Covalent Molecular Probes for Class A G Protein-Coupled Receptors

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Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications Dietmar Weichert and Peter Gmeiner* Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstraße 19, 91052 Erlangen, Germany ABSTRACT: Covalent modification of G protein-coupled receptors (GPCRs) by employing specific molecular probes has for decades provided a successful strategy to facilitate the elucidation of the structure and function of this pharmacologically important class of membrane proteins. The ligands typically comprise a pharmacophore that generates affinity for a given GPCR and contain a reactive functionality that may form a covalent bond with a suitably positioned amino acid residue. Covalent ligands have been successfully applied to circumvent poor affinity of compounds when stable labeling of receptor populations was required, and they have been used in the isolation, purification, and pharmacological characterization of specific subtypes of GPCRs. Recently, structural studies have demonstrated that covalent molecular probes are effective at stabilizing GPCRs to obtain X-ray crystal structures, thus providing valuable insights for the development of novel therapeutics. Herein, we review covalently binding molecular probes for class A GPCRs with a focus on ligands comprising cross-linking groups that do not require photoactivation and further highlight their significant and diverse applications.

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protein-coupled receptors (GPCRs) are membrane proteins that translate extracellular signals, including ions, hormones, and peptides, into intracellular responses, thus mediating important physiological and pathophysiological processes. Receptor activation by endogenous agonists triggers binding of an intracellular protein, like G proteins, that addresses downstream signaling pathways. Being among the pharmacologically most relevant family of proteins, GPCRs have been subjected to extensive investigations, culminating in the development of a large number of drugs that cover more than one-third of the current market.1,2 To date, studies on this class of membrane proteins still provide a major challenge because of their poor availability due to low expression in native tissue, their inherent flexibility and instability once they are extracted from the membrane into detergent solution, and the low affinity of their native hormones.2 Over the past decades, a diverse array of bifunctional molecular probes helped to overcome these obstacles. A bifunctional molecular probe is a small molecule comprising an antagonist or agonist pharmacophore that displays affinity for a given GPCR, connected to a tag or encompassing functional group that exhibits specific properties. For instance, radioactive or fluorescent tags3 can be employed to enable the quantification and visualization of GPCRs, while bivalent tags4 containing two pharmacophores have been used to study dimerization (Figure 1). To circumvent poor affinity of endogenous and commercial ligands, covalently binding molecular probes were developed. Such probes, also termed affinity labels, were originally defined as compounds that irreversibly bind to the active site of an enzyme and feature a reactive cross-linking moiety. The efficiency of the ligation reaction is ultimately dependent on © XXXX American Chemical Society

Figure 1. General design of bifunctional molecular probes. The antagonist or agonist pharmacophore is directly fused to either a tag or a connecting linker moiety is employed.

the affinity of the pharmacophore for the target protein, the reactivity of the electrophilic cross-linking moiety, and its proximity to an appropriate nucleophilic amino acid residue. An ideal electrophilic function displays a low nonspecified reactivity toward nucleophiles in solution; however, once the pharmacophore accommodates the ligand in the binding pocket of the receptor, it is reactive toward a suitably positioned amino acid residue. The residence time of the covalent ligand enhances the concentration of the electrophilic moiety around the nucleophile, triggering the formation of a covalent bond. This review covers covalently binding molecular probes that feature reactive groups of electrophilic nature. Photoaffinity moieties, requiring a photoactivation step to generate carbenes Received: January 30, 2015 Accepted: April 10, 2015

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DOI: 10.1021/acschembio.5b00070 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ligands, it is shown that there is still a need for highly specialized tool compounds to further guide the elucidation of the structure and function of these important drug targets.

