (IC50 = 0.4 µM) featuring a trifluoromethyl thioether group. Since compound 81b possessed limited ATP competitivity and docking suggested close proximity between the Cys67 side chain and the C4-aryl carbon atom, covalent binding via formation of a Meisenheimer complex was considered as the potential mechanism of action. It is worth mentioning that Meisenheimer complex formation has already been described earlier as the mode of action of the glutathione S-transferases inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4ylthio)hexanol.235 The stability of such negatively charged σ-complexes can be rationalized by the very electron-deficient nature of the (hetero)aryl system combined with the very low anionic stability of the hydride ion, preventing its departure. By using n-butylamine and 1-butanethiolate as surrogate nucleophiles in NMR and UV-VIS experiments, potency was roughly correlated with the propensity of the compounds to form Meisenheimer-type adducts. As expected, the reaction was reversible upon removal of excessive n-butylamine. Consequently, it can be assumed that such inhibitors would be subjected to rapid but reversible GSH addition in vivo. Moreover, inhibitor 81a was completely inactive (IC50 > 100 µM) against the PLK1 C67S mutant. Screened at 100 µM, 81a showed good selectivity in two small panels (38 kinases) including 3 kinases with an equivalently positions cysteine. No details on cellular permeability, metabolic stability and binding kinetics were provided and no ligand-bound X-ray crystal structure was solved to confirm covalent modification of Cys67. Although being highly interesting from a conceptual point of view, it remains to be seen to what extend molecules capable of forming stable Meisenheimer complexes,


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which typically contain one or several nitro groups to stabilize the negative charge, will enrich our current medicinal chemistry toolbox of covalent-reversible warheads.

Figure 33: Meisenheimer complex electrophiles as putatively covalent-reversible PLK1-inhibitors. Mechanism and selected compounds.

2.16 Reversible cysteine targeting by disulfide tethering It is well known that cysteine thiols form disulfide bridges. To this end, they can be addressed by strained or unstrained disulfides via thiol–disulfide exchange, by different reagents possessing electrophilic sulfur atoms or even with free thiols under oxidizing conditions (see scheme Figure 34A for some examples). Interesting recent applications include disulfide tethering for identifying low affinity fragments,182,236 thiol-mediated uptake approaches237 and glutathione-responsive prodrugs.238 Disulfide tethering can be very useful in structural biology.239 For example, covalent trapping of the of the β2 adrenergic receptor featuring an engineered cysteine (H93C) with the disulfide-based ligand FAUC50 (82, Figure 34B) enabled the first X-ray crystallographic structure determination of this GPCR in an agonist-bound state.240 Although many medicinal chemistry campaigns, especially in academia, have exploited disulfide formation for covalent cysteine targeting (for some recent examples see Ref.241–243), it remains questionable if this linkage strategy


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is suitable for targeted covalent inhibition due to the complexity of physiological thiol-disulfide equilibria and redox systems.244,245 Therefore, no further discussion is provided.

Figure 34: Reversible cysteine targeting by disulfide bonds. A) Selected examples of headgroups known to generate disulfide bonds. B) FAUC50, a covalent ligand which enabeled crystallographic structure determination of the β2 adrenergic receptor. 3. NUCLEOPHILIC TARGETING OF OXIDIZED CYSTEINES WITH CARBON ACIDS Cysteine oxidation is known as an important regulator of protein function.57 Oxidation of the thiol group with reactive oxygen species (ROS) furnishes sulfenic acids, which can further react following a variety of reversible and irreversible reaction pathways.246 Reversible S-sulfenylation is involved in the regulation of phosphatases, kinases, ion channels and a plethora of other proteins.247 For example, sulfenylation of Cys797 in the EGFR kinase domain increases kinase activity while rendering the target insensitive to covalent EGFR inhibitors.248 In contrast to thiols, sulfenic acids are only weak nucleophiles while holding a more pronounced electrophilic


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reactivity. The latter has been capitalized for sulfenic acid detection with nucleophilic carbon acids.249 The most prominent sulfenic acid sensors are based on 5,5-dimethyl-1,3cyclohexanedione (dimedone, 83, Figure 35) and similar cyclic 1,3-dicarbonyl scaffolds. Other electrophilic groups that have been shown to trap sulfenic acids include norbornenes250 and strained cycloalkynes251, the latter having simultaneous electrophilic reactivity towards cysteine thiols.247

Figure 35: Carbon acids adressing cysteine sulfenic acids. A) Model system used by the Carroll group to assess sulfenic acid labeling with C-nuclophiles. A possible alternative reaction pathway


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is highlihted by the dashed arrow. B) Cysteine sulfenic acid formation by ROS and trapping with dimedone. C) Reactivities of cyclic C-nucleophiles. D) and E) Reactivities of linear Cnucleophiles. F) Tofacitinib, an approved JAK inhibitor shown to react with sulfenic acids. Reactivity is expressed by pseudo-first order rate constants derived from an LC-MS assay using the model system shown in A). The most notable contributions in targeting oxidized cysteine have come from the group of Kate Carroll, who made extensive use of α-CH-acidic β-dicarbonyls and similar compounds for sulfenic acid sensing. For example, they used redox-based probes to monitor oxidation of the phosphatases active site cysteine in living cells.249 As the reaction rate of sulfenic acids with dimedone is relatively low, the Carroll group developed a convenient LC/MS-based assay for screening the reactivity of C-nucleophiles towards a dipeptide-derived sulfenic acid under aqueous conditions. The principles relies on the in situ generation of the sulfenic acid from a more stable cyclic sulfenamide (Figure 35A).247 A large number of cyclic247 and linear252 C-nucleophiles were screened and an increase in reactivity could be achieved by replacing one of the ketones by a sulfone group, by adding electron-donating substituents to the ring system or by forcing non-planar ring conformations. The most reactive C-nucleophiles outperformed the reactivity of dimedone more than 200-fold as indicated by their pseudo-first order rate constants determined in the abovementioned assay. (see Figure 35B for selected examples). The reaction products were stable against reduction with DTT, GSH and TCEP and no cross-reactivity with protected cysteine, cystine, serine, lysine or sulfinic acid was observed. However, it should be mentioned at this point that recent evidence suggests, that sulfenamides can react faster with dimedone analogs than sulfenic acids.253 Therefore, the reactivities observed in the aforementioned assay may at least in part arise from direct reaction with the employed sulfenamide precursor. In a model study, selected


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compounds showed near-quantitative labeling of the C64S/C82S mutant of the glutathione peroxidase (GPX) 3 at a single oxidized cysteine residue. Linear C-nucleophiles could also be tuned, however within a narrower range (Figure 35C and D). Good reactivities were obtained by attaching electron-withdrawing groups to methyl sulfones. Interestingly, the reaction products of the most reactive derivative 85 could be readily cleaved under reducing conditions, a finding that was confirmed in GPX3 and HeLa cell lysates. In contrast, nitro analog 84 formed stable products withstanding reductive cleavage for several days. Intriguingly, the cyanoacetamide group of the approved reversible JAK inhibitor tofacitinib (86, Figure 35E) was also shown to react with cysteine sulfenic acids raising the question about potential off-target modification. In a subsequent study, the same group investigated the cysteine "sulfenylome" of RKO colon adenocarcinoma cells.254 To this end, five of the C-nucleophiles from the above studies were equipped with alkyne handles (87–91, Figure 36) to enable click-chemistry-based tagging and enrichment. By employing MS-proteomics, 1283 S-oxidation sites in 761 proteins were identified. Since sulfenamides, which can be formed from sulfenic acids, but also from mixed disulfides and amides, are quite abundant in biological systems, it seems likely that the observes oxidation sites account for a combination of the two former oxidized species.253 Nevertheless, little overlapping was observed with respect to the labeling sites of different nucleophiles suggesting significant discrimination even at this low level of structural complexity. Despite the relatively slow reaction kinetics, these data indicate that selective targeting of cysteine sulfenic acids and sulfenamides with more optimized compounds should in principle be possible. Further investigations, including the application of such molecules in vivo, are highly anticipated.


