Toward Fluorescent Probes for G-Protein-Coupled Receptors (GPCRs

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Perspective

Towards Fluorescent Probes for G-Protein Coupled Receptors (GPCRs) Zhao Ma, Lupei Du, and Minyong Li J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401823z • Publication Date (Web): 01 Jul 2014 Downloaded from http://pubs.acs.org on July 2, 2014

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Journal of Medicinal Chemistry

Towards Fluorescent Probes for G Protein-Coupled Receptors (GPCRs) Zhao Ma, Lupei Du and Minyong Li* Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China

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ABSTRACT G-protein coupled receptors (GPCRs), a superfamily of cell-surface receptors that are the targets of about 40% of prescription drugs on the market, can sense numerous critical extracellular signals. Recent breakthroughs in structural biology, especially in holo-form X-ray crystal structures, have contributed to our understanding of GPCR signaling. However, actions of GPCRs at the cellular and molecular level, interactions between GPCRs, and the role of protein dynamics in receptor activities still remain controversial. To overcome these dilemmas, fluorescent probes of GPCRs have been employed, which have advantages of in vivo safety and real-time monitoring. Various probes that depend on specific mechanisms and/or technologies have been used to study GPCRs. The present perspective focuses on surveying the design and applications of fluorescent probes for GPCRs that are derived from small molecules, or using protein-labeling techniques, as well as discussing some design strategies for new probes.


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INTRODUCTION G protein-coupled receptors (GPCRs) are a versatile superfamily of cell-surface receptors characterized by a heptahelical structure with an extracellular N-terminus and intracellular Cterminus. Considering several intercellular messenger molecules (such as hormones and neurotransmitters) and sensory messages (such as photons and organic odorants) are sensed by GPCRs, these membrane receptors mark a critical position in intercellular communication.1 The transmembrane signal transmission mediated by GPCRs is realized by activating G proteins and arrestins, which are coupled with these receptors.2 Therefore, the association of GPCRs with a series of physiopathological alterations in the body ensures they are the targets of approximately 40% of all current medicinal drugs.3 Recently, structural biology studies of GPCRs using crystallography, mutagenesis and biophysical approaches have significantly contributed to our understanding of GPCR signaling.2 However, the complicated interaction of ligands with allosteric sites on GPCRs and the existence of 150 orphan GPCRs with unknown ligands hampers our understanding.2 To advance our understanding at the molecular level, further advances in real-time monitoring of ligand-receptor and receptor-receptor interactions in cellulo and/or in vivo are urgently required. Fluorescence techniques have been extensively applied as powerful biophysical tools for analysis of the structure and dynamics of proteins.4 In comparison to isotope-labeled methods, fluorescence-based technology becomes a reasonable choice to “watch” receptors in living cells or in vivo, because of its biocompatibility, affordability and feasibility in a variety of strategies. In parallel with the rapid progress in fluorescence technologies, efficient fluorescent probes, including fluorescent-labeled ligands, antibodies, proteins and amino acids, have been widely applied in the general areas of medicine, chemistry, biology and genomics. Among these probes,


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fluorescent ligands efficiently facilitate the real-time monitoring of ligand-receptor interactions, as well as the visualization and location of GPCRs. On the other hand, fluorescent antibodies, proteins and amino acids directly contribute to receptor protein-oriented studies, such as receptor-receptor interactions. Importantly, fluorescent probes play a pivotal role in studying orphan GPCRs.5 This perspective article focuses on the advances in GPCR fluorescent probes and their application in detecting various GPCRs for live-cell imaging. A brief discussion of the design strategy of fluorescent probes in GPCR studies is also presented at the end of this article. LIGAND-BASED FLUORESCENT PROBES FOR GPCRs Ligand-based probes for GPCRs, also known as fluorescent ligands, have been studied and used for a few decades.6, 7 Ligand-based fluorescence detection methods have gained popularity, because of their applications in visualizing receptor-ligand interactions and evaluating drug candidates.8 Using fluorescence microscopes with nanoscale spatial resolution, fluorescent ligands have demonstrated significant power in single particle analysis.8 For example, fluorescence correlation spectroscopy (FCS), based on measuring fluorescence fluctuations caused by diffusion of fluorescent particles, gives access to information about chemical kinetics in receptor-ligand interactions at nanomolar concentrations.8-10 Total internal reflection fluorescence (TIRF) microscopy is a wide-field imaging method that enables the visualization of proteins on or near the plasma membrane.11 Using this approach, Hern et al. tracked the position of the M1 muscarinic receptor, which is highly expressed in exocrine glands and in the central nervous system (CNS).12 Currently, instead of the conventional labeling of ligands with fluorophores, the fluorescent ligands are designed by rationally conjugating an agonist or antagonist of the GPCRs to various fluorophores (Figure 1). Using this strategy, GPCR fluorescent ligands with high affinity and selectivity have been reported.7


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The primary factors highlighted in probe design are physicochemical properties and pharmacological activities of the final probes.13 Because of the large mass of fluorophores, their conjugation to ligands or pharmacophores targeted at GPCRs may have an impact on the properties of the final conjugate, especially the affinity and selectivity to the receptor. If the affinity and selectivity strongly decrease, the fluorescent conjugate will be of no use. The physicochemical properties of fluorescent conjugates should be considered as well, because these hydrophobic fluorophores may cause non-specific binding. Thus, the choice of a fluorophore is predicated on retention of affinity of the ligand to the receptor, and the positional attachment of a fluorophore to the ligand structure must be particularly suitable to minimize the influence on receptor-binding affinity. In general, a classical fluorescent ligand contains three segments (Figure 1): the pharmacophore, the fluorophore and the linker.8 The former two moieties, as the receptor and reporter group, respectively, are essential components for a fluoroligand, while the linker between the pharmacophore and fluorophore provides the appropriate space to prevent the loss of pharmacological activity of the desired receptor. Usually, the linker is a carbon chain that ends with heteroatoms such as nitrogen atoms. The terminal heteroatom groups are used to couple with the fluorophores or pharmacophores. It has been reported that the high lipophilicity would increase the non-specific binding of a fluoroligand.8 Therefore, polyamide, polyethylene glycol and peptide hydrophilic linkers are viewed favorably. Besides the compatibility of the pharmacological properties, physicochemical properties and fluorescent properties (fluorescence yield or resistance to photobleaching) should be considered when a fluorophore is employed. To this end, some common fluorescent groups, including coumarin, xanthene, 7-nitro-2,1,3benzoxadiazole (NBD) and dansyl fluorophores, have been introduced.


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Coumarin derivatives have been used widely because of their marked Stokes shift and high quantum yield. The activated 3-carboxyl or -amino or -azido derivatives are synthesized to be coupled to the linkers or pharmacophores. Electron-donating groups at the 7-position may increase the redshift in the absorption and emission wavelengths. There are several commercially available coumarin derivatives, for instance Alexa Fluor 350, Alexa Fluor 430, ATTO 390 and ATTO 425 (structures not disclosed).14 These coumarin-based fluorophores have low photochemical stability, which limits their usefulness. Xanthene dyes, like fluoresceins and rhodamines, can absorb and emit in the wavelength region of 500 to 700 nm with high fluorescence quantum yields. Even if the Stokes shift of xanthene is small (about 20-30 nm), their derivatives perform very well in bioanalytical application. Most of the commercially available Alexa and ATTO dyes (structures not disclosed) belong to the xanthene derivatives.14 These derivatives, carrying an unesterified carboxyl group, display a pH-dependent optical spectrum, and this unesterified carboxyl group is not suitable for a covalent conjugation with a linker or ligand because of the steric effect. The fluorescence of esterified xanthene dyes does not rely on pH. Conjugation between these fluorophores and pharmacophores is achieved via an additional reactive group at the carboxyphenyl ring including maleimides, iodoacetamides, isothiocyanates, and sulfonyl or carboxyl groups. NBD and dansyl fluorophores are famous for their small size, green-yellow emission wavelength (550 nm), and large Stokes shift (about 200 nm). Their emission spectrum is highly sensitive to solvent polarity. These fluorophores are widely used to label proteins and are especially, conducive to polarization measurement. Their amino-reactive halides, such as NBDCl and dansyl chloride, are used to achieve a covalent coupling between the fluorophore and


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pharmacophore. For dansy fluorophores, the chemical modification of aromatic amino groups can also be considered. Furthermore, the red-emitting fluorophores, such as cyanine and BODIPY dyes, are preferred in light of their suitable fluorescent properties and feasible low-background imaging characteristics. Some environment-sensitive fluorophores, such as 6-(((4,4-difluoro-5-(2thienyl)-4-bora-3a,4a-diaza- s -indacene-3-yl)styryl-oxy)acetyl)amino-hexanoic acid (BODIPY 630/650 or BY630), which are often quenched in aqueous solution and activated to emit fluorescence only when bound to receptors, have advantages in kinetic and receptor studies.15, 16 However, difficult synthesis and instability limits the application of these red-emitting fluorophores. Covalent coupling with linkers or pharmacophores is carried out via acylation reactions. Herein, according to the classification of the GPCR ligands, these probes are categorized into peptide and nonpeptide probes. Peptide fluorescent probes. As there are numerous peptide receptors in the GPCR family (e.g., neuropeptide Y, galanin, chemokine, secretin and glucagon), peptide-based probes are important fluorescent probes for GPCRs. Compared with a large peptide, a bulky conjugated fluorophore constitutes a relatively small part of the final molecule.8 In this case, these peptide probes may retain a similar affinity compared to their parent molecules. The peptides’ N- or C-terminus and some side chains of peptides can be targeted as the conjugation site between the fluorophores and GPCRs’ pharmacophores. Amino-reactive fluorophores or linkers are conjugated to the Nterminus by a simple condensation reaction, and the hydrazide derivatives are used to achieve the C-terminal labeling. In the side-chain conjugation, the fluorophores or linkers, conjugated with a thiol-reactive group such as maleimide, are introduced easily through a nucleophilic reaction. For example, a fluorescein-N-galanin probe (1, Scheme 1), generated by directly conjugating