or nitrenes, which mediate irreversible ligation of the ligand and the protein, are not discussed herein.5 The development of covalent molecular probes was inspired by the photoreceptor rhodopsin and its native ligand retinal. The isomer 11-cis-retinal binds in the orthosteric binding pocket of the receptor and subsequently forms a covalent bond with lysine 233 via a protonated Schiff base, thus providing the archetypical example of a covalent ligand−receptor complex. In 2000, Palczewski et al. solved the first GPCR crystal structure of rhodopsin covalently bound to 11-cis-retinal.6 Upon the absorption of a photon, 11-cis-retinal isomerizes to all-transretinal, thereby switching the receptor from an inactive to an active conformation. The crystal structure of the active form metarhodopsin II, covalently bound to the full agonist all-transretinal and in complex with a peptide derived from the Gα subunit of the G protein transducin, was solved in 2011.7 These studies corroborated the notion that covalent ligands might be useful in the crystallization of GPCRs. After the first structure of an aminergic GPCR was solved in 2007, when the β2-adrenoceptor (β2AR) was crystallized in its inactive state and in complex with the inverse agonist carazolol,8 the crystallization of GPCRs has been marked by groundbreaking advances.9 More recently, a number of inactive-, intermediate-, and active-state GPCR structures, including one of a β2AR-G protein complex,10 have been crystallographically solved, thus greatly improving our structural understanding of these membrane proteins and the process of signal transduction. Because of their inherent flexibility, however, obtaining diffraction-quality crystals of GPCR complexes is still a major hurdle. In particular, active states of GPCRs are notoriously difficult to crystallize, owing to enhanced dynamic behavior and conformational heterogeneity of an agonist-bound receptor. To fully stabilize the active state, a high-affinity agonist and an intracellular binding partner, such as a G protein or a G protein mimetic antibody fragment obtained from lamas (nanobody), is required.11 Covalent ligands have recently proven to constitute excellent and useful tools to promote structural studies on GPCRs.12−14 Moreover, covalent molecular probes had been frequently employed in a number of biochemical studies that required the formation of stable ligand−receptor complexes. Thus, chemoreactive antagonists and agonists were used to study the physiological function and prevalence of receptors, their subtypes, and the pharmacological effects mediated by various drugs.5 The purification of GPCRs from native tissue was facilitated by covalent ligands that were additionally endowed with a radioactive label. Subsequent enzymatic digestion and investigation of the radiolabeled peptide fragments provided information about the localization of the orthosteric binding site.15,16 Site-directed mutagenesis and subsequent investigation of receptor mutants and covalent ligands in radioligand binding studies facilitated the mapping of the orthosteric binding site.17,18 Furthermore, covalent probes enabled the study of receptor reserve and turnover19 and the evaluation of the relationship between receptor occupancy and a tissue or cellular response.20 Very recently, an irreversibly binding agonist was used in a directed evolution approach to optimize the properties of a state-specific nanobody (Nb) for application in structural studies.21 This review discusses recent advances and applications of covalently binding molecular probes for biochemical investigation and structural studies on class A GPCRs. Despite the growing development of high-affinity and subtype-selective

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COVALENT MOLECULAR PROBES FOR CLASS A GPCRS Reactive Thiols. Protein crystallography, the most powerful tool to study GPCR structure, requires the formation of stable and homogeneous ligand−receptor complexes. This is a particular challenge for the agonist-bound low-affinity state and the high-affinity ternary complex between receptor, agonist, and G protein. Strongly binding ligands with dissociation constants in the low- to subnanomolar range and low off-rates are necessary to stabilize the protein throughout the process of expression, purification, and crystallogenesis. To overcome rapid dissociation rates of conventional diffusible agonists, a covalent ligand strategy can be employed. The concept depends on a combination of a functionalized agonist or antagonist incorporating a specific pharmacophore and a linker moiety that targets a reactive chemical group to a specific residue of the receptor. A disulfide-based strategy, originally described by Buck and Wells for fragment-based drug discovery,22,23 has proven to be highly suitable to produce functional and stable GPCR-ligand complexes. Cross-linking depends on a disulfide exchange between a reactive disulfide group of the ligand and the sulfhydryl function of a proximate cysteine residue of the receptor. This reaction, which is chemoselective over other nucleophilic amino acids, is promoted by the inherent affinity of the pharmacophore of the covalent ligand for the orthosteric binding pocket. Thus, if the activated thiol is positioned in close proximity to a cysteine anchor, the enhanced local concentration is the driving force for the conjugation of ligand and receptor. Because reductive cleavage of byproducts formed at readily accessible cysteine residues at the surface of the receptor protein is possible, the reversible disulfide transfer reaction displays high specificity for the cysteine group in the binding pocket showing an enriched concentration of ligand. Taking advantage of a covalent ligand strategy, disulfidebased agonists for the stabilization of aminergic GPCRs were recently developed.12,14 This strategy gave access to the first structure of an agonist-bound GPCR, providing diffractionquality crystals for the agonist-bound low-affinity state (binary complex) of the β2-adrenoceptor (β2AR).12 The low-affinity state is characterized by enhanced dynamics and rapid dissociation of the agonist-receptor complex, rendering crystallization particularly challenging. Covalent agonists can thus be used to restrain the receptor in a predefined conformation (Figure 2). In general, disulfide-functionalized covalent ligands comprise a pharmacophore unit facilitating specific binding for a given GPCR, and a linker moiety that is optimized in its nature and length for high binding affinity, and its ability to target the disulfide function into a position that allows cross-linking. Thus, the linker positions the disulfide in close proximity to a mutated cysteine residue (Figure 2). Docking studies, using the structure of the β2AR bound to the inverse agonist carazolol as a template,8 and previous biochemical experiments15 helped to identify histidine 93 in the upper part of transmembrane helix 2 (TM2; position 2.64 according to Ballesteros Weinstein nomenclature)24 of the β2AR as a suitable position to introduce a cysteine anchor but also facilitated the design of the linker moiety. The disulfidebased ligand FAUC50 was developed and found to efficiently form a covalent complex with a β2ARH93C mutant, capable of G B