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Figure 36: Alkyne-tagged C-nucleophilic probes for chemical proteomics studies and their reactivity expressed by second order rate constants derived from the model system shown in Figure 35A. 4. TARGETING THE LYSINE SIDE CHAIN Much effort has recently been put into developing selective warheads for lysine residues, and key studies up to early 2017 have been summarized in a recent mini-review.39 Lysine, together with cysteine, has been the most common residue addressed in peptide and protein labeling chemistry.41 Perhaps the most prominent labeling reagents for the lysine ε-amino group are highly activated N-hydroxysuccinimide (NHS)-esters, which are generally considered too reactive for application in TCIs. Lysine offers certain critical advantages over cysteine as it has a much higher abundance (5.8 % vs. 1.9 %)59 and is typically found on protein surfaces, on interfaces mediating protein-protein interactions as well as in binding cavities. Lysine residues play a role in catalysis e. g. by acting as a base or as a nucleophile.255 Moreover, lysine in active sites can assist catalysis by positioning reactive residues or activating them via (charge-assisted) hydrogen bonds, (e.g. in kinases). In such cases, lysine residues are indispensable and resistance mutations, as they may


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occur for non-catalytic cysteines,65 unlikely. However, lysine has been much less frequently considered as a residue to be addressed by TCIs. The latter fact can be attributed to the special challenges associated with the ε-amino group as a nucleophile, especially when compared to cysteine. At physiological pH, surface-exposed lysine (pKa ≈ 10.5)256,257 is almost entirely protonated (99.9%) rendering the side chain poorly nucleophilic. However, the pKa largely depends on the chemical environment and buried lysines can undergo a pKa shift of up to 5 units making them addressable by electrophiles.257 As shown in several example above, the ligand itself may also contribute to pKa perturbation. Besides protonation, post-translational modifications (most notably acylation) can make the lysine side chain unsusceptible towards electrophilic reagents. Another special feature of lysine is the long and linear side chain causing a high degree of conformational freedom. Whether this property is to be considered favorable or not cannot be generalized and depends on the respective context. By employing a similar approach as previously used for assessing the reactivity of cysteine in native biological systems,13,156 the Cravatt group set out to identify ligandable lysine residues by means of chemical proteomics.258 A highly activated ester probe equipped with an alkyne handle (compound 92, Figure 37) was used to quantify the reactivity of lysine residues in proteomes. The study recovered over 9000 reactive lysine residues from different human cell lines, a much higher number than previously found for cysteine with a iodoacetamide probe, that roughly reflects the relative abundance of these amino acids.13,156 Moreover, experiments at different probe concentrations and competition experiments with less reactive fragments (general structures 93– 95, Figure 37) identified (hyper)reactive lysine residues, that have a high probability of being addressable by small molecules in a selective manner. A fact particularly worthy of note is that such lysines were identified mainly in proteins not found in the drug bank, including many


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challenging targets such as transcription factors and scaffolding proteins. However, no conserved motifs could be associated with lysine hyper-reactivity and further insights are required to deepen our understanding of lysine reactivity and ligandability.

Figure 37: Probes and warheads used to indentify ligandable lysines by chemical proteomics. Lysine side chains can react covalently with Michael acceptors but aza-Michael additions with lysines have often been discovered by chance rather than implemented by design. For example, the natural product wortmannin (96, Figure 38) labels Lys833 in phosphoinositide 3-kinase γ (PI3Kγ) by aza-Michael addition and concomitant opening of the ligand's furan ring (Figure 38 and PDB: 1E7U).259,260 More recently, the Cheeseman group serendipitously discovered that an adenosine-derived inhibitor designed to target a cysteine (Cys17) residue in the heat shock 70 kDa protein 1 (HSP72) was in fact bound to a lysine side chain (Lys56).261 In general, most Michael acceptors are more reactive towards the "softer" cysteine thiols (vide infra), which can be a disadvantage as the lysine amino group may require more activated electrophiles. The latter are more likely to exhibit cysteine-mediated off-target reactivity. The aza-Michael reaction with lysine furnishes Mannich-type bases which can re-eliminate the (protonated) amino group to recover the reactive electrophile. Thus, adduct formation can in principle be considered reversible.262 The


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extent of the reverse reaction, however, largely depends on the chemical nature of the reaction product (i.e. α-CH acidity of β-amino ketones vs. esters or amides and the basicity of the amino group) and will require further consideration.

Figure 38: Reaction mechanism of wortmannin with Lys833 in PI3Kγ. 4.1 Reactivities of Michael-acceptors and nitriles towards lysine versus cysteine Adam Gilbert and colleagues from Pfizer have recently assessed the reactivity of a large set of common electrophiles including α,β-unsaturated amides, α,β-unsaturated sulfones, α,βunsaturated sulfonamides, cyanamides and nitriles towards N-α-acetyl lysine and compared these with the corresponding GSH reactivities.263 In the case of N-α-acetyl lysine, the assay was conducted at pH 10.2 (≈ 20% of unprotonated amine) to mimic the pKa perturbation in binding pockets while reactions with GSH where performed at pH 7.4. Although it is clear that this model system does not fully reflect the real situation in binding sites, it provides a reasonable picture on reactivity trends towards thiolate and amino nucleophiles. Remarkably, no reaction was observed for N-α-acetyl lysine at pH 7.4 even with the most reactive electrophiles, pointing out the poorly


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nucleophilic nature of the protonated amine and confirming that the carboxylate group also remains inert. As expected, this model system recapitulated the same general trends as the previous GSH reactivity studies mentioned above (for selected examples, see Figure 39).263 Reactions of α,β-unsaturated amides proceeded 2–5 times faster with glutathione than with N-α-acetyl lysine. Similarly, the GSH addition to a cyanamide was twice as fast compared to N-α-acetyl lysine while the reaction with 2-cyanopyrimidine was even 9 times faster. Interestingly, an opposite trend was observed for α,β-unsaturated sulfones and the corresponding sulfonamides. For example, α,βunsaturated sulfonamides reacted 1.4–4-fold faster with N-α-acetyl lysine compared to GSH while an even 9 times faster reaction was measured for methyl E-prop-2-enyl sulfone. This striking difference can be rationalized with the Pearson HSAB theory,54 suggesting that the strongly electron-withdrawing sulfonyl group makes the β-carbon atom less polarizable (i.e. harder) favoring the reaction with a harder nitrogen nucleophile rather than with the soft thiol group. Although α,β-unsaturated sulfones and sulfonamides have a higher general reactivity compared to the corresponding carbonyl-derived Michael acceptors, sterical tuning of these electrophiles might be harnessed to generate selective lysine-targeted probes with a low degree of promiscuity


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Figure 39: Reactivity of different Michael acceptors and activated nitriles towards lysine and GSH. Compounds with a preference for GSH are shown the upper row, while such preferably reacting with N-α-acetyl lysine are depicted in the lower row. Half-lives were determined in the presence of 50 mM N-α-acetyl lysine at pH 10.2 and 37 °C or 10 mM GSH at pH 7.4 and 37 °C. The higher reactivity of α,β-unsaturated sulfones towards lysine was used by the groups of Bernhard Golding and Jane Endicott to generate the first irreversible inhibitor of cyclin-dependent kinase (CDK) 2.264 The key compound of this study, vinyl sulfone NU6300 (98a, Figure 40A), was identified while optimizing sulfonamide NU6102 (97). Binding of 98a was reversible on short time scales and an equilibrium binding constant could be determined by SPR (KD = 1.31 µM) pointing out the only moderate affinity. However, inhibition was time-dependent and 20 h of incubation with 98a abolished 50 % of the apparent binding capacity of the slightly more potent saturated analog NU66310 (98b, KD = 0.72 µM). Mass spectrometry and washout experiments confirmed the covalent modification. The binding to non-conserved Lys89 was suggested by studies with the CDK2 K89V mutant which did not show adduct formation in MS experiments and recovered activity upon removal of the compound by dialysis. X-ray crystallography finally


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confirmed the binding mode (Figure 40B). The compound inhibited 13 out of 113 kinases in a panel at 1 µM but only two of those in a time-dependent manner. The rather slow inactivation kinetics might be rationalized by the surface exposed location of Lys89 suggesting that the ε-amino group is almost completely protonated and therefore unreactive. However, it remains questionable if such slow inactivators would be useful in an in vivo setting as they only partially benefit from key advantages of covalent labeling (e.g.

decoupling of pharmacodynamics from

pharmacokinetics) while comparatively rapid off-target modification might be an issues, especially since a quite reactive electrophile was employed.