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fluorescein to the NH2 of the neuropeptide galanin, binds to galanin receptor 1 (GalR1).17 Galanin receptors modulate several physiological functions including food intake, nociception, nerve regeneration, memory, neuroendocrine release, and gut secretion and contractility. The fluorescent agonist 1 has been used successfully in flow cytometry to probe the molecular nature of the interaction between galanin and its receptors, and the internalization of the galanin/rGalR1 complex after binding. NPY receptors are involved in the control of a diverse set of behavioral processes including appetite, circadian rhythm and anxiety. Schneider et al. have reported a fluorescent agonist of NPY receptors, cyanine-dye-labeled neuropeptide Y at Lys-4 (Dy630-LysNPY, 2, Scheme 1)18, and confirmed its applications for determining the affinity, selectivity, and activity of NPY receptor ligands. This powerful approach can be replicated for investigating other GPCRs.18 In general, the conjugating mode or site should be set up based entirely on structure–activity relationship analysis of fluorescent peptides, which are labeled at different positions. It has been confirmed that this site should not appear in the receptor-binding domain of the parent peptide. Moreover, sometimes a spacer is used to achieve the optimal affinity and efficacy, although a direct conjugation may be adequate. For example, Oishi et al. have reported the synthesis and modification of probes for chemokine receptor 4 (CXCR4) based on the CXCR4 antagonist, 3 (Table 1), a T140 derivative optimized for receptor binding and stability.19 CXCR4 correlates with cancer metastasis and inflammatory autoimmune disorders such as rheumatoid arthritis. CXCR4 also serves as the second receptor for cellular entry of T-tropic strains of human immunodeficiency virus (HIV). Although compounds (6 and 7, Table 1)19 modified at the Nterminus of the peptide have been proven unfavorable as ligands for CXCR4, modification of the ε-amino group of D-Lys8 in the parent peptide 3 provided another promising approach for the


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production of labeled CXCR4 antagonists.19 Fluorescein-modified peptides 8 and 9 and Alexa Fluor 488-labeled peptide 11 all exhibited specific and high affinity to CXCR4.19 In a follow-up study, Nomura et al. have designed and synthesized the fluorophore-labeled 3 (Figure 2) molecules, such as TAMRA-3(13, Figure 2) and fluorescein-3 (14, Figure 2), which were used in confocal microscopy imaging of CXCR4 and in the exploration of novel pharmacophores for CXCR4-specific ligands with high-throughput screening.20 Another example is probing the binding domain of the neurokinin 2 (NK2) receptor with fluorescent ligands designed by Turcatti et al.21 NK2R is involved in human airway inflammatory diseases, but little is known about the residues in its binding domain. Binding assays with several fluorescent antagonists ANT-1~6 (16~21, Table 2) of NK2 receptors, which were labeled on the first residue of the heptapeptide GR94800 (15, Table 2) with NBD using spacers of different lengths, have revealed that the optimal spacer length is approximately 5–10 Å.21 The receptor affinity assay of the fluorescent peptides AGO-1~3 (23~25, Table 2) pointed out that 23 and 24 bound the NK2 receptor stronger than 25, which indicated that the N-terminal labeling was needed.21 It needs to be underlined that native peptides may not be used directly to develop probes, for several reasons such as excessive residues, steady metabolism and low selectivity. The basic principles of modifying such a peptide are to preserve the pharmacophoric domain and condense the peptide chain as much as possible. In this case, replacement of amino acid residues frequently occurs in peptide structure reformation. For example, cholecystokinin (CCK), consisting of totally 33 amino acids, is a peptide hormone that relates with wide-ranging physiologic actions such as controlling of nutrient assimilation. The C-terminal octapeptide


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fragments of CCK have a stronger influence on the biological activity than intact CCK.22 Following the replacement of Met with Nle, the CCK-8 probe (eight amino acid residues) was prepared by attaching Alexa dye to the amino terminus of the CCK-8 analogs, which showed similar selectivity for both subtype A and B receptors. In contrast, the CCK-4 probe (four amino acid residues), in which the fluorophore was located closer to the pharmacophoric moiety, had an exclusive selectivity for the subtype B receptor. In another case, considering the amidated Cterminus sequence homology of seven amino acids, -Trp-Ala-Val-Gly-His-Leu-Met-NH2, was shared by Gastrin-Releasing Peptide (GRP) and Bombesin (BBN), Smith et al. have synthesized an Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate by tethering Alexa Fluor 680 succinimidyl ester to the N-terminal primary amine of H2N-Gly-Gly-Gly-Gln-Trp-Ala-Val-GlyHis-Leu-Met-NH2, which revealed high affinity and high selectivity for the GRP receptor.23 In this probe, three Gly residues play a role as a spacer and the rest of the peptide chain belongs to the receptor-binding domain of the ligand. Finally, in respect to fluorophores, traditional NBD, cyanine dyes, fluorescein and other groups have been accepted as described above. In addition, fluorescent amino acids provide alternative methods to probe peptides and proteins.24, 25 For instance, Fernandez et al. have reported that α-melanocyte stimulating hormone (α-MSH) analogues, containing the aromatic fluorescent amino acid 52 (Scheme 5)26, have high affinity and selectivity for the melanocortin (MC)-4 receptor, which is very important for central regulation of weight homeostasis. These fluorescent peptides have been used for structural analysis of melanocortin peptides.26 Small-molecule fluorescent probes. Except for peptide ligands, many ligands of GPCRs are small molecules. Small-molecule fluorescent probes, derived from these ligands, are named for their low molecular weight. Compared with peptide-based probes, these small-molecule probes


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have become more promising tools for the study of GPCRs, owing to their tremendous advantages in stability, solubility, reasonable cell permeability, subtype-selectivity, application in high-throughput screening, and prevention of receptor desensitization.6, 27, 28 Even for peptide receptors, much work has been done to develop nonpeptide probes.29, 30 Unlike peptides, small-molecule ligands usually do not equal fluorophores in size. The conjugated site of these receptor groups with reporter groups would usually be close to their binding domains. As a consequence, the design and synthesis of small-molecule fluorescent probes, which retain the receptor-binding affinity and efficacy, are challenging. In smallmolecule probes, the linker moiety is seemingly inseparable to provide fluorophores with some flexibility to reduce the influence on the ligand-receptor binding. Additionally, the aromatic moiety in ligands can be replaced with similar fluorophores, if appropriate. As well as drug design and discovery, development of successful probes for GPCRs depends on a series of derivatives of lead compounds and the study of their structure-activity relationship (SAR). This process typically begins with a known ligand or drug. If the crystal structures of receptors, the SAR of drugs and ligand-receptor-binding mode have been clarified, then developing an ideal probe will be easier,31-33 as it will only require choosing an appropriate lead compound, adopting the right tag and a proper linker, and finding an appropriate site. The most important step in designing a small-molecule fluorescent probe for GPCR is the choice of a strong ligand. This ligand must have a high affinity and selectivity to the receptor, as well as an easy-to-handle modification site in its chemical structure. Based on the SAR, a reactive group, such as amine, hydroxyl, alkynyl or carboxyl group, is introduced into the ligand molecules. Condensation reactions and click reactions are widely used to achieve the conjugation between the pharmacophores and linkers or fluorophores.


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Cannabinoid receptor 2 (CB2 receptor) is highly expressed on immune cells and has become a particularly attractive target for drug development to treat neurological diseases. It is essential to develop a probe to precisely map CB2 receptor in tissues. Bai et al. have reported a conjugated ligand , 27 ( Scheme 2A), for CB2 receptor imaging in 2008, which was derived from the inverse selective agonist, 26 (Scheme 2A).34 In contrast, modification of another selective agonist, 28 (Scheme 2B)35, was beset with difficulties to generate an excellent probe. The conjugation of NBD and 28, even with an additional 3-atom spacer (29, Scheme 2B), strongly decreased the affinity of the ligand to CB2 receptor.35 Additionally, a novel and successful fluorescent probe, compound 31(Scheme 2C), was designed simply by substituting the morpholine moiety of the new potent antagonist, compound 30 (Scheme 2C), with NBD and a methylene.36 Considering both the fluorophore and the associated linker may critically influence the pharmacological or physicochemical properties of a fluorescent ligand, the selection of a suitable fluorophore and a proper linker is crucial for designing an ideal GPCR fluorescent probe. It is worth choosing a fluorophore that is favorable for kinetic studies for fluorescent ligands. Therefore, sufficient consideration of the fluorophore and linker selection should be well conducted. For example, the hydrophobic BODIPY630/650 or 4-amino-1,8-naphthalimide could increase the non-specific binding.37-39 As reported by Rose et al.,37 a commercial mepyramineBODIPY630/650 conjugate showed high affinity to histamine H1 receptor (H1 receptor), which is accountable for the allergic reactions. This conjugate can reveal whether H1 receptor is localized on the cell membrane by a competitive binding assay, but in cell imaging, many fluorescent ligands are caught in the non-specific uptake to the cytosol. To reduce the influence of uptake of such lipophilic ligand, FCS is used to measure receptor-ligand binding in the cell membrane. Likewise, similar events have happened in the study of adenosine receptors (A1AR


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and A3AR).40, 41 As adenosine receptor subtypes that signal to inhibit adenylyl cyclase (cAMP), A1ARs are responsible for mediating physiological effects including vasoconstriction, lipolysis, sleep, acupuncture and analgesia, while A3ARs are related to mast cell activation, airway contraction, inflammation and white cell chemotaxis. In combination with FCS, a fluorescent A1AR antagonist, (E)-3-(4-(2-((6-((2-(2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin8-yl)phenoxy)acetamido)ethyl)amino)-6-oxohexyl)amino)-2-oxoethoxy)styryl)-5,5-difluoro-7(thiophen-2-yl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (XAC-BY630), has been used to quantify antagonist-receptor binding at the single cell level;40 and a fluorescent A3AR agonist, ((2S,3 S,4 R,5 R,E)- N-ethyl-3,4-dihydroxy-5-(6-(4-(6-(2-(4-(2-(4,4-difluoro4,4a-dihydro-5-(thiophen-2-yl)-4-bora-3a,4a-diaza- s-indacene-3yl)vinyl)phenoxy)acetamido)hexan-amido)butylamino)-9 H-purin-9-yl)tetrahydrofuran-2carboxamide (ABEA-X-BY630), has revealed that agonist-occupied A3ARs exist in heterogeneous complexes in membrane microdomains of individual living cells.41 In another study by Baker et al. about fluorescent ligands for A1AR, the effect of the linker on the pharmacology of ligands was well discussed.42 Fluorescent derivatives of the antagonist xanthine amine congener (XAC) and the agonist 5-(N-ethylcarboxamido) adenosine (NECA) with an equilong spacer but different tags have shown significant differences in their binding to A1AR. BODIPY 630/650 was a good fit for both fluorescent agonists and antagonists. Additionally, as an environment-sensitive fluorophore, BODIPY630/650, whose fluorescence is quenched in aqueous solution, facilitates the kinetic study of A1- and A3-AR ligands at the single cell level, such as real-time monitoring of the dissociation rate.43, 44 However, the impact of the linker length on the agonist potency differs between fluorescent agonists with different tags: when the linker is extended, higher agonist potency could be achieved with dansyl derivatives, but lower