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Figure 2. General strategy for the generation of covalent GPCR-ligand complexes. The pharmacophore (shown as a blue circle) generates affinity for a given GPCR, and a flexible linker (green box) positions a reactive thiol function (red) close to a native or engineered cysteine side chain in position X2.64C of the receptor. The formation of a mixed disulfide stabilizes the binary complex in its low-affinity state, while the addition of a G protein or a G protein mimetic nanobody (Nb, shown in yellow) leads to the formation of a ternary complex, inducing the high-affinity state of the receptor. Structures of the low-affinity state of the β2ARH93C (blue ribbons) bound to FAUC50 (light blue carbons) and structure of the active state β2ARH93C (gray) bound to FAUC37 (orange). Both ligands form a disulfide bond with the engineered cysteine 932.64.

overall shape and contraction of the adrenaline binding pocket unaltered. Covalent ligands that feature disulfide groups have also been developed to target native cysteines in opioid receptor subtypes (ORs), namely μ, δ, κ, and nociceptin receptors (MOR, DOR, KOR, and NOR, respectively). To evaluate the accessibility of cysteine residues, small sulfhydryl alkylating reagents like iodoacetamide25 or agents that form mixed disulfides can be employed.26 Subsequent ligand-binding experiments indicated if a thus modified cysteine is positioned in close proximity to the binding pocket and whether it might serve as an anchor for affinity labels. These studies led to the design of various covalently binding peptides derived from endogenous hormones like enkephalins and dynorphins containing activated thiol groups.27−29 Moreover, a covalent ligand based on the natural product salvinorin A, an agonist selective for the κsubtype, has recently been reported.30 The authors convincingly demonstrated that the 22-thiocyanato-derivative of salvinorin A (RB-64) forms a disulfide bond with C3157.38 by nucleophilic substitution of the cyanate group in a highly κselective manner, and it was found to be about 20-fold more potent than the parent compound (Figure 3A). Clopidogrel is a marketed antiplatelet drug that is used in the prevention of thrombotic events by antagonizing the response of adenosine at P2Y12 G protein coupled-receptors on platelets. Investigation of its mode of action revealed that clopidogrel acts as a prodrug, which is activated in the liver by oxidative ring opening (Figure 3B).31 Convincing evidence has been gathered indicating that the free thiol of the active metabolite reacts with cysteine C973.25 in the P2Y12 receptor, thereby mediating a covalent binding mode.32,33 This demonstrates the relevance of disulfide-based covalent GPCR-ligand complexes, even for clinical drug efficacy. Michael Acceptors. Substituted or unsubstituted acrylamides, fumarates, and related α,β-unsaturated carbonylderived functions are commonly employed in affinity labels.