Figure 40: Vinyl sulfones for targeting a lysine in the solvent-exposed front region of CDK2. A) Covalent and non-covalent inhibitors. B) X-ray crystal structure of vinyl sulfone 98a covalently bound to Lys89 flanking the solvent-exposed front region of CDK2 (PDB 5CYI). The purine NH is hydrogen-bonded to the backbone carbonyl atom of Glu81 while the N3-atom and the diaryl NH are anchored to the backbone of Leu83 by two additional hydrogen bonds. Another water-bridged hydrogen bond links the purine N7-atom to the backbone carbonyl group of Asp145 in the DFG


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motif. Further direct and water-mediated hydrogen bonds are established by the sulfonyl group. The N-terminal lobe was omitted for clarity. 4.2 Reactivities of sulfonyl fluorides and related sulfur (VI) fluorides towards lysine, tyrosine and cysteine Sulfonyl fluorides are amongst the most prominent electrophiles for addressing the ε-amino group of lysine residues, but also for tyrosine, activated serine or threonine and to a lesser extent cysteine and histidine side chains.265 The potential of sulfonyl fluorides, which have been known as insecticides for more than 100 years,266 in drug discovery was first recognized by Bernhard. A. Baker in the late 1960s.267 However, until its recent revival by Sharpless and co-workers, sulfur (VI) fluoride chemistry has played a subordinate role while sulfonyl chlorides have frequently been used as reagents in organic synthesis.266 The high reactivity of sulfonyl chlorides, as well as their instability towards hydrolysis and reduction have hampered their use for biological applications. In contrast, sulfur (VI) fluorides are thermodynamically stable and typically resist hydrolysis and reduction. In contrast to their chloride counterparts, they are quite demanding electrophiles requiring sufficiently strong nucleophiles and proper solvation of the fluoride group for reaction. While sulfonyl chlorides may also be attacked on the chlorine atom, sulfonyl fluorides exclusively react at the sulfur atom. Therefore, sulfur (VI) fluoride exchange (SuFEx) has recently manifested itself as a useful click-type chemical reaction266 with favorable properties for labeling biomolecules.45 Sulfonimidoyl fluorides, the imino-analogs of sulfonyl fluorides, possess similar favorable properties and an additional handle to attach substituents allowing for modulation of reactivity by the moiety attached to the nitrogen atom. Fluorosulfates, and the analogous sulfamoyl fluorides are even less reactive (Figure 41). It has been further suggested that fluorosulfates become only activated if the departing fluoride ion is stabilized by the protein surrounding, e.g. by


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a hydrogen bond donor or suitable electric field effects.268 Therefore, this functional group has been termed a "latent electrophile".269

Figure 41: Sulfur (VI) fluorides for lysine and tyrosine targeting. Early examples of the application of sulfonyl fluorides in chemical biology and drug discovery include protease labeling reagents such as benzenesulfonyl fluorides (99a and b, Figure 42)270,271 and dansyl fluoride (100),272 enzyme inhibitors exemplified by dihydrofolate reductase inhibitors (e.g. NSC 127755, 101) and nucleotide-derived probes (e.g. 5'-p-fluorosulfonylbenzoyl adenosine, 5'-FSBA, 102).273 Sulfonyl fluorides have also been used to generate irreversible GPCR ligands274– 276

and the tyrosine-targeted covalent adenosine A1 receptor antagonist DU172 (103) has recently

enabled the crystal structure determination of the latter receptor.277 Methanesulfonyl fluoride (MSF, 104) is known as an irreversible acetylcholinesterase (AChE) inhibitor for more than 50 years.278 Based on the rationale that AChE in the CNS has a substantially lower re-synthesis rate than in peripheral tissue, clinical phase I and II studies have been conducted assessing the potential of MSF in the treatment of Alzheimer's disease.279,280 MSF doses up to 0.18 mg/kg three times a week were well tolerated while effectuating the desired AChE inhibition in vivo and improving cognitive performance. Further applications of sulfonyl fluorides have been reviewed recently.39,265,266,281


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Figure 42: Early examples for the application of sulfonyl fluorides in medicinal chemistry and chemical biology. A detailed assessment of the reactivity of sulfonyl fluorides and analogous sulfur (VI) fluorides (Figure 43) was provided by Grimster and colleagues from AstraZeneca.282 They found that arylsulfonyl fluorides readily react with N-acetylcysteine (NAC) at pH 7.5. Reaction rates of most derivatives were slightly higher compared to analogous N-aryl acrylamides. However, the reactivity of sulfonyl fluorides could be tuned over a wider range with a variety of electronwithdrawing and electron-releasing groups (Figure 43A), the rate following a log-linear relationship with the Hammett value of the aryl substituent. The higher sensitivity to the electronic properties of the aromatic ring system compared to α,β-unsaturated amides can be rationalized by


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the direct attachment of the sulfonyl fluoride group. The thiol substitution products, however, were unstable under the assay conditions suggesting this functionality unsuitable for durable cysteine modification. This finding is not unexpected since the thiosulfonate S-esters formed by fluoride displacement are known to be unstable towards hydrolysis and react with thiols to form disulfide bonds (see the mechanism in Figure 43D).283 In cells, an analogous reaction would predominantly produce glutathione disulfide (GSSG) due to the high intracellular GSH concentrations. It can also be assumed that thiosulfonate S-esters within protein binding sites would be cleaved by reductive sample preparation emphasizing that cysteine labeling in living systems would be difficult to capture. The reactivity towards N-acetyl tyrosine and N-α-acetyl lysine was considerably lower but stable substitution products were obtained in both cases. While the reaction rate of phenylsulfonyl fluoride (105a) with the tyrosine side chain was 3.4 times lower compared to NAC, a 10-fold slower reaction was observed for N-acetyl lysine at pH 7.4. In contrast, reaction rates increased substantially in both cases when the pH was increased reaching a similar level for N-αacetylated tyrosine and lysine at pH 10. However, no reaction other than hydrolysis was observed for N-acetylated serine. Warhead hydrolysis was rapid for electron-deficient arylsulfonyl fluoride derivatives (t1/2 ≈ 5–15-minutes) while analogs bearing electron-donating substituents were stable up to several days. Nevertheless, hydrolysis was typically 1000–5000-fold slower than reaction with the tyrosine side chain. As shown for N-acetyl tyrosine, the more electron-rich alkyl sulfonyl fluoride 106 was 9 times less reactive than aryl analog 105a. The benzylic derivative phenylmethane sulfonyl fluoride (PMSF, 107), a reagent commonly used in biochemistry to label active site serine residues or to prevent protein degradation in cell lysates,284 was rapidly hydrolyzed, presumably due to the increased α-CH acidity favoring the reaction via a sulfene-like intermediate. Plasma stability reflected the trend from the hydrolysis assay with 4-dimethylamino


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derivative 105d showing a favorable half-life (t1/2 = 25.1 h). Clearance data was obtained for compounds 105b−d (165, 14 and 170 µL/106 cells min, respectively) and several 5'-FSBA analogs (vide infra) in rat hepatocytes. In these series, metabolic clearance correlated rather with the lipophilicity of the compounds than with the Hammett value of the aryl substituents suggesting that inherent warhead reactivity is not the key determinant of hepatic metabolism.