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potency could be achieved with BODIPY 630/650 derivatives. The linker length change is accomplished by varying not only the number of carbon atoms but also the type of atoms. With low lipophilicity, a polymer-based linker provides an alternative alkyl linker to increase hydrophilicity and get specific binding. Polyamide45 and polyethylene glycol46 have been used as linkers to form powerful probes for quantitative analysis of A3AR and visualization of ligandhuman β-adrenoceptors interactions, respectively. In addition, insertion and optimization of peptide-based linkers between the adenosine receptor pharmacophore and the fluorophore turns the non-selective GPCR adenosine receptor antagonist, XAC, into a selective fluorescent probe for A3AR, which is a potential target for treating cancer, inflammation, glaucoma and asthma.47 Finding an appropriate conjugated site in the design of a GPCR fluorescent probe is imperative. A proper site is generally determined in terms of the SAR study. Based on extensive SAR studies and computational models of human 5-HT1A receptor-ligand interactions, Alonso et al. have chosen two potent agonists 32 (Scheme 3) and 33 (Scheme 3) as scaffolds, to develop fluorescent 5-HT1A receptor ligands.48 5-HT1A receptor is involved in the regulation of neurological processes, such as excitotoxicity, pain and anxiety. Two possible modifications were implemented: (i) replacing the aromatic residue connected to the piperazine moiety with fluorophores, and (ii) incorporating the fluorophore at position 7a of the bicyclohydantoin through a spacer. After synthesis and activity evaluation, most compounds demonstrated nanomolar affinity for the h5-HT1AR, and derivative 36 deserves special attention as it enables direct observation of the h5-HT1AR in cells.48 Overall, these sections in the process are inseparable. Only organized “teamwork” can eventually develop good probes. Compared with the study of the 5-HT1 receptor, a similar study on the 5-HT6 receptor was not as successful. The 5-HT6 receptor may be associated with central


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nervous system (CNS) diseases, including cognition and feeding. The latest research on fluorescent 5-HT6 receptor ligands by Henar et al. was based on the scaffold of SmithKline Beecham 46 (Scheme 4), which was confirmed to be a 5-HT6 receptor antagonist with high affinity, good selectivity and oral bioavailability.49 A set of probes was prepared by attaching different tags to (a) the piperazine ring, (b) the methoxy group, or (c) the sulfonamide moiety of the scaffold with or without a spacer. However, all compounds based on this strategy demonstrated either low affinity or weak fluorescence. Accordingly, the possibility of replacing the benzothiophenesulfonyl moiety with the dansyl group was envisioned, and the synthesized compound 49 has shown high affinity (Ki = 8 nM), but with no fluorescence yet.49 The fluorescence intensity depends on the distance between the dansyl and piperazine groups. If the distance was long enough, strong fluorescence should have been acquired. Therefore, it has been proposed that the fluorescence of these compounds may be quenched by piperazine through the photoinduced electron transfer (PET) effect.50 Next, a dansyl group was attached to the piperazine ring for significant fluorescence, to get an optimum balance between affinity and fluorescence. Compound 50 was confirmed to specifically label the human 5-HT6R in cells.49 In addition, compound 51, which exhibits better properties both in fluorescence and receptor affinity, was designed by attaching a biotin moiety to the piperazine ring.49 Perspective. As safe and sensitive kits, ligand-based fluorescent probes contribute largely to the knowledge of GPCRs or receptor-ligand complexes. Currently, they are widely used in drug discovery and screening. Fluorescent peptides are regarded as the earliest fluorescent ligands for GPCRs. These probes are reasonable for studying interactions of GPCRs with their endogenous ligands. Application of the fluorescent amino acids would greatly accelerate their development. Small-molecule fluorescent probes are fast-growing fluorescent ligands. They are very popular


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because of their advantages in synthesis, receptor subtype selectivity and high throughput screening feasibility. However, challenges still exist. The autofluorescence and noise could perturb the results. Common non-specific binding of fluorescent ligands to cellular components limits their applications in cellular-based imaging, because of large signal-to-noise (S/N) ratio. This non-specific binding may be related to the probes’ physicochemical properties. Especially, hydrophobic fluorophores could increase penetration into cell membranes. The advent of novel fluorescence microscopes, such as FCS and TIRF, which may significantly reduce the influence caused by non-specific binding, further reveals the great power of fluorescent ligands in the study of GPCRs at the single cell level. Several potential directions for future fluorescent ligand studies could be considered: 1) to wash away the non-specific binding probes, hydrophilic fluorophores or linkers are adopted to generate fluorescent conjugates on the premise of not influencing receptor affinity; 2) vastly improving the receptor affinity or activity of fluorescent ligands; 3) introducing the fluorescence on/off strategy such as PET or FRET and designing switchable fluorescent ligands. These methods will be introduced in detail later. PROTEIN-BASED FLUORESCENT PROBES FOR GPCRs Because GPCRs comprise a large family of cell-surface signaling proteins, powerful fluorescent detection technology for proteins has also been applied to GPCRs. These probes could be classified into two categories, fluorescent antibodies and protein tag-based probes, according to the function of the protein they are based on. Antibody-based probes rely on an antigen-antibody specific reaction, which is similar to the mode of ligand-receptor binding. Protein tag-based probes are generated by fusing the receptor protein with a protein tag.


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Antibody-based fluorescent probes. As receptor proteins, GPCRs not only bind specific ligands, but also exist as specific antigens in immunohistochemistry. Antibodies are very specific and efficient molecules produced by the body’s humoral immune system. The introduction of antibodies with attached fluorescent tags has significantly contributed to the study of GPCRs. Targeting GPCRs by an antibody with a fluorophore enables the receptor to be visualized (Figure 3A).51 In most studies, immunofluorescence is used as a method to evaluate the receptor binding of ligands or drugs by a competitive assay (Figure 3B).52 To imitate the ELISA method, a sandwich immunoassay (Figure 3C) can be developed using a labeled secondary antibody. Because the GPCR’s structure, poor immunogenicity and low receptor density make it difficult to generate GPCR antibodies, the development of fluorescent antibodies is limited. Furthermore, antibody targeting of intracellular proteins normally requires cell fixation and permeabilization, which limits their usage in fluorescent imaging in vivo.53 Protein tag-based fluorescent probes. Protein tags, including SNAP, fluorescent protein (FP), His and Flag, are peptide sequences genetically grafted onto a receptor protein by generecombination techniques. These tags are attached to the receptors for studying the visualization, mobility, internalization and other actions of GPCRs. (1) SNAP-tag. In the SNAP-tag technology, a 20-kDa O6-alkylguanine-DNA alkyltransferase (SNAP) is fused to the N-terminus or C-terminus of GPCRs. SNAP reacts with O6benzylguanine derivatives carrying a fluorophore (Figure 4).54 After the reaction, the fluorophore can be covalently attached to the receptor via SNAP, and then the receptor is visualized by fluorescence techniques. Furthermore, the combination of SNAP-tag technology and the Homogeneous Time-Resolved Fluorescence (HTRF) detection method creates a powerful technology, named Tag-lite. According to ligand-binding assays of different GPCRs, Jurriaan et


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al. have clearly demonstrated that the new technology is highly suitable for high-throughput screening (HTS) applications with great advantages in terms of flexibility, rapidity, userfriendliness and easy miniaturization.55 (2) FP-tag. Fluorescent proteins (FP) are typical of genetically encoded fluorophores, including green fluorescent proteins (GFP), yellow fluorescent proteins (YFP), and cyan fluorescent proteins (CFP). Because of the stable self-fluorescence, this tag is fused to the C-terminus of proteins to label receptors without other substrates.56 FP is large size; for example, GFP is a 27kDa protein, which is greater than 50% of the size of the majority of class 1 and 2 GPCRs.57 When they are used, a key initial issue is to ensure that the FP would not alter the basic features of the GPCRs. Fusion of FP to the N-terminus of GPCRs is unsuitable because it would involve in the transmembrane transport of FP.57, 58 Using these tags, location, mobility, kinetics and protein-protein interactions of GPCRs can be easily studied. Sen et al. have reported that the ARGFP fusion protein enabled them to monitor the expression and subcellular distribution of α2Badrenergic receptor.59, 60 Using a CB1-GFP fusion protein, Skretas et al. have screened genes that cause a large enhancement of production of membrane-integrated CB1 receptor, which serves as a treatment target of obesity and tobacco addiction as well as Parkinson’s and Alzheimer’s disease.61 Using fluorescent proteins, a GPCR “pathway” screening assay for vasopressin-2 receptor (V2R) antagonists, which have aquaretic effects on the kidney for the treatment of hyponatremia, has been established by Yangthara et al.62 The principle of this assay is diagrammed in Figure 5C. The fluorescence of YFP-H148Q/I152L, a yellow fluorescent protein-based halide sensor, can be quenched by I–. Before an antagonist binds to V2R, it emits fluorescence, and after