protein activation. Consequently, covalent ligation prevented the dissociation of the low-affinity binary ligand receptor complex, which greatly facilitated the solution of the first agonist-bound crystal structure of a GPCR (Figure 2).12 Low binding affinity of native neurotransmitters and their synthetic congeners with rapid association and dissociation rates leads to conformational heterogeneity that limits the formation of diffraction-quality crystals. Employing the disulfide-based strategy, a crystal structure of a fully activated ternary complex between β2AR, a covalent neurotransmitter, and a G protein mimetic antibody fragment could be determined. The introduction of a cysteine mutation in position X2.64 into aminergic GPCRs provided a universal anchor for the formation of covalent agonist-receptor complexes. On the basis of the prototypic β2AR agonist FAUC50, a general synthetic strategy enabled the conversion of the low-affinity neurotransmitters noradrenaline, dopamine, serotonin, and histamine into covalent molecular tools. It was shown that the covalent neurotransmitter analogs efficiently bind to and activate representative subtypes like the β2AR, the dopamine D2 receptor, the serotonin 5-HT2A receptor, and the histamine H1 subtype, after X2.64C mutation was conducted. Only for 5-HT2A, a second mutation was necessary to improve the yield of the disulfide exchange. As a proof of concept, an active-state crystal structure of β2ARH93C in complex with the covalent noradrenaline analog FAUC37 and a state-specific antibody fragment (nanobody) verified that these ligands facilitate crystallization and stabilize a receptor conformation identical to the endogenous hormone adrenaline (Figure 2).14 Comparison of this structure with a corresponding ternary complex of the same nanobody and the β2AR bound to adrenaline thus demonstrated that the catecholamine head group of FAUC37 adopts a position identical to adrenaline, while the linker accommodates to an extended pocket that is unoccupied in the adrenaline-bound structure, leaving the C


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Figure 3. (A) Structure of the 22-thiocyanato-Salvinorin A (RB-64) forming a covalent bond with C3157.38 in the KOR. (B) CYP enzymes in the human liver account for the conversion of the prodrug clopidogrel into its active metabolite. The metabolite binds to the P2Y12 receptor and subsequently forms a mixed disulfide bond with C973.25.

These electrophilic groups display a pronounced reactivity toward cysteines, but also undergo a Michael-type addition with nucleophilic lysine or histidine residues (Figure 4).34 In solution, Michael acceptors react slowly with nucleophiles, for instance with the cysteine residue in glutathione.35 As covalent ligation is accelerated by the affinity of a ligand for the target protein, adequately designed Michael acceptor-based probes offer a high degree of selectivity for their target receptor. In general, the addition of nucleophiles to acrylamides is a reversible reaction; however, the equilibrium between covalently bound and diffusible acrylamide-derived ligands can be tuned by the use of more electron-deficient acceptor functions.36 To develop subtype-selective covalent agents for opioid receptors, Portoghese and co-workers exchanged the functional group in position 6 of the reversible opioid antagonist naltrexone by a fumaroylamido unit, giving β-funaltrexamine (β-FNA).37 Although β-FNA showed affinity for μ, δ, and κ opioid subtypes, covalent ligation was MOR-specific via lysine 2335.39, as verified by mutagenesis experiments.38 This molecular probe was subsequently used in a number of pharmacological studies, thus promoting the elucidation of the function of opioid receptors and effects mediated by opioid drugs (see ref 5 for review).5 Further covalently binding cinnamoyl- and fumaroyl-substituted MOR antagonists were reported.39 Structural studies on the MOR have been hampered by the lack of antagonists that effectively stabilize the receptor. Although they display affinities in the low nanomolar range, μ-opioid receptor antagonists share very low dissociation halflives. By employing the covalently binding opioid derivative βFNA, Manglik et al. were able to obtain a stable and conformationally homogeneous ligand−receptor complex that yielded well-diffracting crystals, allowing determination of the inactive structure of the MOR at a resolution of 2.8 Å (Figure

Figure 4. Mechanism of the Michael addition reaction. The amino group of a lysine reacts with the electrophilic β-position of the Michael acceptor (red) in β-FNA. The structure of the binary β-FNA-MOR complex (ligand in gray, receptor in orange) was subsequently solved. β-FNA covalently binds to the MOR via Lys2335.39.

4).13 It was found that the ligand-binding pocket is widely exposed to the extracellular surface, which most likely accounts for the short residence time of MOR ligands. Continuous electron density could be observed between the ligand and Lys2335.39, indicating covalent ligation via a Michael-type reaction and confirming previous mutagenesis experiments (Figure 4). A structure-based approach guided the design of VUF14480, a covalently binding partial agonist for the histamine H4 receptor (Figure 5). 40 Docking studies, employing a pyrimidine-derived reversible agonist and a homology model of the H4 subtype, revealed that Cys983.36 might be in close proximity to the orthosteric binding pocket. Consequently, the introduction of a vinyl function in position 2 of the pyrimidine ring established a Michael acceptor moiety that mediated D


