Small-molecule fluorescent probes for the aminergic GPCRs -- What new tool compounds do we have post-2014 and what can they do?

Aminergic GPCRs are heavily involved in the physiological functions of the human body and are prominent drug targets for numerous diseases. Despite the extensive work that has been done over the years surrounding the aminergic GPCRs, knowledge gaps persist as not all aminergic GPCRs are currently drugged. This gap could potentially be addressed with the use of small molecule fluorescent probes in wide-ranging assay formats and experiments to obtain deeper insights into the molecular pharmacology of these receptors. This review highlights key developments from 2014 to 2024 in the design and application of fluorescent probes targeting the aminergic GPCRs, featuring a total of 37 fluorescent ligands, spanning all prominent aminergic GPCR subfamilies: adrenergic, dopaminergic, histaminergic, muscarinic, and serotonergic receptors. The review provides a detailed overview of the approach towards probe design, their pharmacological profiles as well as reported utilities, mainly in ligand binding and/or imaging studies, and more recently, the ex vivo and in vivo studies of aminergic GPCRs in both mice and human tissue models. With the integration of cutting-edge image processing and recognition technologies, fluorescent probes will continue to be indispensable tools for exploring the molecular pharmacology of aminergic GPCRs, playing a crucial role in driving forward drug discovery in this field. Small-molecule fluorescent probes for the aminergic GPCRs – What new tool compounds do we have post-2014 and what can they do? Christopher Zi Qian Choo 1, Joey Yun Xuan Ching 1, Myra Mazhar Ud Deen 1, Zhi Yuan Kok 1 1 School of Pharmacy, University of Nottingham Malaysia, Semenyih, Malaysia Correspondence Zhi Yuan Kok, School of Pharmacy, University of Nottingham Malaysia, Semenyih, Malaysia. Email: [email protected] Funding information This study was financially supported by the Ministry of Higher Education Malaysia (Grant Number FRGS/1/2024/STG01/UNIM/02/1)

Aminergic GPCRs are heavily involved in the physiological functions of the human body and are prominent drug targets for numerous diseases. Despite the extensive work that has been done over the years surrounding the aminergic GPCRs, knowledge gaps persist as not all aminergic GPCRs are currently drugged. This gap could potentially be addressed with the use of small molecule fluorescent probes in wide-ranging assay formats and experiments to obtain deeper insights into the molecular pharmacology of these receptors. This review highlights key developments from 2014 to 2024 in the design and application of fluorescent probes targeting the aminergic GPCRs, featuring a total of 37 fluorescent ligands, spanning all prominent aminergic GPCR subfamilies: adrenergic, dopaminergic, histaminergic, muscarinic, and serotonergic receptors. The review provides a detailed overview of the approach towards probe design, their pharmacological profiles as well as reported utilities, mainly in ligand binding and/or imaging studies, and more recently, the ex vivo and in vivo studies of aminergic GPCRs in both mice and human tissue models. With the integration of cutting-edge image processing and recognition technologies, fluorescent probes will continue to be indispensable tools for exploring the molecular pharmacology of aminergic GPCRs, playing a crucial role in driving forward drug discovery in this field. Word count: 7992 words (exclude abstract, reference, Table 1-5)