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binding, the fluorescence is quenched by iodide ions inflowing to cells via open cystic fibrosis transmembrane conductance regulator (CFTR) ion channels. Fluorescent proteins are also extensively applied in bimolecular fluorescence complementation (BiFC) analysis,63, 64 which has the tremendous advantage of allowing the detection of the subcellular localization of GPCRs’ interactions. Fluorescent proteins are divided into two fragments, protein N- (PN) and C-termini (PC), which are fused to the carboxyl-termini of two receptors. When the interaction of the receptors of interest happens, the protein fragments irreversibly reconstitute a functional protein, which will emit fluorescence that enables the visualization of the GPCRs’ interactions (Figure 5B). Additionally, fluorescent proteins are used as fluorescent sensors in fluorescence resonance energy transfer (FRET). However, because of the irreversible reconstitution, studies on dynamic changes in GPCRs’ interactions are difficult. (3) His-tag. His-tag proteins containing different numbers of histidine residues can be inserted into the N- or C-terminus of target proteins. His-tag-fused GPCR proteins are recognized by probes based on the Ni (II)–nitrilotriacetic acid complex [Ni (II)–NTA; Figure 6]65, 66 or the Zn (II) complex [Zn (II)–Ida].67 These are widely applicable tools for immobilization, purification, handling and detection of GPCR proteins. Amino acid-based fluorescent probes. As mentioned above, fluorescent amino acids can be used as the fluorophore to label a peptide.24 However, their application as fluorescent probes is limited, because of their poor fluorescent properties (e.g., low quantum yield, small Stokes shift and excessive sensitivity to local environment)68 and inaptitude for nonpeptide ligands. Hence, side-chain modifications of natural amino acids are required. Here, we introduce the unnatural amino acids (UAAs). The UAA mutagenesis of GPCRs makes it possible to chemically modify


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the protein of interest directly and uniquely.69 Without fluorescence, site-directed UAA mutagenesis can determine the ligand-binding pockets of GPCRs.70 Combined with fluorescent methods, fluorescent UAAs yield unusually good results in the study of GPCRs, because UAAs can harbor diverse fluorophores directly or indirectly. In this approach, one method is the attachment of UAAs to a fluorophore before the incorporation of UAAs into GPCRs. This case is similar to fluorescent ligands or antibodies. Receptor proteins are visualized and located after the incorporation of UAA-fluorophores into the receptor sequence. Pantoja et al. have reported the single-molecule imaging of the muscle nicotinic acetylcholine receptor, nAChR.71 In another method, chemoselective UAAs are initially incorporated into GPCRs, and then fluorescent receptors are generated by the reaction of the UAA with specially modified fluorophores or ligands. This method is analogous to the SNAP-tag approach. Through reactions mediated by special UAAs, GPCRs will link to the fluorophore or the probe. Some representative UAAs, such as p-Acetyl-L-phenylalanine (AcF), p-Azido-L-phenylalanine (azF) and p-Benzoyl-L-phenylalanine (BzF), enable the incorporation of fluorophores with biologically active ligands for selective chemical modifications, which facilitate real-time protein dynamics and interaction studies.72 It should be noted that these coupling reactions are regular reactions in organic chemistry. For instance, the reaction for identifying ketone with organic hydrazine is used in AcF-fluorophore coupling (Scheme 5). Hence, more and more UAAs are designed based on various chemical reactions. For example, Daggett et al. have demonstrated the application of UAAs in the site-specific labeling of GPCRs,73 and Siderius et al. have reported the study of the chemokine receptor structure and dynamics with UAAs.69


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Perspective. As GPCRs are proteins in nature, well-developed protein-based fluorescence approaches are applicable to GPCRs. Fluorescent antibodies accurately bind to GPCRs by an antigen-antibody specific reaction. A disadvantage that cannot be ignored is that the necessary fixation procedure may change or denature the receptor protein. Poor immunogenicity of GPCRs brings challenges to the development of antibody-based fluorescent probes. Protein tag-based fluorescent probes play indispensable roles in a series of studies on GPCRs, such as visualization, localization, dynamics, oligomerization, and interactions with ligands or other protein. These unique approaches rely on gene-recombination techniques. The issue is that the fused large tag may make the final receptor different from its parent receptor. When the tags like SNAP are used, the non-specific binding of fluorophores should be noticed. Amino acid-based fluorescent probes provide another site-specific labeling of GPCRs. The incorporation of fluorescent amino acids into receptor proteins is not feasible because of their poor fluorescent properties. Genetically encoding UAAs are widely used to introduce small unique bioorthogonal tags into GPCRs of interest, which facilitates cell-based studies of receptor proteins using fluorescence spectroscopy or single-molecule imaging. UAAs have an advantage over SNAP in size. Site-direct UAA mutagenesis is a potent approach to study the ligand-binding pockets of GPCRs. Protein tag- and amino acid-based fluorescent methods are irreversible, and the irreversible reconstitution may influence the native features of GPCRs. Combination of these protein-based approaches and ligand-based fluorescence by advanced mechanisms, such as FRET, will be favored. NEW STRATEGIES FOR FLUORESCENT PROBES To date, more and more fluorescent probes based on ligands, proteins and amino acids have been developed. However, for the current fluorescent probes, some intrinsic issues such as the imbalance between affinity and fluorescence, interference of autofluorescence and noise, false-


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positive or negative results caused by non-specific binding, and the difficulty to operate will limit their general application. To overcome these restrictions, some new or hybrid strategies are being developed for cell imaging, with rapid progress in molecular biology, organic chemistry and materials science. Novel functional groups as fluorophores. (1) Quantum dots. Quantum dots (QDs) are semiconductor crystalline nanoparticles characterized by water-solubility broad excitation, tunable narrow emission spectra, resistance to photobleaching and ultrahigh brightness.56, 74 As new-type fluorophores, QDs have been chemically conjugated to specific cellular components, such as ligands and antibodies, to enable biological imaging and therapeutics. For example, Zhou et al. have prepared peptide-labeled QDs that are selectively coupled to peptide ligands targeting GPCRs through an amine or thiol linkage, and demonstrated their utility in whole-cell and single-molecule imaging.75 Das et al. have explored QD conjugates by coupling them with an A2aAR agonist through a polyamidoamine linkage for characterization of the GPCR. Like common fluorescent ligands, the structure modification of pharmacophores or spacers will be needed to make useful QD probes with a high receptor affinity.76 QDs have proven their value as powerful inorganic fluorescent probes for ligand screening, and detecting or visualizing GPCRs.77, 78 However, this QD fluorophore is limited, because of its large size and high toxicity.56, 78 These bulky QDs may get inside cells through intracellular endocytic processes, which may cause ligand- and receptor-independent cell uptake, thus resulting in non-specific binding. Moreover, QDs contain heavy metals,and their crystals are generally greater than the renal excretion limit, thus narrowing their application in bioimaging because of their high toxicity.


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(2) Environment-sensitive fluorophores. As described above (see BODIPY 630/650), these probes are named environment-sensitive fluorophores, because their spectroscopic behavior varies with the physicochemical properties of the surroundings. Such fluorophores have been employed to develop fluorescent GPCR ligands. The incorporation of the environment-sensitive chromophore, 6-N,N-dimethylamino-2, 3-naphthalimide, into δ-selective opioid peptides makes it possible to study membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.79 Tan and coworkers have established new fluorescent turnon probes by conjugating another environment-sensitive fluorophore, 4-sulfamonyl-7aminobenzoxadiazole (SBD), and protein-specific ligands (Figure 7A), which can be used to identify hydrophobic ligand-binding sites.80, 81 (3) Avidin/streptavidin-Biotin. Biotin is a water-soluble B-vitamin with small size and low toxicity. Biotin and its analogs have been used to label proteins and nucleic acids for purification, detection and visualization.82, 83 Essentially, biotin is not a fluorophore, as it has no fluorescent properties. The application of biotin as a fluorophore depends on its high affinity to anti-biotin antibodies or avidin/streptavidin, which act as second probes. For GPCRs, biotinligand conjugates bind to their receptor, followed by the addition of avidin to trap the biotin for fluorescence. Unlike the bulky fluorophores, biotin does not affect the biological activity of the protein or the ligand, because of its small size. For example, in the design of probes for the abovementioned 5-HT6 receptor, Henar et al. have introduced biotin into the modified ligand in the piperazine ring to obtain the probe, compound 28 (Scheme 4), which showed both high affinity and good optical properties. In imaging assays, cells were incubated in the presence of the biotinylated probe, followed by a streptavidin-Alexa Fluor 488 conjugate. Then, the receptors were observed by fluorescence microscopy.49


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(4) Biarsenical-tetracysteine.84, 85 In the biarsenical-tetracysteine system, a tetracysteine motif (TCM) such as Cys-Cys-Pro-Cys-Cys is fused to the protein of interest at C- or N- termini as well as in alpha helical regions to produce a recombinant protein that can be site-specifically labeled with a membrane-permeable biarsenical dye. As has been reported, FlAsH and ReAsH are almost non-fluorescent until bound to the TCM to form a covalent complex.85 This system is a promising alternative to the fluorescent proteins in the FRET approach. Initially, the donor CFP and the acceptor YFP were employed in the FRET approach for GPCRs. However,the large protein incorporated into the intracellular receptor loop may reduce the GPCR activity. Thus, Ziegler et al. have used FlAsH as the acceptor to replace the large YFP.86 These FRET-based sensors have been used to study the activation and signaling of human M1-, M3- and M5acetylcholine receptors, including monitoring concentration-dependent effects of receptor modulation in real-time and analyzing receptor kinetics in living cells. Switchable fluorescent probes. The fluorescent intensity of a switchable fluorescent probe can obviously increase or decrease when some conditions change, such as binding of ligands to the receptor, chemical reactions with probes or irradiation. Besides the environment-sensitive turnon/off fluorescent probes mentioned above, the switchable fluorescent probes depend on ingenious mechanisms. (1) Fluorescence quenching. Fluorescence quenching has been widely used to design switchable fluorescent probes in many fields except GPCRs.87, 88 Fluorescence quenching mainly relies on PET,89, 90 FRET,91 and internal charge transfer (ICT). For example, Maurel et al. have designed photosensitive fluorophores for SNAP-tagged probes used for protein labeling.92 The state-of-the-art probe is the introduction of the donor along with the acceptor and the departure of the acceptor alone at the right time. At first, the fluorescence is quenched, because of the


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energy transfer between the donor and the acceptor. The fluorescence emitted by the donor can be absorbed by the acceptor. When irradiated at 365 nm, the acceptor leaves automatically, and the fluorescence of the donor is observed. (2) Ratiometric fluorescent probes. The fluorescent ratiometric methods focus more on the variation of the fluorescence intensity ratio emitting at two wavelengths under the same experimental conditions.93 These endow the ratiometric fluorescent probes with high temporal and spatial resolution in both chemical and biochemical imaging. Masharina et al. have demonstrated the first FRET-based ratiometric fluorescent sensor (named GABA-Snifit, Figure 7B) for measuring γ-aminobutyric acid (GABA) concentrations on the cell membrane.94, 95 As the main inhibitory neurotransmitter in the mammalian nervous system, GABA plays a critical role in neuronal communication, intercellular communication outside the nervous system, embryonic development and adult neurogenesis. The Snifit is a fusion protein containing a SNAP-tag, a CLIP-tag and a receptor protein (RP) of interest. The CLIP-tag is located between the SNAP-tag and the RP. A ligand is linked to the SNAP-tag via a fluorophore (the acceptor). The CLIP-tag is tagged by a second fluorophore (the donor). When the analyte is absent, the ligand binds to the acceptor, making the acceptor close to the donor, and then FRET happens. When the analyte is present, it competes with the ligand on receptor binding, and then FRET will weaken or disappear. Multifunctional probes. Considering the complicated GPCRs’ actions, a single-function fluorescent probe can hardly sense or identify the receptor proteins, especially in the case of orphan receptors. Therefore, there is an urgent demand for developing a multifunctional probe in the research area of GPCRs by multidisciplinary techniques.