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aziridinium ions toward amines, sulfides, and carboxylates, which may lead to unspecific binding, a number of studies reported the introduction of mustard groups to successfully convert ligands for aminergic GPCRs into specific irreversibly binding molecular probes. Acetylcholine mustard (AchM) represents an archetypical example for a mustard-based ligand at muscarinic receptors. This compound was found to alkylate all five muscarinic subtypes and was initially described to maintain agonist efficacy after covalent binding.44 However, it was shown that the aziridinium analog of the AchM activates the receptor before cross-linking but yields inactive binary complexes after formation of the covalent ligand−receptor bond. AchM was described to irreversibly bind the carboxylate of Asp3.32, which was identified as a highly conserved residue that forms a salt bridge with the ammonium cation of aminergic neurotransmitters.16 Mutational studies revealed that this particular residue is necessary to promote agonist-induced receptor activation.45 Very recently, a mustard based on the agonist iperoxo, namely FAUC123, was employed in a directed evolution approach to obtain a G protein mimetic camelid antibody fragment (nanobody) for the M2 muscarinic receptor (Figure 6A).21 Nanobodies (Nbs) can be used as crystallization chaperones that stabilize active states of GPCRs by binding into the same intracellular cavity as G proteins.46 They are generated by immunization of lamas using phospholipid vesicles that contain agonist-bound receptor. In an effort to further optimize a thus identified M2-binding nanobody, the irreversible ligand FAUC123 was used to construct stable agonist-receptor complexes, enabling state-specific staining of

Figure 5. Chemical structures of the described histaminergic and αadrenergic covalent molecular probes VUF14480 and SLZ-49, respectively, featuring Michael acceptor groups (red).

efficient formation of a covalent bond between VUF14480 and Cys983.36. Interestingly, this particular cysteine residue is conserved among a wide range of aminergic GPCRs (24 out of 42 receptors),41 thus potentially providing a universal nucleophilic anchor for the design of covalent molecular probes. Further work demonstrated that exchange of the furyl moiety of the α-adrenergic antagonist prazosin with a bicyclic Michael acceptor system resulted in the alkylation agent SLZ-49, selective for α1A/B-adrenoceptors (Figure 5).42 Nitrogen Mustards. Nitrogen mustard analogs or 2haloalkylamines are frequently termed mustards and represent an important class of cross-linking functionalities. In aqueous solution, they undergo cyclization to the corresponding aziridinium ion, which is the reactive species that accounts for the alkylation of amino acid side chains (Figure 6A). The rate of formation of an aziridinium ion is dependent on the halogen atom that is employed. In general, 2-bromo mustard analogs cyclize rapidly compared to the corresponding 2chloro-derived mustards.43 Despite the high reactivity of

Figure 6. (A) The 2-chloroethylamine moiety (red) of the irreversible agonist FAUC123 forming an aziridinium ion in solution. This reactive species ligates with a nucleophilic amino acid residue in the muscarinic M2 receptor, endowed with a fluorescent tag (yellow) to form a binary ligand−receptor complex. (B) For the conformational selection of nanobodies, a library of nanobody clones (dark green) was generated and displayed on yeast cells. The fluorescently labeled covalent FAUC123-M2 receptor complexes, which were purified and solubilized in detergent solution, were employed to stain yeast cells expressing the nanobody clones. The cells were selected for nanobodies that specifically bind to agonistbound receptor and recognize the active state of the M2 receptor by fluorescence-activated cell sorting (FACS). Thus, the state-specific high-affinity nanobody Nb9−8 was identified and subsequently used to obtain a crystal structure of the agonist-bound muscarinic M2 receptor. E