Figure 43: Compounds used for assessing the reactivity of sulfur (VI) fluorides with different amino acids. A) sulfonyl fluorides. B) sulfonimidoyl fluorides. C) aryl fluorosulfates. D) Mechanism of the reaction between phenylsulfonyl fluoride and NAC. A series of related sulfonimidoyl fluorides (represented by 108a–f, Figure 43B) with different substituents at the nitrogen atom was also probed for reactivity towards N-acetyl tyrosine. As expected, these compounds featured a lower reactivity compared to the analogous sulfonyl fluorides and electron withdrawing N-substituents were required for obtaining substitution products at the given conditions. Among the activated derivatives, N-acylated derivatives 108a


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and b reacted slightly faster than sulfonyl fluoride 105a, while the N-Boc analog 108c reacted at a similar rate. N-Methylated analog 108f showed no reaction under the same assay conditions. Interestingly, hydrolytic stability was increased compared to 105a even for the more activated derivatives suggesting this structural class as superior warheads in terms of stability, versatility and tunability. However, it should not remain unmentioned, that electron-rich N-aryl derivatives (e.g. compound 108d), in contrast to p-CF3-phenyl-substituted analog 108e, degraded rapidly, presumably via an oxidative decomposition pathway. Notably, NAC also reacted with the Nacylated derivatives to form unstable products in while N-alkylated derivatives remained inert under the same conditions. Aryl fluorosulfates 109 and 110 (Figure 43C) were completely stable at pH 7.5 and neither hydrolysis nor substitution with N-acetyl lysine or tyrosine occurred within a 24 h timeframe. Reaction with NAC was very slow but extended incubation (4 days) resulted in the formation of the corresponding phenol, presumably via cystine formation in analogy to Figure 43D and subsequent elimination of SO2. It is worth mentioning that the intrinsic reactivity of fluorosulfates is so low, that fluorosulfate-L-tyrosine could recently be genetically encoded and incorporated in proteins as latent nucleophile for inter- and intra-protein crosslinking in mammalian cells.285 No sulfamoyl fluorides were evaluated in this study but it can be assumed that these compounds would even be less reactive than the corresponding aryl fluorosulfates.266

4.3 Lysine targeting with sulfur (VI) fluorides In the second part of the last-mentioned study, the reactivity of sulfonyl fluorides in a protein binding pocket was probed. To this end, analogs of the above-mentioned inhibitor 5'-FSBA (m-5'FSBA; general structure 111, Figure 44) were prepared with different substituents ortho to the warhead. A crystal structure of prototype inhibitor 111a in complex with FGFR1 (PDB: 5O49)


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and MS experiments confirmed the expected binding to the conserved Lys514 and suggested that an ortho-substituent could be accommodated by the binding site. The rate of covalent modification was evaluated by an LC-MS method showing that steric bulk in the ortho position did not significantly influence the reaction rates, which roughly correlated with the substituents Hammett parameters. As expected, the two analogous aryl fluorosulfate derivatives showed a very low reactivity with only traces of covalent modification after extended reaction times.

Figure 44: Lysine-targeted m-5'-FSBA analogs for the evaluation of the relationship between warhead reactivity and FGFR1 inhibitory activity. An interesting application of sulfonyl fluorides as CRGs was provided in 2017 by Jack Taunton and co-workers who generated a lysine-targeted broad spectrum kinase inhibitor as an active-site probe for chemical proteomics applications.286 A promiscuous pyrimidine 3-aminopyrazole scaffold was selected based on X-ray crystal structures of such ligands complexed to the ATP pockets of various kinases. Structural data indicated that a linker attached to the 2-position of the pyrimidine ring would position a phenylsulfonyl fluoride proximal to the conserved active site lysine, a residue that is involved in the positioning and activation of the ATP triphosphate moiety. Using SRC as a model kinase, it was shown by MS and X-ray crystallography that optimized and clickable probe XO44 (112, Figure 45A) specifically modified the expected lysine (Lys295, Figure 45B) while none of the other 16 lysines and 13 tyrosines were labeled by a 3-fold excess of the compound. Another X-ray crystal structure (PDB: 5U8L) confirmed a similar binding mode for


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the EGFR receptor tyrosine kinase. In Jurkat T-cells the inhibitor captured 133 protein kinases (of which 50 are not covered by the kinobead technology287) and even 219 kinases were inhibited > 50 % at 1 µM in a panel containing 375 kinases. Notably, 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 non-physiological conditions in in vitro screening panels or arise from non-covalent inhibition that cannot be captured in the cellular labeling experiment. Although some non-kinase 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.

Figure 45: A) Promiscuous kinase probe XO44. B) X-ray crystal structure of XO44 covalently bound to the conserved Lys295 in the kinase SRC (PDB: 5K9I). The 3-aminopyrazole is anchored to the hinge region by three hydrogen bonds involving the backbone of Glu339 and Met341. The sulfonyl group forms two additional hydrogen bonds with Phe278 and Gly279 in the glycine-rich loop while the propargyl amide tag is oriented towards the bulk solvent without being involved in specific interactions.


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As an application example of aryl fluorosulfates, these warheads were used by Jeffrey Kelly and co-workers in fluorogenic probes to image transthyretin in living cells and Caenorhabditis elegans.288 Cell permeable inhibitors 113a and b (Figure 46) were designed to target the pKaperturbed 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 b, respectively. These findings comply with the known low reactivity of the fluorosulfate warhead, 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 no general reactivity towards the proteome could be detected. Unexpectedly, the amino group was only present as a free sulfamate without the ligand attached indicating that the substitution product had been hydrolyzed. Although the authors stress the probable catalytic effect of the target protein on hydrolysis, these results demonstrated the limited stability of the covalent sulfamate complex that will have to be evaluated on a case-by-case basis. In proteins where the lysine residue is required for catalytic activity, the ε-sulfamate 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 anticipated. Examples for the use of fluorosulfates in tyrosine and serine targeting can be found in the chapters 5.1 and 6.1.


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Figure 46: Fluorosulfates for lysine targeting. A) Aryl fluorosulfates for adressing Lys15 in human transthyretin. B) X-ray crystal structure of 113b bound to in human transthyretin (PDB: 4YDM). Unexpectedly, the Lys15 ε-sulfamate was found instead of the covalently bound ligand. The ligand is located in a relatively shallow pocket on the protein surface forming only a single hydrogen bond between the hydroxy group of the dichlorophenol moiety and the side chain of Ser117. 4.4 Lysine acylation by activated esters The potential of activated esters for targeting lysine side chains was recently emphasized by a team around Sebastian Campos at GSK.290 With the aim of generating a potent and selective covalent PI3Kδ inhibitor, they modified the clinical candidate GSK2292767 (115), 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−g) would be a promising strategy to address this moiety. A variety of phenolic esters with electron-withdrawing or releasing substituents in the para position was prepared and tested for their ability to covalently inactivate PI3Kδ. Isoform selectivity and activity


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in human whole blood was assessed in parallel. Activity in the isolated kinase assay correlated roughly with the Hammett values 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 and d maintained low nanomolar potency, high isoform selectivity (ca. 2–3 orders of magnitude) and excellent activity (≈ 10 nM) in human whole blood. Kinetic analysis revealed that the rate of covalent inactivation was similar for all compounds (kinact = 5.8–7.5 x 10-3 s-1) and did not correlate with the leaving group properties of the phenol group, 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 non-activated methyl ester 116g bound only reversibly to the enzyme. Compound 116d was stable against hydrolysis and reaction with N-αBoc-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 was 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 clean chemoproteomic profile in Ramos cells. Washout experiments in CD4+ Tcells 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 was 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 fumarate esters, 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 protection,291 might be used in a similar manner as these activated esters.