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(1) Ligand-based receptor capture. Ligand-based receptor capture (LRC) described by Wollscheid and colleagues is a method for purifying receptor peptides for identification by quantitative mass spectrometry.96, 97 LRC relies on a trifunctional chemical probe called 54 (Figure 8), which contains a biotin for peptide enrichment, a hydrazine for covalent crosslinking and an N-hydroxysuccinimidyl ester for ligand attachment.96 This technology is successful in indirect identification of ligand-GPCR interactions in living cells and tissues. (2) Single-molecule pull-down. In single-molecule pull-down (SiMPull), cellular protein complexes are pulled down directly to the imaging surface of the single-molecule fluorescence microscope.98 This approach is carried out in a flow chamber constructed by Jain et al. 98 When the cell extracts are infused in the flow chamber, a surface-tethered antibody, such as anti-YFP, captures the bait protein (for example, YFP), which brings along its binding partner, the protein complex of interest. Hitherto, this SiMPull approach has been successfully applied with β2adrenergic receptor (β2-AR; Figure 7C), a prototypical GPCR that mediates the relaxation of smooth muscles in non-cardial tissues, glycogenolysis and glucogenesis in liver, and cell metabolism in skeletal muscle. Perspective. Nowadays, single fluorescent ligands and tags cannot meet the requirements for a reasonable tool for GPCRs. Accordingly, some new and hybrid strategies are being developed. New functional groups with good fluorescent properties or high sensitivity are developed to provide novel fluorophores, which will be applied to GPCR studies. These fluorophores produce good ideas to develop fluorescent ligands and antibodies. However, the use of these fluorophores cannot completely address the issue of non-specific binding, and some of them are toxic to various degrees. Based on ingenious mechanisms, switchable fluorescent probes are acceptable for receptor binding without excessive background noise. Multifunctional probes are established


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on transdisciplinary techniques, which is beneficial to research the complicated actions of GPCRs simultaneously. Switchable fluorescent probes and multifunctional probes are designed intellectually. The main difficulty for these two types of probes is how to apply them to other GPCRs, as some of them need special steps, like oxidation with NaIO4 in LRC, which is unfavorable for tissues. CONCLUSION Over the years, the fluorescent probe has become a necessary toolkit for studying GPCRs at the single cell level, even up to the molecular level. Information acquired by fluorescent probes made it clear where GPCRs spread and how they behave in receptor-ligand and receptor-receptor interactions. Following the development of fluorescence technologies, fluorescent probes used with GPCRs have been involved in various fields. In this article, we summarized the development of fluorescent probes targeted at GPCRs. It is well known that most ligand-based fluorescent probes belong to fluorophore-tagged GPCR ligands. Therefore, the process of exploring a new ligand-based probe is very similar to drug design and discovery. The key question in developing fluorescent ligands is how to make the final probe retain the high binding affinity and efficacy. Extensive knowledge and experience with probe design was discussed and reported herein. Fluorescent ligands have provided significant data about GPCRs and receptor-ligand complexes, which are invaluable for discovering new drugs. Therefore, these probes are generally used in high-throughput screening of GPCR agonists or antagonists. Fluorescent antibodies are mainly used in subsidiary research, competitive assays and screening antibodies. Beyond that, the fluorescent probes based on proteins and amino acids depend on genetic technologies. Although these methods are difficult to


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execute, they manifest huge potential for the direct study of GPCRs, especially the interactions among proteins. Some specific chemical reactions are used to achieve the coupling of fusion proteins and fluorophores. It should be highlighted that some novel fluorophores and hybrid systems have been reported to develop better fluorescent probes for GPCRs. Meanwhile, switchable and multifunctional fluorescent probes hold the attention of scientists who are studying GPCRs. There is no doubt that these new strategies, by overcoming the limitations of traditional probes, will substantially promote the development of fluorescent probes. Therefore, in the near future, great achievements in studying GPCRs with fluorescent probes are expected. AUTHOR INFORMATION Corresponding Author * Tel./fax.: +86-531-8838-2076. E-mail: [email protected] Notes The authors declare no competing financial interest. Biographies Zhao Ma is currently studying for the Ph.D. in the Department of Medicinal Chemistry, School of Pharmacy, Shandong University since 2011. His research area involves design, synthesis and bioactivity study of novel fluorescent probes for α1-adrenoceptors and reactive oxygen species (ROS). Lupei Du is an associate professor at the School of Pharmacy, Shandong University. She received her Ph.D. from China Pharmaceutical University in 2006 and conducted her


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postdoctoral research at the Department of Chemistry, Georgia State University from 2006 to 2009. She joined the Shandong University in 2009. Her research interests mainly focused on the rational design and synthesis of medicinal molecules and bioactive probes. Minyong Li is a professor at the School of Pharmacy, Shandong University. He received his Ph.D. degree from China Pharmaceutical University in 2005. He conducted postdoctoral research in the laboratory of Dr. Binghe Wang at Georgia State University from 2005 to 2007, and then became a research assistant professor of the Department of Chemistry at Georgia State University. In 2009, he moved to his current institution as a full professor. His research interests are in the general areas of medicinal chemistry and chemical biology. ACKNOWLEDGMENTS We acknowledge Professor Xiaodong Shi (Department of Chemistry, West Virginia University, USA) for his assistance in manuscript writing. Financial support from the Fok Ying Tong Education Foundation (No. 122036), the New Century Excellent Talent Project (No. NCET-110306), the Shandong Natural Science Foundation (No. JQ201019) and the Independent Innovation Foundation of Shandong University, IIFSDU (No. 2012JC002 and 2014JC008) is acknowledged. ABBREVIATIONS GPCRs, G-protein coupled receptors; NBD, 4-nitro-7-aminobenzoxadiazole; FCS, fluorescence correlation spectroscopy; TIRF, total internal reflection fluorescence; GalR1, galanin receptor 1; NPY, neuropeptide Y; CCK, cholecystokinin; GRP, Gastrin-releasing peptide; BBN, Bombesin; SAR, structure-activity relationship; CB, cannabinoid; A1AR, A1 adenosine receptor; 5-HT, 5hydroxytryptamine; FP, Fluorescent protein; CFTR, cystic fibrosis transmembrane conductance


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regulator; UAAs, unnatural amino acids; BiFC, bimolecular fluorescence complementation; HTS, high-throughput screening; FRET, fluorescence resonance energy transfer; QDs, Quantum dots; SBD, 4-sulfamonyl-7 aminobenzoxadiazole; TCM, tetracysteine motif; FlAsH, a fluorescein derivative modified to contain two arsenic atoms; ReAsH, a derivative containing two arsenic atoms based on resorufin; LRC, Ligand-based receptor capture; SiMPull, singlemolecule pull-down; PET, photo-induced electron transfer; ICT, intramolecular charge transfer; ELISA, enzyme linked immunosorbent assay. REFERENCES 1.

Bockaert, J.; Philippe Pin, J. Molecular tinkering of G protein-coupled receptors: an

evolutionary success. EMBO J. 1999, 18, 1723-1729. 2.

Granier, S.; Kobilka, B. A new era of GPCR structural and chemical biology. Nat. Chem.

Biol. 2012, 8, 670-673. 3.

Filmore, D. It's a GPCR world. Mod. drug discovery 2004, 7, 24-28.

4.

Lakowicz, J. R. Principles of fluorescence spectroscopy. Springer Science + Business

Media LLC: New York, USA, 2006; Vol. 1. 5.

Hara, T.; Hirasawa, A.; Sun, Q.; Koshimizu, T. A.; Itsubo, C.; Sadakane, K.; Awaji, T.;

Tsujimoto, G. Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1). Mol. Pharmacol. 2009, 75, 85-91. 6.

Kuder, K.; Kiec-Kononowicz, K. Fluorescent GPCR ligands as new tools in

pharmacology. Curr. Med. Chem. 2008, 15, 2132-2143. 7.

Vernall, A. J.; Hill, S. J.; Kellam, B. The evolving small-molecule fluorescent-conjugate

toolbox for class A GPCRs. Br. J. Pharmacol. 2014, 171, 1073-1084.


30

Page 31 of 58

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8.

Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R. Developments in fluorescent probes

for receptor research. Drug Discovery Today 2009, 14, 706-712. 9.