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likely, did not yield active irreversible ligand−receptor complexes.50,51 Nitrogen mustard analogs were developed for a variety of aminergic receptor ligands. Thus, N-benzyl-N-(2-chlorethyl)-1(phenoxy)-2-aminopropane (phenoxybenzamine mustard) was extensively applied in biochemical studies to inactivate α2adrenoceptors, muscarinic receptors, and histaminergic and dopaminergic subtypes.52,53 It was reported to alkylate Cys1173.36 in the α2A receptor.54 This particular Cys3.36 was mentioned above as being highly conserved among aminergic GPCRs, which may explain the promiscuity of phenoxybenzamine mustard in irreversibly labeling this class of receptors. Chlornaltrexamine (β-CNA) that features a bis-chloroethylsubstituted amino group in position 6 of the tetracyclic opioid scaffold was the first specific affinity label that was found to alkylate opioid receptor subtypes (Figure 7B).55 Compounds that employ bis-mustard groups had also been developed for adenosine receptors.56 The mustard analog of the α2-adrenergic selective agonist clonidine (chloroethylclonidine, CEC) is an irreversible antagonist at α1B/D receptors and acts as an irreversible agonist at α2A/C subtypes, but not at α2B (Figure 7B).57 In an elegant approach, the substituted cysteine accessibility method (SCAM)58 was used to probe the binding site of α2 receptors around TM5. The authors systematically mutated amino acid residues in TM5 (197−201) to cysteine and tested the mutants for their ability to covalently ligate with CEC.18 Thus, the residues that are exposed to the ligand-binding pocket could be identified. In addition, the authors could confirm that the native Cys5.44 determines covalent binding of CEC to α2A/C receptors, whereas an analogous Ser5.44 accounts for the resistance of the α2B subtype to CEC-mediated alkylation. Furthermore, nitrogen mustard-based covalent ligands were also developed for dopaminergic59−61 and serotonergic receptors.62 Isothiocyanates. The synthetic accessibility of isothiocyanates constitutes a major advantage since they can be easily prepared from primary amines.63 Because of their pronounced reactivity toward amine nucleophiles and sulfhydryl groups of cysteines, but poor tendency to react with alcohols or water,64 isothiocyanates found broad application for covalent molecular probes for GPCRs. Very recently, Davie et al. reported the first irreversibly binding allosteric ligand for GPCRs, more specifically, the M1 muscarinic subtype.65 Cross-linking was promoted by an isothiocyanate group linked to the benzyl quinolone carboxylic acid (BQCA) pharmacophore. The covalent ligand displays identical pharmacological activity compared to the parent compound BQCA; however, the binding mode has yet to be fully elucidated (Figure 8A). Because it is less conserved than the orthosteric binding site, addressing the allosteric pocket of muscarinic receptor subtypes may provide a valuable strategy to develop more subtype-selective pharmacological agents. Unfortunately, the low affinity of allosteric ligands makes them inherently difficult to study. The authors therefore provided a framework for the development and characterization of irreversible allosteric ligands that may facilitate the elucidation of ligand−receptor interactions in the allosteric region. To compensate for the lack of high-resolution structural data for both subtypes of cannabinoid receptors (CBRs), an approach termed “ligand assisted protein structure (LAPS)” was applied by Mercier et al.17,66 This study investigated the binding and functional properties of isothiocyanate-based covalent ligands at CBRs containing cysteine to serine/alanine

yeast that expresses a library of Nb clones. Simultaneous staining of yeast cells with an agonist- or antagonist-bound receptor population, both labeled with distinct fluorophores, guided the conformational selection by fluorescence-activated cell sorting (FACS; Figure 6B). In contrast to AchM, irreversible FAUC123-receptor complexes were found to fully activate G proteins. The strategy yielded high-affinity and active-state specific Nb9−8 that was subsequently used in crystallization trials to obtain the first active-state structure of the M2 receptor and the first structure of a GPCR in complex with an allosteric modulator. Because the dissociation of ligand and receptor during the immunization of lamas may prevent successful generation of state-specific nanobodies,46 covalent molecular probes that yield stable ligand−receptor complexes provide attractive tools to circumvent this particular problem. Mustard-type muscarinic receptor antagonists based on benzilylcholine (BCM) or propylbenzilylcholine (PrBCM) were also developed (Figure 7A).47,48 The lack of truly

Figure 7. (A) Structures of the described 4-DAMP and benzilylcholine mustard (BCM) that are based on a substituted benzilate ester moiety and that irreversibly bind to muscarinic receptors. The closely related propylbenzilylcholine mustard (PrBCM) features propyl function at the mustard nitrogen, instead of the methyl group in BCM. (B) Chemical structures of the irreversible muscarinic ligand acetylcholine mustard (AchM), the opioid receptor antagonist chlornaltrexamine mustard (β-CNA), and the irreversible α-adrenergic ligand chloroethylclonidine mustard (CEC). The nitrogen mustard groups are highlighted in red.