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Figure 47: Development of activated esters covalently targeting Lys779 in PI3Kδ. 4.5 Acylation of surface-exposed lysines by N-acyl-N-alkyl sulfonamides Another structure class that has recently been used as a warhead for lysine side chains are Nacyl-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 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 (KI = 20 nM), was attached 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 x 104 M-1s-1, kinact = 6.1 x 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 where present at


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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 the importance of potent and selective reversible binding for target specificity, especially when highly reactive electrophiles are employed. Notably, similar experiments were conducted with an analogous trimethoprim-derived probe (not shown) specifically labeling Lys32 in E. coli dihydrofolate reductase (kinact/KI ≈ 9.3 x 103 M-1s-1, kinact = 1.3 x 10-2 s-1). In an inverse 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 x 104 M-1s-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 suitable 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 in spatially restricted binding sites. Finally, despite the good cellular selectivity demonstrated for compound 120, the intrinsic reactivity of this warhead class towards other nucleophiles remains yet to be assessed.


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Figure 48: N-acyl-N-alkyl sulfonamides addressing surface-exposed lysine side chains. A) Biotintransferring probes. B) Covalent ligand design and validation. The transferable residue is highlighted in red. Further examples of electrophiles that have been used to target lysine residues include natural product-derived spiro-epoxides addressing Lys100 in phosphoglycerate mutase 1 (PGAM1)293,294 and quinazolin-4(3H)-one hydroxamate esters modifying non-active 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 may be referred to recent reviews.41–43,46


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4.6 Condensation of lysine with aldehydes forming stabilized Schiff-bases Schiff bases (imines), which are formed by the reversible condensation of a lysine side chain 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 co-factors 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 the 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 chapter 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 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 well-known 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 stabilize


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the iminoboronate generated upon Schiff-base formation 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-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 minutes.

Figure 49: 2-formylbenzenboronic acid reversibly forming stabilized Schiff bases with amines. Applications of this chemistry include the labeling Gram-positive bacteria by targeting aminepresenting 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 Quibin 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. Formylboronic acids 122a and b (Figure 50) as well as the analogous acetophenone 122c featured low nanomolar IC50 values in a TR-FRET based binding assay while derivatives lacking either the boronic acid or the orthocarbonyl group were substantially less active. The improved potency translated into superior cellular activity and the best compound (122b) 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


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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 c, respectively). It is worth mentioning that the warhead alone did not generate covalent complexes. Although no X-ray was included, the targeting of Lys234 was supported by binding data of covalent and non-covalent analogs to the K234A mutant. According to this data, 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 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 mentioned susceptibility of aldehydes and ketones towards redox biotransformation, will have to be assessed.

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


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5. TARGETING THE TYROSINE SIDE CHAIN Neutral tyrosine has a relatively low intrinsic nucleophilicity when compared to cysteine or the (unprotonated) lysine ε-amino group. Therefore, the selective modification of non-activated 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 towards 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 to react with hard Lewis acids. Besides the nucleophilic properties of the phenolic hydroxy group, the electron-rich nature of the phenol ring has been exploited for tyrosine-selective bioconjugation reactions e.g. with in situ generated Mannich reagents,308 diazonium salts309 or via ene-like reactions with triazoline diones.310,311

5.1 Tyrosine targeting with sulfur (VI) fluorides Since the inherent reactivity of tyrosine and lysine towards sulfur (VI) fluorides has already been discussed extensively in chapter 4.2, only examples for their application are provided in this section. Sulfonyl fluoride warheads have recently been used by Lyn Jones and colleagues from Pfizer to address the mRNA-decapping scavenger enzyme DcpS.

307

In a structure-based design

approach, they modified known diaminoquinazoline-derived 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 two orders of magnitude more potent than the reversible parent compound D153249 (123, Figure 51). X-ray and MS experiments showed that the ortho-


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and meta-substituted inhibitors 124a and b selectively modify Tyr113, while compound 124c with the CRG in the para-position reacts exclusively with Tyr143. However, all of these inhibitors left Lys142 and His139 untouched indicating that precise positioning of the warhead in conjunction with the appropriate intrinsic reactivity of the sulfonyl fluoride group for tyrosine confers specificity. Notably, however, analogous inhibitor with a silent click-tag that was used in the study revealed some off-targets that were not further disclosed.

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

Figure 52: Distinct binding modes of 124a–c in the respective X-ray crystal structure in complex with DcpS. The ligand is completely embedded in the protein environment and the 2,4diaminquinazoline 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


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group and the Asp205 side chain. The second proton of the quinazoline 4-amine forms an intramolecular H-bond to the ether linker. 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. 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. The overall orientation resembles that of 124a, but Tyr143 is labeled instead of Tyr113. A similar conformation of Lys142 is present in B) and C) and no interactions with Tyr273 and His139 are observed in these structures (these residues were omitted for clarity). No covalent modification of the proximal nucleophiles Lys142 and His139, which are also highlighted, was observed in any of the experiments. 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 kinase ligand covalently targeting 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 towards the SR-protein kinase SRPK1 (IC50 = 11 nM on isolated SRPK1). A co-crystal 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 head group conserved 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


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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 Tyr277 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.

Figure 53: Design of sulfonyl fluoride SRPKIN-1, the first tyrosine-targeted covalent kinase inhibitor. The less reactive aryl fluorosulfates have been suggested as privileged tyrosine-targeted warheads 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 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), it was demonstrated that this compound class


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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 analog 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. Cell-permeable probe 128 showed a very low background proteome labeling in HET293 T-cells. An X-ray crystal structure in complex with CRABP2 (Figure 55) confirmed covalent bond formation with Tyr143. In contrast to the above-mentioned complex with transthyretin-Lys15, 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, these inhibitors were too unstable to determine the metabolic turnover in human liver microsomes precluding their application as in vivo probes.316

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


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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 a water-mediated hydrogen bond to the Arg111 side chain. The PEG-linker is not resolved and a second, slightly deviating conformation of the ligand and the Tyr134 side chain was omitted for clarity. Subsequently, the same group 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 fluorosulfates for covalent lysine or tyrosine binding.269 By capitalizing three distinct alkyne-labeled probes of intermediate complexity (130133, 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 or reaction with thiols (compare chapter 4.2) might also hamper product detection. Labeling at a specific site was validated for 11 targets by


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MS, mutagenesis and X-ray crystallography using the recombinant proteins. As expected, covalent modification occurred only at tyrosine and lysine residues. Interestingly, probe 132 was reactive towards a largely different set of proteins compared to probes 130 and 131. 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 56. Alkyne-tagged aryl fluorosulfate-based probes used in an "inverse drug discovery" approach. 5.2 Tyrosine targeting by SNAr reactions LAS17 (133, Figure 57), an inhibitor with an 4,6-dichloro-1,3,5-triazin warhead, was recently shown by Lisa Crawford and Eranthie Weerapana to target Tyr108 in glutathione S-transferase Pi (GSTP1).317 After having discovered by chemical proteomics that dichlorotriazines preferably label lysines while p-chloronitrobenzenes favor cysteines,130 a library of 20 N,N-disubstituted 4,6dichloro-1,3,5-triazin-2-amines equipped with a click handle was synthesized and tested against HeLa cells at 1 µM. Protein enrichment from cell lysates revealed that one compound, LAS17,


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selectively modified a 25 kDa protein that was identified as GSTP1. This selectivity is quite surprising since 4,6-dichloro-1,3,5-triazin-2-amines chemically react with amino nucleophiles even at ambient temperature.318 LAS17 inhibited GSTP1 in a concentration-dependent manner and activity was not comprised in the presence of the protein background from HeLa cell lysates. The second-order rate constant of inactivation (kinact/Ki) was determined as 3.12 x 104 M-1s-1 and intact protein MS proved selective mono-labeling. Further MS studies revealed Tyr108 as the only site of modification despite the presence of two highly reactive cysteine residues in GSTP1. It cannot be fully excluded that the reaction proceeds via an initial reaction with one of the more nucleophilic cysteines followed by transfer on Tyr108 to form the thermodynamically favored product. However, due to the distance between these residues (≥ 11 Å between the cysteine sulfur atoms and the phenolic oxygen in PDB: 5X79) and their arrangement, such a low energy transfer pathway seems unlikely in this case. Mutation of Tyr108 to Phe prevented covalent modification and LAS17 possessed negligible activity on the mutant protein. It should be mentioned at this point that Tyr108 had previously been shown to be reactive towards sulfonyl fluoride based probes. 319 Although this study highlights the ability of SNAr warheads to efficiently target tyrosine residues, it should be kept in mind that the employed electrophile features a high intrinsic reactivity. It is therefore remarkable that LAS17 only marginally labeled other proteins at a concentration of 1 µM 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.