Hegener, O.; Jordan, R.; Häberlein, H. Dye-labeled benzodiazepines: development of

small ligands for receptor binding studies using fluorescence correlation spectroscopy. J. Med. Chem. 2004, 47, 3600-3605. 10. Hess, S. T.; Huang, S.; Heikal, A. A.; Webb, W. W. Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 2002, 41, 697-705. 11. Boyer, S. B.; Slesinger, P. A. Probing novel GPCR interactions using a combination of FRET and TIRF. Commun. Integr. Biol. 2010, 3, 343-346. 12. Hern, J. A.; Baig, A. H.; Mashanov, G. I.; Birdsall, B.; Corrie, J. E.; Lazareno, S.; Molloy, J. E.; Birdsall, N. J. Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. P. Natl. Acad. Sci. USA 2010, 107, 2693-2698. 13. Daly, C. J.; McGrath, J. C. Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol. Ther. 2003, 100, 101-118. 14. Sauer, M.; Hofkens, J.; Enderlein, J. Handbook of Fluorescence Spectroscopy and Imaging: From Ensemble to Single Molecules. WILEY-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2011; Vol. 12. 15. Banks, P. Impact of a red-shifted dye label for high throughput fluorescence polarization Asays of G Protein-Coupled Receptors. J. Biomol. Screen. 2000, 5, 329-334. 16. Höltke, C.; von Wallbrunn, A.; Kopka, K.; Schober, O.; Heindel, W.; Schäfers, M.; Bremer, C. A fluorescent photoprobe for the imaging of endothelin receptors. Bioconjugate Chem. 2007, 18, 685-694.


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17. Suke Wang, A. C., Catherine Strader, and Marvin Bayne. Evidence for hydrophobic interaction between galanin and the GalR1 galanin receptor and GalR1-Mediated Ligand Internalization: fluorescent probing with a fluorescein-galanin. Biochemistry (Mosc) 1998, 37, 9528-9535. 18. Schneider, E.; Mayer, M.; Ziemek, R.; Li, L.; Hutzler, C.; Bernhardt, G.; Buschauer, A. A simple and powerful flow cytometric method for the simultaneous determination of multiple parameters at G protein-coupled receptor subtypes. Chembiochem 2006, 7, 1400-1409. 19. Oishi, S.; Masuda, R.; Evans, B.; Ueda, S.; Goto, Y.; Ohno, H.; Hirasawa, A.; Tsujimoto, G.; Wang, Z.; Peiper, S. C.; Naito, T.; Kodama, E.; Matsuoka, M.; Fujii, N. Synthesis and application of fluorescein- and biotin-labeled molecular probes for the chemokine receptor CXCR4. Chembiochem 2008, 9, 1154-1158. 20. Nomura, W.; Tanabe, Y.; Tsutsumi, H.; Tanaka, T.; Ohba, K.; Yamamoto, N.; Tamamura, H. Fluorophore labeling enables imaging and evaluation of specific CXCR4-ligand interaction at the cell membrane for fluorescence-based screening. Bioconjugate Chem. 2008, 19, 1917-1920. 21. Turcatti, G.; Vogel, H.; Chollet, A. Probing the Binding Domain of the NK2 Receptor with Fluorescent Ligands: Evidence That Heptapeptide Agonists and Antagonists Bind Differently. Biochemistry 1995, 34, 3972-3980. 22. Harikumar, K. G.; Clain, J.; Pinon, D. I.; Dong, M.; Miller, L. J. Distinct molecular mechanisms for agonist peptide binding to types A and B cholecystokinin receptors demonstrated using fluorescence spectroscopy. J. Biol. Chem. 2005, 280, 1044-1050. 23. Ma, L.; Yu, P.; Veerendra, B.; Rold, T. L.; Retzloff, L.; Prasanphanich, A.; Sieckman, G.; Hoffman, T. J.; Volkert, W. A.; Smith, C. J. In vitro and in vivo evaluation of Alexa Fluor


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680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide Receptor. Mol. Imaging 2007, 6, 171–180. 24. Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Fluorescent analogs of biomolecular building blocks: design, properties, and applications. Chem. Rev. 2010, 110, 2579-2619. 25. K Maity, S.; Bera, S.; Haldar, D. Synthetic strategies for the development of fluorescent amino acids as optical probe. Curr. Org. Synth. 2013, 10, 525-546. 26. Fernandez, R. Structural study of melanocortin peptides by fluorescence spectroscopy: identification of β-(2-naphthyl)-D-alanine as a fluorescent probe. BBA-Gen. Subjects 2003, 1623, 13-20. 27. Lavis, L. D.; Raines, R. T. Bright ideas for chemical biology. ACS Chem. Biol. 2008, 3, 142-155. 28. Eggert, U. S.; Mitchison, T. J. Small molecule screening by imaging. Curr. Opin. Chem. Biol. 2006, 10, 232-237. 29. Schneider, E.; Keller, M.; Brennauer, A.; Hoefelschweiger, B. K.; Gross, D.; Wolfbeis, O. S.; Bernhardt, G.; Buschauer, A. Synthesis and characterization of the first fluorescent nonpeptide NPY Y1 receptor antagonist. Chembiochem 2007, 8, 1981-8. 30. Höltke, C.; Waldeck, J.; Kopka, K.; Heindel, W.; Schober, O.; Schäfers, M.; Bremer, C. Biodistribution of a nonpeptidic fluorescent endothelin A receptor imaging probe. Mol. Imaging 2009, 8, 27-34. 31. Davis, A. M.; Teague, S. J.; Kleywegt, G. J. Application and limitations of X-ray crystallographic data in structure-based ligand and drug design. Angew. Chem. Int. Ed. Engl. 2003, 42, 2718-2736.


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 58

32. Anderson, A. C. The process of structure-based drug design. Chem. Biol. 2003, 10, 787797. 33. Jacobson, K. A. Functionalized Congener Approach to the Design of Ligands for G Protein-Coupled Receptors (GPCRs). Bioconjugate Chem. 2009, 20, 1816-1835. 34. Bai, M.; Sexton, M.; Stella, N.; Bornhop, D. J. MBC94, a Conjugable Ligand for Cannabinoid CB2 Receptor Imaging. Bioconjugate Chem. 2008, 19, 988-992. 35. Yates, A. S.; Doughty, S. W.; Kendall, D. A.; Kellam, B. Chemical modification of the naphthoyl 3-position of JWH-015: in search of a fluorescent probe to the cannabinoid CB2 receptor. Bioorg. Med. Chem. Lett. 2005, 15, 3758-3762. 36. Petrov, R. R.; Ferrini, M. E.; Jaffar, Z.; Thompson, C. M.; Roberts, K.; Diaz, P. Design and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in immune cells. Bioorg. Med. Chem. Lett. 2011, 21, 5859-5862. 37. Rose, R. H.; Briddon, S. J.; Hill, S. J. A novel fluorescent histamine H(1) receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands. Br. J. Pharmacol. 2012, 165, 1789-1800. 38. Lacivita, E.; Masotti, A. C.; Jafurulla, M.; Saxena, R.; Rangaraj, N.; Chattopadhyay, A.; Colabufo, N. A.; Berardi, F.; Perrone, R.; Leopoldo, M. Identification of a red-emitting fluorescent ligand for in vitro visualization of human serotonin 5-HT1A receptors. Bioorg. Med. Chem. Lett. 2010, 20, 6628-6632. 39. Berque-Bestel, I.; Soulier, J.-L.; Giner, M.; Rivail, L.; Langlois, M.; Sicsic, S. Synthesis and characterization of the first fluorescent antagonists for human 5-HT4 receptors. J. Med. Chem. 2003, 46, 2606-2620.


34

Page 35 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40. Briddon, S. J.; Middleton, R. J.; Cordeaux, Y.; Flavin, F. M.; Weinstein, J. A.; George, M. W.; Kellam, B.; Hill, S. J. Quantitative analysis of the formation and diffusion of A1adenosine receptor-antagonist complexes in single living cells. P. Natl. Acad. Sci. USA 2004, 101, 4673-4678. 41. Cordeaux, Y.; Briddon, S. J.; Alexander, S. P.; Kellam, B.; Hill, S. J. Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells. FASEB J 2008, 22, 850-860. 42. Baker, J. G.; Middleton, R.; Adams, L.; May, L. T.; Briddon, S. J.; Kellam, B.; Hill, S. J. Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands. Br. J. Pharmacol. 2010, 159, 772-786. 43. May, L. T.; Briddon, S. J.; Hill, S. J. Antagonist selective modulation of adenosine A1 and A3 receptor pharmacology by the food dye Brilliant Black BN: evidence for allosteric interactions. Mol. Pharmacol. 2010, 77, 678-686. 44. May, L. T.; Bridge, L. J.; Stoddart, L. A.; Briddon, S. J.; Hill, S. J. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics. FASEB J 2011, 25, 3465-3476. 45. Stoddart, L. A.; Vernall, A. J.; Denman, J. L.; Briddon, S. J.; Kellam, B.; Hill, S. J. Fragment screening at adenosine-A3 receptors in living cells using a fluorescence-based binding assay. Chem. Biol. 2012, 19, 1105-1115. 46. Baker, J. G.; Adams, L. A.; Salchow, K.; Mistry, S. N.; Middleton, R. J.; Hill, S. J.; Kellam, B. Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-sindacene-labeled fluorescent ligands for human beta-adrenoceptors. J. Med. Chem. 2011, 54, 6874-6887.


35


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 58

47. Vernall, A. J.; Stoddart, L. A.; Briddon, S. J.; Ng, H. W.; Laughton, C. A.; Doughty, S. W.; Hill, S. J.; Kellam, B. Conversion of a non-selective adenosine receptor antagonist into A3selective high affinity fluorescent probes using peptide-based linkers. Org. Biomol. Chem. 2013, 11, 5673-5682. 48. Alonso, D.; Vázquez-Villa, H.; Gamo, A. M.; Martínez-Esperón, M. F.; Tortosa, M.; Viso, A.; Fernández de la Pradilla, R.; Junquera, E.; Aicart, E.; Martín-Fontecha, M.; Benhamú, B.; López-Rodríguez, M. L.; Ortega-Gutiérrez, S. Development of fluorescent ligands for the human 5-HT1A receptor. ACS Med. Chem. Lett. 2010, 1, 249-253. 49. Vazquez-Villa, H.; Gonzalez-Vera, J. A.; Benhamu, B.; Viso, A.; Fernandez de la Pradilla, R.; Junquera, E.; Aicart, E.; Lopez-Rodriguez, M. L.; Ortega-Gutierrez, S. Development of molecular probes for the human 5-HT6 receptor. J. Med. Chem. 2010, 53, 7095-7106. 50. Guy, J.; Caron, K.; Dufresne, S.; Michnick, S. W.; Skene, W.; Keillor, J. W. Convergent preparation and photophysical characterization of dimaleimide dansyl fluorogens: elucidation of the maleimide fluorescence quenching mechanism. J. Am. Chem. Soc. 2007, 129, 11969-11977. 51. Wang, X.; Zhang, S. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3. PLoS One 2011, 6, e23076. 52. Tardieu, J. L. Shedding a new light on GPCRs. Drug Discovery & Development. September 13, 2010. 53. Giepmans, B. N.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 2006, 312, 217-224. 54. Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 2003, 21, 86-89.