selective ligands made it difficult to specifically label this class of receptors. However, a mustard that was derived from the antagonist 4-diphenylacetoxy-N-(2-cholorethyl) piperidine (4DAMP; Figure 7A) exhibited good affinity for all subtypes except M2, thus allowing selective inactivation of M2 in the presence of M3 receptors and vice versa, if one receptor was protected with a respective subtype-preferring reversible ligand.49 As in the case of AchM, further effort to develop an irreversible agonist resulted in mustard derivatives that, most F


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Figure 8. (A) A nucleophilic amino acid residue in the M1 muscarinic receptor reacts with an isothiocyanate (red) of the irreversible allosteric modulator NCS-BQCA. (B) Chemical structures of isothiocyanate-containing molecular probes including the CBR agonist AM-841, the DOR selective ligands FIT and SUPERFIT, and the dopaminergic antagonist NIPS; reactive moieties are shown in red.

Figure 9. (A) A nucleophilic amino acid residue reacting with the bromoacetamide (red) of the irreversible antagonist pBABC to form a covalent complex with the β2AR. (B) Reaction of the o-phthalaldehyde moiety (red), which was introduced into the opioid receptor ligand naltrexone to yield PGNA that forms a fluorescent indole upon reaction with a lysine and an adjacent cysteine residue in opioid receptors.

adrenergic,76 melatonin,77 muscarinic,78,79 serotonin,80 and vasopressin receptors.81 Halomethylketones. Halomethylketones have been frequently employed in affinity labels for a variety of soluble and membrane-embedded proteins. Their reactivity ranges from the least reactive fluoro- to chloro-, bromo-, and the highly reactive iodo-substituted analogs. In comparison to a related Michael acceptor moiety, chloromethylketones display a slightly higher rate of glutathione alkylation.35 While fluoro- or chlorosubstituted methylketones are selective for cysteine residues, their congeners that feature bromo and iodo substituents are more promiscuous and have been shown to react also with nucleophilic residues of histidine or lysine.82,83 Halomethylketones were primarily used to label βARs, leading to the development of irreversibly binding β-antagonists based on the drugs alprenolol,84 pindolol,85 and carazolol.86 In a landmark study, the radioiodinated para-(bromoacetamidyl)benzylcarazolol (pBABC) was the first ligand to be employed for direct identification of the region representing the orthosteric binding site of a GPCR (Figure 9A).15 After specific irreversible labeling of the receptor, the protein was

mutations in proximity to the orthosteric binding pocket. Supported by computational modeling and mass spectrometry,67 the interaction of a covalent agonist (AM-841) and antagonists with the receptor mutants provided insights for the structure-based design of CBR-ligands (Figure 8B). Aiming to discover novel covalent molecular probes, additional compounds based on subtype-selective opioid receptor ligands and featuring isothiocyanate groups were generated.64,68−71 The fentanyl isothiocyanate (FIT) and the more potent and closely related (+)-cis-methyl-analog (SUPERFIT) were among the first selective irreversible ligands for the δ subtype and displayed partial agonist activity in activation assays (Figure 8B).69 N-(p-isothiocyanatophenylethyl)spiperone (NIPS) (Figure 8B) was described to selectively label dopamine D2, D3, and D4 receptor subtypes. The labeling of D3 receptors can be prevented in the presence of nanomolar concentrations of dopamine, which might explain the resistance of these receptors toward NIPS-mediated alkylation in vivo.72 Introduction of an isothiocyanate moiety further led to the development of irreversibly binding molecular probes for adenosine,73−75 αG


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tool box of nanobodies, since they do not dissociate during the immunization of lamas and they have been shown to guide a successful directed evolution and conformational selection approach. Therefore, this strategy may provide useful crystallization chaperones, facilitating the solution of GPCR conformations. Besides the applications described in this review, covalent molecular probes may be useful for the investigation of the dimerization of GPCRs. By employing a system that coexpresses one of the above-described X2.64C-GPCR mutants (β2AR-, D2R-, 5HT2A-, and H1R-X2.64C mutants) and a putatively interacting wild-type receptor, the disulfide-based tethering strategy enables selective formation of a covalent ligand−receptor complex. Thus, this approach allows the investigation of the functional properties of the wild-type receptor in the presence or absence of a covalent agonist. Covalent molecular probes may further aid the investigation of receptor trafficking and endosomal signaling. The covalent agonist-GPCR complex assures stable and prolonged activation of the receptor, even after its transition to endosomal and subsequent compartments, thereby maybe further elucidating the processes occurring after internalization of agonist-bound receptors. In conclusion, structural studies, involving complex biophysical and biochemical investigations create a demand for novel covalently binding molecular probes or may lead to a revival of existing compounds, making the development of such probes a vital field of research with a high potential to promote structural and functional studies on GPCRs.