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Figure 57: LAS17, a tyrosine-targeted dichlorotriazine-derived GSTP1 inhibitor. 6. TARGETING NON-CATALYTIC SERINE AND THREONINE RESIDUES Serine and threonine are abundant as the key catalytic residues in the binding sites of proteases and other hydrolase enzymes. In these active sites, 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 this enzyme class and as mentioned already, many of the CRGs described in this Perspective had initially been developed to target the active sites of hydrolases.7,321 A detailed description of covalent inhibitors of proteolytic enzymes, however, is far beyond the scope of this article. In contrast, non-active site 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 non-catalytic serine modification, namely the acetylation of Ser530 in cyclooxygenases by aspirin.322 However, reports on compounds targeting non-catalytic serine and threonine residues in a proximity-driven manner are very rare. In general, hard Lewis acids, such as sulfur (VI) fluorides or oxophilic phosphorous (V) compounds, but also boron-based reagents may be suited best for addressing such hydroxy groups.


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6.1 Targeting non-catalytic serine by fluorosulfates In a follow up of the study on diaminoquinazolines targeting tyrosine residues in DcpS (see chapter 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 towards 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 = < 0.02 nM, cf. Figure 51). Unexpectedly, however, compound 134a modified neither of the expected tyrosine residues nor the adjacent lysine. Instead, reaction with the non-catalytic Ser272 was observed in peptide mapping experiments. LC-MS experiments using the full protein and MS-based analysis after tryptic digestion did not reveal the labeled protein as the predominant species but the dehydroalanine (Dha) elimination product (see the mechanism in Figure 58B). In contrast, native ESI-MS323 detected the labeled protein along with minor amounts of the dehydroalanine product, which slowly increased over time suggesting that the latter species is mainly an artefact and not dominant in protein's binding site on a short time scale. The covalent engagement of Ser272 was especially surprising since this residue is not functionally relevant as the S272A mutant retains activity. The authors argued, that the additional oxygen spacer would position the sulfur atom in an unfavorable position for being attacked by one of the more nucleophilic tyrosine or lysine side chains, while the positive electrostatic nature of the binding pocket featuring an adjacent histidine triad may depress the pKa of the serine and/or facilitate the departure of the fluoride leaving group. One implication of the limited stability of these sulfate products is that classical strategies for monitoring (off)-target modification, e.g. the click-chemistry based capturing approaches discussed above, would not be suitable for this


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warhead type. Alternative strategies will be required to examine the scope of protein modification by such compounds in proteomes. Further investigation will also be required to show whether the formation of dehydroalanine-containing proteins could be a safety issue in vivo as these are electrophilic themselves and may react with other physiological nucleophiles to generate haptencarrier adducts. On the other hand, the underlying elimination reaction might be optimized and serve as a handle for specific in vivo protein derivatization.

Figure 58: A) Aryl fluorosulfate-based inhibitor FS-p1 targeting Ser272 in DcpS. B) Proposed mechanism for the formation of the dehydroalanine elimination product.

7. TARGETING GLUTAMATE AND ASPARTATE SIDE CHAINS Covalent modification of carboxylate residues as present in aspartate and glutamate side chains poses a special challenge due to the weakly nucleophilic nature of carboxylate group. Although carboxylates can react with many of the electrophiles discussed above (e.g. with epoxides or αhaloacetamides), these reactions are not specific to carboxylates and stronger nucleophiles react preferentially. Some methods for the selective covalent trapping of carboxylates in proteins such as the photoclick-reaction with tetrazole-based reagents324,325 or the reaction with α-diazo carboxamides326 have been described as methods for bioconjugation. Cyclitol epoxides and aziridines as well as fluorinated glycosides have further found application as carboxylate-targeted


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electrophiles in activity-based probes and inhibitors of retaining glucosidases, an enzyme class featuring aspartate or glutamate residues as the catalytic nucleophile.165 Sulfonate esters were shown to react preferentially with aspartate and glutamate in proteomes, but this behavior seems to be mediated by the respective protein environments as no such selectivity could be observed in solution.157 On the other hand, it was shown that aziridines and stabilized diazo groups react with N-Boc-aspartate and benzoic acid in solution, but these warheads failed to significantly label Asp12 in the K-Ras G12D mutant.182 Boronic acids have further been suggested to interact with an aspartate in the front pocket of the EGFR kinase domain via reversible covalent bond formation327 but this interaction was not confirmed experimentally and is not specific for carboxylates. Therefore, biocompatible warheads with a specific intrinsic reactivity for carboxylates are largely underdeveloped. However, selective and tunable warheads for this functionality would be particularly desirable since Asp and Glu constitute more the 10 % of the protein sequence space59 and are thus frequently found in or around binding pockets.

7.1 Targeting carboxylates with isoxazolium salts In a recent proof of principle study, Herbert Waldmann and colleagues employed isoxazolium salts derived from Woodward's reagent K (WDK)328 as site-specific covalent warheads for a glutamic acid residue in the lipoprotein binding chaperone phosphodiesterase (PDE) 6δ.329 Since previous sub-nanomolar bis-sulfonamide-based inhibitors (exemplified by 135, Figure 59A) suffered from ligand displacement by allosteric release factor (ARL) 2/3 proteins,330 covalent labeling was considered. Since no cysteine or lysine residues are available in the PDE6δ prenyl binding pocket, tosylate tags331 were initially introduced for covalently addressing Glu88 but labeling efficiency was low. An alternative targeting strategy was inspired by Woodward's


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isoxazolium-derived carboxylic acid activation reagents. N-methyl isoxazolium salts (exemplified by general structure 136, Scheme Figure 59B) undergo ring opening in a base-promoted manner furnishing ketenimides (137), which readily react with carboxylic acids to form O-acylated 1,2enolized β-ketoamides (138). The latter slowly rearrange to form the corresponding β-enol esters (139). It was hypothesized that initial ring opening could be initiated by proximal basic groups in the binding pocket furnishing the reactive ketenimide species. Starting from 135 the benzyl moiety was replaced by a 3-unsubstituted N-methyl isoxazolium warhead attached via an ethyl spacer in the C5-position of the isoxazole ring (140a). The piperidine substituent was further replaced by cyclohexyl in this series. As predicted, compound 140a and its homologue 140b covalently labeled PDE6δ at Glu88 as shown by mass spectrometry, covalent inactivation however, was incomplete (80% after 30 min) and PDE6δ got reactivated within 24 h due to the limited stability of the reaction products. Shifting the attachment point to the C4-position of the isoxazole ring and the introduction of an additional methyl substituent at the C5-position (compounds 140c and d) increased labeling efficiency (> 95 % in less than 10 min) and only marginal cleavage was observed within 24 h. Finally, re-introduction of the piperidine nitrogen atom from 135 furnished 141, the most promising compound of this series. Interestingly, removing the methylcyclohexyl or 4-methyl piperazine substituent from the sulfonamide nitrogen atom yielded a compound which did not show any labeling pinpointing the critical role of accurate warhead positioning. The optimized compounds possessed low reactivity towards isolated amino acids such as Glu, Asp, Lys, Cys and Ser and did not label the E88A mutant of PDE6δ. 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 in the crystal structure, the ligand appears to


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be bound in the less stable 1,2-enolized β-ketoamide form. 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.

Figure 59: Glutamate-targeted PDE6δ inhibitors. A) Attachment of N-methyl isoxazolium warheads to reversible inhibitors exemplified by 135. B) Mechanism of the reaction between the N-methyl isoxazolium group and carboxylates.