36

Page 37 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55. Zwier, J. M.; Roux, T.; Cottet, M.; Durroux, T.; Douzon, S.; Bdioui, S.; Gregor, N.; Bourrier, E.; Oueslati, N.; Nicolas, L.; Tinel, N.; Boisseau, C.; Yverneau, P.; Charrier-Savournin, F.; Fink, M.; Trinquet, E. A fluorescent ligand-binding alternative using Tag-lite(R) technology. J. Biomol. Screen. 2010, 15, 1248-1259. 56. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 2010, 110, 2620-2640. 57. Milligan, G. Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein. Br. J. Pharmacol. 1999, 128, 501-510. 58. Palmer, E.; Freeman, T. Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays. Comp. Funct. Genom. 2004, 5, 342-353. 59. Sen, S.; Jaakola, V. P.; Heimo, H.; Engstrom, M.; Larjomaa, P.; Scheinin, M.; Lundstrom, K.; Goldman, A. Functional expression and direct visualization of the human alpha 2B-adrenergic receptor and alpha 2B-AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors. Protein Expres. Purif. 2003, 32, 265-275. 60. Sen, S.; Jaakola, V. P.; Pirila, P.; Finel, M.; Goldman, A. Functional studies with membrane-bound and detergent-solubilized alpha2-adrenergic receptors expressed in Sf 9 cells. BBA-Biomembranes 2005, 1712, 62-70. 61. Skretas, G.; Georgiou, G. Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor. Biotechnol. Bioeng. 2009, 102, 357-367.


37


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Page 38 of 58

62. Yangthara, B.; Mills, A.; Chatsudthipong, V.; Tradtrantip, L.; Verkman, A. S. Smallmolecule vasopressin-2 receptor antagonist identified by a g-protein coupled receptor "pathway" screen. Mol. Pharmacol. 2007, 72, 86-94. 63. Vidi, P. A.; Ejendal, K. F.; Przybyla, J. A.; Watts, V. J. Fluorescent protein complementation assays: new tools to study G protein-coupled receptor oligomerization and GPCR-mediated signaling. Mol. Cell. Endocrinol. 2011, 331, 185-193. 64. Vidi, P. A.; Chemel, B. R.; Hu, C. D.; Watts, V. J. Ligand-dependent oligomerization of dopamine D2 and adenosine A2A receptors in living neuronal cells. Mol. Pharmacol. 2008, 74, 544-551. 65. Lata, S.; Gavutis, M.; Tampé, R.; Piehler, J. Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex F formation. J. Am. Chem. Soc. 2006, 128, 2365-2372. 66. Nonaka, H.; Fujishima, S.-h.; Uchinomiya, S.-h.; Ojida, A.; Hamachi, I. Selective covalent labeling of tag-fused GPCR proteins on live cell surface with a synthetic probe for their functional analysis. J. Am. Chem. Soc. 2010, 132, 9301-9309. 67. Fujishima, S. H.; Nonaka, H.; Uchinomiya, S. H.; Kawase, Y. A.; Ojida, A.; Hamachi, I. Design of a multinuclear Zn(II) complex as a new molecular probe for fluorescence imaging of His-tag fused proteins. Chem. Commun. (Camb) 2012, 48, 594-596. 68. Callis, P. R.; Vivian, J. T. Understanding the variable fluorescence quantum yield of tryptophan in proteins using QM-MM simulations. Quenching by charge transfer to the peptide backbone. Chem. Phys. Lett. 2003, 369, 409-414.


38

Page 39 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


69. Siderius, M.; Wal, M.; Scholten, D. J.; Smit, M. J.; Sakmar, T. P.; Leurs, R.; de Graaf, C. Unnatural amino acids for the study of chemokine receptor structure and dynamics. Drug Discovery Today: Technologies 2012, 9, e301-e313. 70. Torrice, M. M.; Bower, K. S.; Lester, H. A.; Dougherty, D. A. Probing the role of the cation-pi interaction in the binding sites of GPCRs using unnatural amino acids. P. Natl. Acad. Sci. USA. 2009, 106, 11919-11924. 71. Pantoja, R.; Rodriguez, E. A.; Dibas, M. I.; Dougherty, D. A.; Lester, H. A. Singlemolecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors. Biophys. J. 2009, 96, 226-237. 72. Ye, S.; Kohrer, C.; Huber, T.; Kazmi, M.; Sachdev, P.; Yan, E. C.; Bhagat, A.; RajBhandary, U. L.; Sakmar, T. P. Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J. Biol. Chem. 2008, 283, 1525-1533. 73. Daggett, K. A.; Sakmar, T. P. Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors. Curr. Opin. Chem. Biol. 2011, 15, 392-398. 74. Wang, Y.; Chen, L. Quantum dots, lighting up the research and development of nanomedicine. Nanomedicine-UK. 2011, 7, 385-402. 75. Zhou, M.; Nakatani, E.; Gronenberg, L. S.; Tokimoto, T.; Wirth, M. J.; Hruby, V. J.; Roberts, A.; Lynch, R. M.; Ghosh, I. Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules. Bioconjugate Chem. 2007, 18, 323-332. 76. Das, A.; Sanjayan, G. J.; Kecskes, M.; Yoo, L.; Gao, Z. G.; Jacobson, K. A. Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists. J. Nanobiotecg. 2010, 8, 11.


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 58

77. Lee, J.; Kwon, Y. J.; Choi, Y.; Kim, H. C.; Kim, K.; Kim, J.; Park, S.; Song, R. Quantum dot-based screening system for discovery of G protein-coupled receptor agonists. Chembiochem 2012, 13, 1503-1508. 78. Barroso, M. M. Quantum dots in cell biology. J. Histochem. Cytochem. 2011, 59, 237251. 79. Vázquez, M. E.; Blanco, J. B.; Salvadori, S.; Trapella, C.; Argazzi, R.; Bryant, S. D.; Jinsmaa, Y.; Lazarus, L. H.; Negri, L.; Giannini, E.; Lattanzi, R.; Colucci, M.; Balboni, G. 6N,N-dimethylamino-2,3-naphthalimide: A new environment-sensitive fluorescent probe in δand µ-selective opioid peptides. J. Med. Chem. 2006, 49, 3653-3658. 80. Zhuang, Y. D.; Chiang, P. Y.; Wang, C. W.; Tan, K. T. Environment-sensitive fluorescent turn-on probes targeting hydrophobic ligand-binding domains for selective protein detection. Angew. Chem. Int. Ed. Engl. 2013, 52, 8124-8128. 81. Liu, Z.; Du, L.; Li, M. Fluorescence triggered by ligand-protein hydrophobic interaction. Sci. China. Chem. 2013, 56, 1667-1670. 82. Jemielity, J.; Lukaszewicz, M.; Kowalska, J.; Czarnecki, J.; Zuberek, J.; Darzynkiewicz, E. Synthesis of biotin labelled cap analogue-incorporable into mRNA transcripts and promoting cap-dependent translation. Org. Biomol. Chem. 2012, 10, 8570-8574. 83. Thomas, N. R.; Yong-qing, Y.; Drewe, W. C. Biomolecular labelling using multifunctional biotin analogues. In US Patent 20120083599: 2012. 84. Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell. Biol. 2002, 3, 906-918. 85. Cooper, T. Fluorescent assay technologies for G-protein interactions. Ph.D. Thesis, University of Adelaide, 2009.


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Page 41 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


86. Ziegler, N.; Batz, J.; Zabel, U.; Lohse, M. J.; Hoffmann, C. FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors. Bioorg. Med. Chem. 2011, 19, 1048-1054. 87. Dumat, B.; Bordeau, G.; Aranda, A. I.; Mahuteau-Betzer, F.; El Harfouch, Y.; Metge, G.; Charra, F.; Fiorini-Debuisschert, C.; Teulade-Fichou, M. P. Vinyl-triphenylamine dyes, a new family of switchable fluorescent probes for targeted two-photon cellular imaging: from DNA to protein labeling. Org. Biomol. Chem. 2012, 10, 6054-6061. 88. Mizusawa, K. I., Y.; Takaoka, Y.; Miyagawa, M.; Tsukiji, S.; Hamachi I.;. Disassemblydriven turn-on fluorescent nanoprobes for selective protein detection. J. Am. Chem. Soc. 2010,, 132, 7291–7293. 89. Zhang, W.; Ma, Z.; Du, L.; Li, M. Design strategy for photoinduced electron transferbased small-molecule fluorescent probes of biomacromolecules. Analyst 2014, 139, 2641-2649. 90. Sparano, B. A.; Koide, K. A strategy for the development of small-molecule-based sensors that strongly fluoresce when bound to a specific RNA. J. Am. Chem. Soc. 2005, 127, 14954-14955. 91. Ogawa, M.; Kosaka, N.; Longmire, M. R.; Urano, Y.; Choyke, P. L.; Kobayashi, H. Fluorophore-quencher based activatable targeted optical probes for detecting in vivo cancer metastases. Mol. Pharm. 2009, 6, 386-395. 92. Maurel, D.; Banala, S.; Laroche, T.; Johnsson, K. Photoactivatable and photoconvertible fluorescent probes for protein labeling. ACS Chem. Biol. 2010, 5, 507-516. 93. Sarkar, D.; Mallick, A.; Haldar, B.; Chattopadhyay, N. Ratiometric spectroscopic response of pH sensitive probes: An alternative strategy for multidimensional sensing. Chem. Phys. Lett. 2010, 484, 168-172.