isolated and subsequently digested. Purification and sequencing of the fragments enabled assignment of the peptide that bound [125I]pBABC. Although the specific amino acid residue accounting for covalent binding could not be explicitly identified, these experiments corroborated the notion that GPCR ligands bind deep within the hydrophobic transmembrane core. To study the interaction between agonists and βARs, a noradrenaline-derived ligand based on a bromoacetamide moiety was developed.87 However, it bound covalently to only about 50% of the total population, and once the covalent bond was formed, the ligand lost its capacity to fully activate the receptor. Nevertheless, a stable catechol-mimicking 8-hydroxylquinolin-2(1H)-one head group was subsequently described to guide the construction of covalent agonists featuring a bromoacetamide moiety, which showed a higher labeling efficiency and was able to persistently activate the receptor after ligation.88−90 Further molecular probes that comprise halomethylketones were developed for melatonin91 and opioid receptors.92 Other Cross-linking Functions. Novel methods to crosslink GPCR-ligand pairs are currently being developed.93 Furthermore, the sulfonylfluoride group has been demonstrated to show superior resistance toward unspecific reaction with nucleophiles due to its low reactivity.94 In spite of its stability, introduction of this group into adenosine receptor ligands yielded efficient and irreversible xanthine-derived95−97 and structurally distinct antagonists.56 In an elegant approach by Portoghese and co-workers, the fumaroylamido moiety in β-FNA was exchanged with orthophthalaldehyde to yield so-called “reporter affinity labels” for opioid receptors.98−100 The resulting fluorescence of a rapidly formed isoindole moiety was used as a reporter signal that provided information about the kinetics of the covalent complex formation (Figure 9B). Furthermore, compared to a ligand that forms a covalent bond to only one amino acid, the mobility of the reporter affinity label is markedly more restricted by ligation to a lysine and a cysteine residue, which may allow modeling of the ligand−receptor complex with greater confidence.100 This review covers recent advances and applications of covalent molecular probes for class A GPCRs, thereby limiting the scope of reactive moieties that are being discussed. The above-described cross-linking functions are among the most commonly employed; however, literature provides a vast array of alternatives that have proven useful in the individual design of covalent ligands by specifically targeting nucleophilic amino acids (e.g., see ref 82 for review). Conclusion and Outlook. Covalently binding molecular probes are important tools that facilitate biochemical and structural investigation of GPCRs, thus substantially contributing to the elucidation of their structure and function. Very recent applications of these molecular probes illustrate that there is a need for efficient and highly specific covalent ligands for GPCRs. This is especially true for structural studies. The production of stable and conformationally homogeneous ligand-GPCR complexes often represents a bottleneck in crystallization trials. Because the crystallization of GPCR-G protein complexes is still highly challenging, the generation and evolution of nanobodies greatly contributed to the solution of active state structures. To date, however, state-specific nanobodies exist only for the β2AR and the M2 muscarinic receptor. Covalent ligand−receptor complexes may help to extend the

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*Tel.: +49 9131 85-29383. Fax: +49 9131 85-22585. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft GRK 1910, Gm13/10). We thank Jeremy Shonberg for helpful discussions.

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GLOSSARY chemical biology, a discipline that combines chemistry and biology, for instance, by employing synthetic compounds to study biological systems; chemical probes, potent and/or selective reagents to manipulate the function of proteins; affinity label, ligands that covalently ligate with amino acids within the active site of an enzyme; covalent ligands, ligands that form a reversible or nonreversible covalent bond with a target protein; irreversible ligands, ligands that form a nonreversible covalent bond with a target protein; structural biology, studies involving biochemistry and biophysical approaches to determine protein structure, for instance, protein crystallography; disulfide tethering, affinity-driven identification or detection of small-molecule compounds containing reactive cysteines reacting with a native or engineered cysteine of a target protein

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NOTE ADDED AFTER ASAP PUBLICATION This paper was originally posted on April 22, 2015. Figure 6 has been updated and the paper was re-posted on April 24, 2015.

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Covalent Molecular Probes for Class A G Protein-Coupled Receptors

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