The chemical stability of the conjugate against 50-fold excess of hydroxylamine334 suggests that the binding pocket shields the enol ester thereby preventing the typical hydrolysis. It is likely that binding to surface-exposed carboxylate residues is reversible and reversibility can also be expected after proteolytic degradation of the target protein, potentially reducing side effects by off-target binding and haptenization. However, whether these CRG can be applied in living organisms remains to be proved. A general problem for in vivo application might be found in the chemical


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stability of the warhead, which is good in acidic solution but only moderate at pH 7.2 and low at pH 9. Although this could offer a kinetic selectivity advantage, chemical instability might be hardly compatible with oral dosing and the comparably slow distribution in vivo. Here, carefully tuning reactivity by modulating steric bulk and electronic properties of the substituents might be the key to success. Although previous isoxazolium-derived activity-based protein profiling (ABPP) probes from the same group were able to penetrate cells,335 it needs to be further investigated to what extend these charged molecules can cross biological barriers and how they are affected by metabolism in vivo. Finally, it will be interesting to see, if other peptide coupling reagents might be tuned to become suitable carboxylate-targeted warheads for application as chemical probes or ultimately in drug discovery.

Figure 60: X-ray crystal structure of compound 141 covalently bound to Glu88 of PDE6δ (PDB: 5NAL). The ligand is predominantly bound in the O-acylated form and deeply buried in the binding site. Hydrogen bonds are formed by both oxygen atoms of the sulfonyl group to the side chains of Arg61 and Gln78.


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8. TARGETING THE HISTIDINE SIDE CHAIN Although the histidine imidazole 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 a readily hydrolyzable sulfonylimidazoles as shown by the covalent modification of His130 in the active site of Salmonella typhimurium ribosephosphate diphosphokinase by FSBA (94, 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-hydroxynoneneal337 or prostaglandin J2338 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 61A) and the derived clinical candidate beloranib (142), which bind to one of the metal-coordinating active site histidines in methionine aminopeptidase (MetAP) 2 via opening of a spiro-epoxide (Figure 61B).340 In order to improve the poor pharmacokinetics of this substance class, Aubry Miller and colleagues designed spiro-epoxytriazoles as drug-like fumagillin analogs (Figure 61C, general structures 144 and 145).341 Several potent inhibitors of human MetAP2 were generated with key compound 145a (Figure 61) 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. Notably, even though the keto and benzoyl derivatives possessed poor stability in plasma and mouse liver microsomes, carbamate analogs were significantly more stable, even when compared with the clinical candidate beloranib. The labeling of His231 was confirmed by X-ray


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crystallography (Figure 62), while solvent-exposed nucleophiles (such as Cys290 and Lys427) remained untouched. These results indicate that histidine residues can be targeted specifically with relatively weak 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 histidine's nucleophilicity and thus on the surrounding protein environment.

Figure 61: Spiro-epoxides as histidine-targeted covalent inhibitors of hMetAP2. A) Clinical 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.


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Figure 62: X-ray crystal structure of the covalent complex between hMetAP2 and key 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. 8.2 Reversible addition of histidine to α-cyanoenones A covalent-reversible approach was recently pursued by Clarissa Jakob 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 analog 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 addition to the α-cyanoenone moiety was


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confirmed by X-ray 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 10-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 non-covalent 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 63: α-Cyanoenones as histidine-targeted covalent-reversible IDH1 inhibitors. A) Hit compound 146 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.


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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, it's 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 straindriven sulfur imidation reagents.344 Incorporating the oxaziridine nitrogen atom into a weakly electron-withdrawing urea (exemplified by compound 147a, Figure 64A) moiety 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 followed by ring opening and release of benzylic aldehydes or ketones to furnish the S-imidation product (Figure 64C). The S-oxidation byproduct is formed 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 (Figure 64B). 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 dose-dependent increase in the number of labeled residues allowing the identification of hyper-


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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 label isolated methionine residues at rates similar to CuAAC reactions. Consistently, all labeled methionine residues identified in this study were solvent-exposed. 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 steric requirements of such warheads may 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 specifically addressing methionine side chains.

Figure 64: Methionine-targeted oxaziridines. A) Urea and carbamate-derived analogs. 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.


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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 asparagine's guanidine group has been exploited for reactions with glyoxal-derived reagents345 forming 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 toxicity issues. A recent method has been described for metal-free tryptophan-selective bioconjugation in proteins.346 Although being conceptually interesting, method requires an organoradical reagent and sodium nitride as an additive precluding applications in TCI design. Addressing phenylalanine or amino acids with non-activated aliphatic side chains is even more challenging. Although catalytic approaches 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 re-purposing warheads for TCI design. A summary of the major warhead classes described in this article is provided in Table 2. It should be noted that the intention of this table is to provide a quick overview. The reader is referred to the respective chapters and the literature cited therein for details. Table 2: Overview of the major warhead classes discussed in this article


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Prim. target residuea

Testing systemsb

Comments

Chapter

Cys

L/P/Y/C/I

Most common CRG in TCIs; warhead of all approved TCI drugs

-

Fumarate ester

Cys

L/P/Y/C/I

Metabolically labile; designed to confer kinetic selectivity

2.1

Allenamide

Cys

L/P/C

Higher reactivity compared analogous acrylamides

2.2

Propiolonitrile

Cys

L

Higher reactivity compared to alkynoic amides; tunable via R

2.3

Vinyl- or alkynylsubstituted heteroaryl group

Cys

L/P/C/I

Motif present in approved drugs

2.4

(Cys)c

n/a

Determinants of reactivity and application potential in small molecule TCI development unknown

2.5

Cys (Lys, Tyr)

L/P/Y/C/I


2.6 (5.2)

Cys

L

Reactivity tunable by aryl substituents; synthetic access can be limiting

2.7

Reactive group

General structure

α,β-Unsaturated amide

Propiolamide

Propargylamide and homologs Electrondeficient (hetero) aryl ring with leaving group (SNAr) Arylsulfonyl bicyclobutane and homologs (strain release)

variable


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Haloalkane L/P/Y/C/I


2.8

Epoxide

Cys (Lys, His)

L/P/Y/C/I

Motif present as warhead in approved drugs

2.9 (4.4, 8.1)

Aziridine

Cys (Glu, Asp)d

L/P/Y/C/I

Motif present in approved drugs; known for DNAalkylation

2.9

Nitroalkane

Cys

P

Masked electrophile; forms electrophilic nitronic acid tautomer

2.10

Cys (His)

L/P/Y/C/I

Reversible; tunable residence time; motif present in approved drugs

2.11 (8.2)

Ketone

Cys, Lys

L/P/Y/C/I

Reversible; motif present warhead in approved drugs

2.12 4.6

Activated nitrile

Cys

L/P/Y/C/I

Reversible; motif present in approved drugs

2.13

Cyanamide

Cys

L/P/C/I

Reversible

2.13

Isothiocyanate

Cys (Lys)

L/P/Y/C/I

Reacts fast & reversible with Cys but slow & irreversible with Lys; present in cruciferous vegetables

2.14

Very electrondeficient (hetero)aryl

Cys (Lys)e

L/P/C

Reversible formation of stable Meisenheimer complexes; typically containing nitro groups

2.15

L/P/C/I

Motif present in approved drugs; forms disulfides; reversible (disulfideexchange, redox transformations)

2.16

α-Halomethyl amide/ester/ ketone

Cys

α-Activated acrylamide α-Cyanoenone Aldehyde

S-electrophile

variable

variable

Cys


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Cyssulfenic acid

L/P/Y/C

Concomitant labeling of sulfenamides

3

Lys, Cys

L/P/Y/C/I

Slight preference for Lys in a model system;

4.1

Sulfonyl fluoride

Lys, Tyrf

L/P/Y/C/I

Common in chemical biology; reactivity/hydrolysis stability depends on R

Sulfonimidoyl fluoride

Lys, Tyrf

L

Less reactive than sulfonyl fluoride; tunable via R and R'

4.2

Aryl fluorosulfate

Lys, Tyr, (Ser)f

L/P/Y/C

Low reactivity; products potentially hydrolysis labile; Dha formation with Ser possible; Tested in C. elegans

4.3 5.1 (6.1)

Activated ester

Lys

L/P/Y/C/I

Motif present as warhead in approved drugs

4.4

N-Acyl-N-alkyl sulfonamide

Lys

L/P/Y/C

Reacts with surface-exposed lysines; bulky

4.5

2-Carbonyl arylboronic acid

Lysg

L/P/C

Reversible; forms stabilized Schiff bases

4.6

N-Methyl isoxazolium

Glu, Asp

L/P/Y/C

Limited stability at neutral pH

7.1

Oxaziridine

Met

L/Y

Reaction via redox mechanism

8.1

C-nucleophile

α,β-Unsaturated sulfone α,β-Unsaturated sulfonamide

4.2 4.3 5.1

a

Excluding catalytic nucleophiles. b L: Protein/peptide (labeling or reactivity assay); P: Protein (activity/binding affinity, kinact/KI); Y: Cell lysate (chemical proteomics); C: Intact cell (functional assays or chemical proteomics); I: In vivo (mammals or human). Data based on the reports discussed in the respective chapters and additional searches in PubMed and the DrugBank.146 c Application restricted to the catalytic cysteine of DUBs and related cysteine proteases so far.