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94. Masharina, A.; Reymond, L.; Maurel, D.; Umezawa, K.; Johnsson, K. A fluorescent sensor for GABA and synthetic GABAB receptor ligands. J. Am. Chem. Soc. 2012, 134, 1902619034. 95. Ma, Z.; Du, L.; Li, M. Lighting up GPCRs with a fluorescent multiprobe dubbed "Snifit". Chembiochem 2013, 14, 184-186. 96. Frei, A. P.; Jeon, O. Y.; Kilcher, S.; Moest, H.; Henning, L. M.; Jost, C.; Pluckthun, A.; Mercer, J.; Aebersold, R.; Carreira, E. M.; Wollscheid, B. Direct identification of ligand-receptor interactions on living cells and tissues. Nat. Biotechnol. 2012, 30, 997-1001. 97. Slavoff, S. A.; Saghatelian, A. Discovering ligand-receptor interactions. Nat. Biotechnol. 2012, 30, 959-961. 98. Jain, A.; Liu, R.; Ramani, B.; Arauz, E.; Ishitsuka, Y.; Ragunathan, K.; Park, J.; Chen, J.; Xiang, Y. K.; Ha, T. Probing cellular protein complexes using single-molecule pull-down. Nature 2011, 473, 484-488.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


= Flouorophore

probe 1 =Pharmacophore

=Linker

GPCRs

probe 2

GPCRs

Figure 1. Visualization of GPCRs with probes based on ligands


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Figure 2. Design of fluorophore-labeled 3. The residues in the red area are critical to CXCR4 binding activity (adapted from Ref.20). Fluorophore is shown as blue spheres.


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Figure 3. The study of GPCRs using antibodies. (A) Recognition of GPCRs with a fluorescent antibody;(B) The “competition assays for screening antibody; (C) The “sandwich” assays with a second probe.


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O

S SNAP

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N N H

S SNAP

N N

NH 2

O N N H

N N

NH 2

Figure 4. The covalent labeling reaction used in SNAP-Tag technology. The fluorophore is introduced into the fusion protein of GPCR-SNAP through the nucleophilic substitution in vitro.


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Figure 5. The use of FP in the study of GPCRs. (A) GPCRs could be labeled with GFP when products encoded by recombinant plasmid are expressed on the cell surface. (B) Principle of BiFC to monitor GPCR interactions. (C) Principle of the assay, showing GFP quenching after V2R binding of vasopressin.


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Figure 6. Principle of recognition of His-tagged GPCRs with Ni(II)-NTA.


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hydrophobic binding site SBD Ligand

A

N

N

O N

ligand-protein binding

O S N

H N

H N

O N O S N O

O Fluorescent proteinligand complex

Protein

SBD-Ligand

FRET CLIP

AP N S

CLIP

SNAP

B ligand

GPCR

GPCR

β2 -AR β2 -AR

YFP- β 2-AR YFP

YFP

C Anti-YFP

Anti-YFP YFP- β 2-AR

Figure 7. (A) The strategy to design the fluorescent turn-on probes for protein of interest based on the environment-sensitive fluorophore, SBD. Fluorescence of SBD-ligand can be released after ligand-protein binding. (B) The mode of a Snifit. When a ligand competes with the tethered antagonist, the Snifit moves into an open state, and FRET happens. (C) A GPCR, β2-AR, was pulled down by SiMPull. YFP serves as the bait protein.


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Figure 8. The structure of 54 (TRICEPS) and LRC work flow. “a” was used to purify the receptor peptides for identification by quantitative mass spectrometry; “b” binds glycosylated receptors on living cells; “c” binds ligands of interest. In the work flow, the mild oxidant sodium metaperiodate oxidizes carbohydrates on the cell surface to aldehydes. Afterward, the 54-ligand recognizes the receptor by ligand-receptor binding firstly, and then a covalent bond between the receptor protein and 54-ligand. The dual binding complex through both covalent bond and ligand-receptor interaction can be identified by quantitative mass spectrometry analysis identifies peptides.


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OH O COOH O S HN NH GWTLNSAGYLLQPHAJDNHRSFAOKHGLT-amide

Glanin

O3S

1 (fluorescein-N-galanin) O

PDNPGEDAPAEDLARYSALYINLITRQRY

N 5

NH K SPY

O NPY N 2 (Dy630-Lys-NPY)

Scheme 1. Examples of probe with fluorophores (green) conjugating to peptides (black) directly.


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SO3 H SO 3H O3 S

A

N

N O

O N H

N

N

N

N H

N

SO3 H

Cl

Cl

O H N

27 (NIR-mbc94)

26 (SR-144528) B

C

O

NO 2 O

N

28 (JWH-015)

O N H

30

N

N N O

N NH

O N

O

29

NO 2 O

N NH

NH

Substitute

N

O

N O N

O

N

NH

O O

N

31

Scheme 2. Structures of CB2 ligands and probes. (A) 26-based probe, 27, shows high affinity to CB2; Three parts of probes are highlighted by different colors: fluorophore (red), linker (green), pharmacophore (blue); (B) 28-based probe, 29, shows low affinity to CB2 with the pharmacophore (blue), linker (black), fluorophore (red); (C) The section in the red sphere (NBD) replaces the section in the black sphere of 30 to obtain 31, which has high affinity to CB2.


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Ds HN n

N

n

N

N

N Ar

34

Ds NH 5 n

N

O N

N

N Ar

35: n=4, Ar=2-MeO- C 6 H4 36 : n=4, Ar=1-Naphthyl 37: n=7, Ar=2-MeO- C6 H4 38: n=7, Ar=1-Naphthyl

O

HN

N

N

O

N O

O N

Fluorophore

O N

N

N Ar

O N Ar

39: n=2, Ar=2-MeO- C6 H4 40: n=4, Ar=2-MeO- C6 H4

O 32 (UCM-310590) Ar=2-MeO-C 6 H 4 33 (UCM-2550) Ar=1-Naphthyl

Z O N

N

SAR 36

Y X

O

O N O

N

42: X= SO2 NMe 2 , Y=H, Z=H 43 : X=H, Y=SO2 NMe 2, Z=H 44: X=H, Y=H, Z= SO2 NMe 2

o re

N

op h

N

N

N

Fluo r

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O

41 N

N

N O

N N S O O

45

Scheme 3. Development of fluorescent ligands for the human 5-HT1A receptor. Compound 36 is suitable for imaging 5-HT1A receptors in cellulo.


53


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Page 54 of 58

O

Cl

NH H

HN O S

S

H N

N

6

N

H

N H

S

O O OMe 51 (Biotin-tagged compound) Cl

Cl

S

c

NH

H N

H N

N

S O O

S

Substitute 46 (SB-271046)

Fail

S O O

OMe

NH N

b OMe

Fail

Cl

N

NH

H N

N

S O O

high affinity nonfluorescent

N

H N

S

N

S O O

47 : n=4; 48: n=8

N

H N

S O O 50

N N

4

N H

nN

Ds

H

OMe

OMe 49

a Fail

low affinity fluorescent

Ds

OMe high affinity fluorescent

Scheme 4. Development of fluorescent ligands for the human 5-HT6 receptor. Any attempt to modify 46 at “a”, “b”, and “c” using fluorophore failed because all final compounds lost the affinity or fluorescence. Compound 49 showed high affinity but weak fluorescence, and compound 47 and 48 did inversely. Compound 50 realizing the balance between the affinity and the fluorescence became an effective probe. Compound 51 is a biotin-tagged compound at “a”.


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Scheme 5. Some representative fluorescent amino acids: 52, 53, AcF (red oval). Compound 52 can emit fluorescence itself; the fluorescence of 53 comes from the section of BODIPYFL, and the reaction between the ketone derivatives and the hydrazine derivative here makes the fluorophore link to GPCRs.


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Table 1. Sequences and receptor affinity of labeled T140 analogues.19

Compound

R1

D-Xaa

R2

IC50[nM]a

3 (Ac-TZ14011)

Ac

D-Lys

H

5.2±0.1

4

Ac

D-Glu

H

6.7±2.6

5

fluorescein

D-Lys

H

24±0.3

6

fluorescein

D-Glu

H

199±26

7

Alexa Fluor 488

D-Glu

H

5700±769

8

Ac

D-Lys

Fluorescein

16±0.8

9

Ac

D-Lys

fluorescein-Acp-

26±2.4

10

Ac

D-Lys

biotin-Acp-

11±0.1

11

Ac

D-Lys

Alexa Fluor 488

8.1±3.5

Ac D-Lys AlexaFluor488-Acp- 267±19 12 [a] IC50 values for the peptides are based on the inhibition of [125I]SDF-1binding to CHO cells that were transfected with CXCR4.


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Table 2. Chemical structures and affinity of fluorescent ligands to NK2 receptor.21 Peptide

Structure

pKi[b]

15

GR94800

PhCO-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2

9.81±0.07

16

ANT-1

PhCO-Dab(γ-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2

8.87±0.11

17

ANT-2

PhCO-Orn(δ-BD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2

8.84±0.07

18

ANT-3

PhCO-Lys(ε-NBD)-Ala-D-Trp-Phe-D-Phe-Pro-Nle-NH2

8.83±0.06

19

ANT-4

PhCO-Lys(ε-GlyNBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2

8.80±0,03

20

ANT-5

PhCO-Lys(ε-ahNBD)-Ala-D.Trp-Phe-D-Pro-Pro-Nle-NH2

8.62±0.17

21

ANT-6

PhCO-Lys(ε-bahNBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2

8.32±0.24

22

NKA

His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2

8.92±0.04

23

AGO-1

N-α(NBD) Asp-Ser-Phe.Val-Gly-Leu-Nle-NH2

8.08±0.09

24

AGO-2

N-a(NBD)His-Lyr.Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2

8.23±0.01

25

AGO-3

Ac-Arp-Ser-Phe-Dap(β-NBD)-Gly-Leu-Nle-NH2

5.71±0.05

Compound

3

[b] Competition for [ H]GR100679 binding in CHO/T cells. Data are mean ± SE


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Page 58 of 58

Table of Contents (TOC) Graphics

ligand-based antibody-based

SNAP-based

new strategy-based

Fluorescent probes for GPCRs

FP-based GFP

His-based

amino acid-based


58

Toward Fluorescent Probes for G-Protein-Coupled Receptors (GPCRs

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