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

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155Sixteenth Street N.W., Washington, DC 20036Published by American Chemical Society. Copyright © American Chemical Society.However, no copyright claim is made to original U.S. Government works, or worksproduced by employees of any Commonwealth realm Crown government in the courseof their duties.

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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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 C-

terminus. 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-2,1,3-

benzoxadiazole (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 NBD-

Cl 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-(2-

thienyl)-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 N-

terminus 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-Lys-

NPY, 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 N-

terminus 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 C-

terminus 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-Gly-

His-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 small-

molecule 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 mepyramine-

BODIPY630/650 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-purin-

8-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-difluoro-

4,4a-dihydro-5-(thiophen-2-yl)-4-bora-3a,4a-diaza- s-indacene-3-

yl)vinyl)phenoxy)acetamido)hexan-amido)butylamino)-9 H-purin-9-yl)tetrahydrofuran-2-

carboxamide (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 ligand-

human 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 gene-

recombination 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 O6-

benzylguanine 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, user-

friendliness 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 27-

kDa 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 AR-

GFP fusion protein enabled them to monitor the expression and subcellular distribution of α2B-

adrenergic 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 turn-

on probes by conjugating another environment-sensitive fluorophore, 4-sulfamonyl-7-

aminobenzoxadiazole (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, biotin-

ligand 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 M5-

acetylcholine 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 turn-

on/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 β2-

adrenergic 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-11-

0306), 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, 5-

hydroxytryptamine; 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, single-

molecule pull-down; PET, photo-induced electron transfer; ICT, intramolecular charge transfer;

ELISA, enzyme linked immunosorbent assay.

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= Flouorophore

=Pharmacophore

=Linker

probe 1

probe 2GPCRs 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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S

N

NN

NH

O

NH2

S

N

NN

NH

O

NH2

SNAP SNAP

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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Anti-YFP

YFP-β2-AR

Anti-YFP

YFP

β2-ARYFP-β2-AR

β2-AR

YFP

NO

N

SHN

O

O

N

NO

N

S

O

O

N

ProteinSBD-Ligand

Ligand

SBD

Fluorescent protein-ligand complex

hydrophobic binding site

ligand-proteinbinding

HN

SNAP

CLIP CLIP

GPCR GPCR

SNAP

FRET

ligand

A

B

C

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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O

COOH

HN

O

OH

GWTLNSAGYLLQPHAJDNHRSFAOKHGLT-amideNH

S

Glanin

PDNPGEDAPAEDLARYSALYINLITRQRY

SPY

K

O

NHN

5

O3S

NPY

O

N

1 (fluorescein-N-galanin)

2 (Dy630-Lys-NPY)

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

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52

Cl

NN

O

NH

ClN

N

O

NH

HN

NHO

N

O3S

SO3H

N

SO3H

SO3H

26 (SR-144528) 27 (NIR-mbc94)

N

O

N

O

NH

O

NHN

O

N

NO2

N

N NH

O

N

O

O

N

N NH

O

O

ON

N O

N

NO2

Substitute

28 (JWH-015) 29 30 31

O

A

B C

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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53

N NN

N Ar

O

O

32 (UCM-310590) Ar=2-MeO-C 6H433 (UCM-2550) Ar=1-Naphthyl

N NN

N Ar

O

O35: n=4, Ar=2-MeO-C6H4

36: n=4, Ar=1-Naphthyl37: n=7, Ar=2-MeO- C6H4

38: n=7, Ar=1-Naphthyl

HN

Ds

n

N NN

N Ar

O

O

39: n=2, Ar=2-MeO- C6H4

40: n=4, Ar=2-MeO- C6H4

HN

n

NH

Ds

5

N NN

N

O

O

N NN

N

O

O

ZY

X

42: X= SO2NMe2, Y=H, Z=H43: X=H, Y=SO2NMe2, Z=H44: X=H, Y=H, Z=SO2NMe2

45

S

O O

N

N NN

N

O

O

N NN

N Ar

O

O

n

Fluorophore

Flu

oro

phore

36SAR

34

41

Scheme 3. Development of fluorescent ligands for the human 5-HT1A receptor. Compound 36 is

suitable for imaging 5-HT1A receptors in cellulo.

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54

N

NH

OMe

HN

SS

Cl

O ON

NH

OMe

HN

SS

Cl

O O

a

b

c

N

N

OMe

HN

SS

Cl

O O

NH

Dsn

46 (SB-271046)

47: n=4; 48: n=8

N

NH

OMe

HN

S

O O

N

49

N

N

OMe

HN

S

O O

N

NH

Ds4

50

NN

OMe

HN

SS

Cl

O O

NH

6

O

S

NHHN

HH

O

51 (Biotin-tagged compound)

Substitute

high affinitynonfluorescent

low affinityfluorescent

high affinityfluorescent

Fail

Fail

Fail

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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55

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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56

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

12 Ac D-Lys AlexaFluor488-Acp- 267±19

[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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57

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

Compound 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

[b] Competition for [3H]GR100679 binding in CHO/T cells. Data are mean ± SE

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58

Table of Contents (TOC) Graphics

GFP

Fluorescentprobesfor GPCRs

ligand-based

antibody-based

SNAP-based

FP-based amino

acid-based

His-based

new strategy-based

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