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d

Modified in solution but aziridines were unable to address Asp12 in the K-Ras G12D mutant. Suggested by experiments with n-butylamine as a model nucleophile. f Forms unstable S-ester reaction products with cysteine. g Hemithioacetal formation with Cys is likely but rapidly reversible. e

Despite the plethora of CRG described in the current literature, selecting the right warhead for a specific application remains a non-trivial task. Especially in vivo, factors to be considered reach far beyond the structure of the protein and nature of the target amino acid and include properties like cell permeability, target turnover, tissue distribution and (sub)cellular location, amongst others. Metabolic stability and chemical reactivity 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 acrylamide-derived TCIs.347 Oxidative metabolism via reactive epoxide intermediates is another well-known biotransformation of α,β-unsaturated amides.348 However, little is known about the metabolic fate of most CRGs discussed here. Reactivity towards glutathione, but also other thiol-containing reagents, has frequently been employed as a surrogate parameter to describe the non-specific reactivity towards 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


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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 head group. Here, alternative acceptors, such as alkenylated or alkynylated heterocycles or the rigid propiolonitriles might come into play. SNAr warheads may offer advantages if spatially defined 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, electron-deficient heterocycles, 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 as alternatives especially if IP claims are an issue. Despite their promising chemical properties, however, such moieties have only been evaluated against isolated peptides so far. Systematic implementation of metabolically labile CRGs, such as fumarate 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


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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, but also vinyl sulfones and the corresponding sulfonamides preferentially react with amino and hydroxy bases in accordance to Pearson's HSAB concept. Although these functional groups 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 towards 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 fluoride 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 tyrosinetargeted 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 non-activated 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 specific glutamate/aspartate or methionine targeted


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CRGs hold the promise that at least some of 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 strategies will be necessary when aiming for clinical applications. Likewise, many issues with the more established warheads remain to be resolved. For example, none of the presented studies on sulfonyl fluorides, SNAr warheads 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 fluoridecontaining 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 since the observed differences in potency can be driven by reversible binding (KI) but also by the rate of covalent bond formation (kinact). In this context, it is important to note that the diffusion-based limit for the rate of ligand association is in the range of 108−109 M-1s-1. A slightly lower value can be expected if other factors, e. g. desolvation or induced fit binding are taken into account.34,350 Since this implies that kinact/KI cannot exceed this rate, both parameters need to be balanced within this limit. A well-designed TCI should be a potent and specific reversible binder to allow for selective target modification at


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low inhibitor concentrations. It should place the reactive residues in favorable positions in terms of distance and angle between the reaction partners. Similarly, the orientation towards activating residues in the binding cleft can play a role. The latter factors are crucial to enable a rapid bond formation between the binding partners. The proper alignment of the reactive residues becomes especially important, when low reactivity warheads are employed since the key properties of covalent binders (see the introduction) would be lost with kinact becoming too slow. In such a case, increased reversible potency could easily be mistaken as covalent binding. In this context, it is interesting to note that 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 MS-proteomics have made the assessment of off-targets in cellular proteomes becoming more and more common. These methods provide powerful tools to determine the fate of reactive ligands. Nevertheless, they also have their limitations. Methods exploiting 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 small 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 ligands. Many other issues are known and a further discussion might be found elsewhere.351

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Combination with emerging techniques, e. g. cellular thermal shift assays (CETSA)332 or thermal proteome profiling333, 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 non-protein off-targets, for example the nucleobases in RNA and DNA. Such investigations would become even more important when employing more versatile warhead chemistries since each functional group features a distinct reactivity against 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,βunsaturated 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,βunsaturated amides cannot satisfy all needs or in 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.

AUTHOR INFORMATION Corresponding Authors * M.G.: Phone: +49 7071 29-72466. E-mail: [email protected]


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

ACKNOWLEDGMENT The authors 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. The authors thank Valentin Wydra and Nathanael Disch for assistance in the preparation of the manuscript and the TOC graphic. M.G. further acknowledges financial support by the Institutional Strategy of the University of Tübingen (Deutsche Forschungsgemeinschaft, ZUK 63) and the Eliteprogramme for Postdocs of the Baden-Württemberg Stiftung. 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 PhD from Tübingen 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 independent research group. His research interests include medicinal chemistry, chemical biology, natural-product synthesis, and innovative drug targeting approaches.


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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 co-founder/spokesman of ICEPHA (Interfaculty center for pharmacogenomics and pharma research), TüCADD (Tübingen Center for Academic Drug Discovery) and co-founder of the two startups CAIR Biosciences and Heparegenix. Three compounds from his lab made it first into man. He is currently (2016-19) president of the German Pharmaceutical Society (DPhG). His research interests are protein kinase inhibitors and eicosanoid modulators. ABBREVIATIONS 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, dimethyl arginine dimethylaminohydrolase; DFT, density functional theory; Dha, dehydroalanine; DMF, dimethyl fumarate; DMPK, drug metabolism and pharmacokinetic; DMSO, dimethyl sulfoxide; DANN, deoxyribonucleic acid; DTT, dithiothreitol; DUBs, deubiquitinating isopeptidases; EC50, half-maximum effective concentration; EGFR, epidermal growth factor receptor; ErbB, Protein-family of four receptor


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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; GSTP, glutathione S-transferase pi; GSTP1, Gluthathion S-transferase 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-go-Related 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, inositol-requiring enzyme; ITAM, immunoreceptor tyrosine-based activation motif; ITK, interleukin-2-inducible T-cell kinase; JAK, Janus kinase; KRAS, p21 GTPase (oncogen first found in Kirsten Rat Sarcoma virus); MAO, monoamine oxidases; MetAP, methionine aminopeptidase; MetAP, methionine aminopeptidase; MGMT, (O6-)methylguanineDNA-methyltransferase; MMF, monomethyl fumarate; MSF, methanesulfonyl fluoride; MSTB, 2-(methanesulfonyl)benzothiazole; MurA, UDP-N-acetylglucosamine enolpyruvyl transferase; NAC, N-acetylcysteine; NBD-dye, nitrobenzoxadiazole-dye; NCI, National Cancer Institute; NHS, N-hydroxysuccinimid; NMR, nuclear magnetic resonance; NS5B, protein

nonstructural

protein 5B; PDB, Protein Data 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 mononuclear blood cells; PMSF, phenylmethane sulfonyl fluoride; PPARs, peroxisome proliferator-activated receptors ; ReACT, redox-activated chemical tagging; ROS, reactive oxygen species; SAR, structure-activity relationship; SH, Scr homology; SNAr, nucleophilic aromatic substitution; SPR, structure-property


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relationship; SRC, steroid receptor coactivator; SRPK1, SR-protein 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; TR-FRET, Time-resolved Förster resonance energy transfer; TRPA1, Transient receptor potential cation channel A1; Ub, ubiquitin; WDK, Woodward's reagent K

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Emerging and Re-Emerging Warheads for Targeted Covalent

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