G protein-coupled receptors (GPCRs) represent one of the largest and most diverse family of membrane receptors in the human genome. Their central role in physiological regulation makes them highly relevant drug targets, with approximately one-third of all marketed drugs acting on GPCRs (Santos et al. , 2017) and till date remain an attractive target for drug discovery (Lorente et al., 2025). Among the GPCR superfamily, Class A aminergic GPCRs are prominent drug targets as they underpin the therapeutic action of a wide array of long-established drugs currently in clinical use i.e. beta-blockers for cardiovascular diseases, antihistamines for allergic conditions, antipsychotics for schizophrenia, bronchodilators for respiratory disorders and more. Despite the extensive studies that have been done on aminergic GPCRs, knowledge gaps persist. Drug discovery efforts involving several aminergic receptors as drug targets have proved to be challenging. For instance, the lack of clinical efficacy in clinical trials (Kollmeier et al., 2018a, Kollmeier et al., 2018b, Werfel et al., 2019) means that the histamine H 4 receptor (H 4 R) remain undrugged despite its clear involvement in immunomodulation (Panula et al., 2015). Additionally, the development of ligands for the muscarinic receptor that may have clinical application have been severely hindered by the lack of subtype selectivity due to the absolute conservation of the orthosteric binding pocket, as well as on-target side effects because of the pleiotropy of signal transduction involving the muscarinic receptor subfamily (Kaoullas et al., 2024). These shortfalls highlight the need for deeper insights into the pharmacology of these receptors, especially allosterism (Conflitti et al., 2025) and biased signaling for drug discovery (Kenakin, 2019). This could be achieved by novel, investigative pharmacological tools such as small molecule fluorescent probes (fluorescent ligands), typically consist of a receptor binding motif (orthosteric/allosteric) covalently linked, often via a flexible linker, to a fluorophore (wide range of selection commercially available). The wide-ranging applications of fluorescent ligands are extensively reported and summarized, providing valuable insight into ligand affinity, ligand binding kinetics and receptor signaling via fluorescent based binding assays (Soave et al., 2020; Hill et al., 2023), as well as receptor localization, expression and organization via advanced imaging techniques such as fluorescence microscopy and fluorescence correlation spectroscopy (Zhang et al., 2021; Wu et al., 2023). Much is known about the design of fluorescent ligands over the years, which mainly revolve around two key principles: a) they must be treated as individual pharmacological entities and not an extension of the parent compound (Vernall et al., 2014); b) both the fluorophore and linker have significant influence on the pharmacological properties of the final fluorescent conjugate (Baker et al., 2010a). For fluorescent ligands to have broad utility, the fluorescence and physicochemical properties of the final fluorescent conjugate are equally important to maximize signal-to-noise ratio in the various experiments they are used in. Red-shifted fluorophores that possess high quantum yield and photostable are often preferred as their strong and stable emission in the red-light region prevents spectral overlap, signal bleed-through during experiments (Goulding et al. 2021) and cell auto-fluorescence, thus improving signal-to-noise ratio and data quality, as well as enabling in vivo imaging applications for many near-infrared shifted fluorophores (Ling et al., 2015; Ma et al., 2016; Wang et al., 2024). The physicochemical properties of the final fluorescent conjugate could be modulated by the fluorophore and linker choices, which could be hydrophilic or lipophilic. Generally, the lipophilicity of the final fluorescent probe should be kept low to minimize non-specific binding and cellular uptake of the probe which can otherwise confound experimental results (Rose et al., 2012). The development of effective fluorescent probes can be realized by considering these different properties in the initial design stage and striving to achieve an optimal balance between them throughout the developmental process. Since the initial development of fluorescent ligands for GPCRs, numerous review articles have chronicled achievements in probe design and applications (Vernall et al., 2014; Iliopoulos-Tsoutsouvas et al., 2018; Conner et al., 2021; Szabo et al., 2025). More importantly, accessibility to these compounds has improved, as many are commercially available through vendors such as HelloBio (https://hellobio.com/) and Celtarys (https://www.celtarys.com/). These have since been successfully employed in diverse pharmacological studies (Kok et al., 2022; Tahk et al., 2023). As the body of knowledge surrounding GPCR fluorescent ligands continues to grow, this review aims to provide a focused and comprehensive overview of the aminergic GPCR fluorescent ligands reported between 2014 to 2024, their key pharmacological profiles and reported application. This review aspires to be a convenient ‘catalogue’ of reported, non-commercially available aminergic GPCR fluorescent ligands for pharmacologists and clinical researchers seeking to leverage these tool compounds for their research and potential collaboration. It is worth noting that the emergence of new innovative approaches such as ligand-directed labelling (Moss et al., 2014; Arttamangkul et al., 2019; Stoddart et al., 2020; Comeo et al., 2024) and photo-switchable ligands (Gomez-Santacana et al., 2018; Hauwert et al., 2018; Wirth et al., 2023; Bérenger et al., 2025) in recent years offer exciting new avenues for receptor labelling, manipulation and visualization, these however, fall outside the scope of this article, which will only cover classical fluorescent ligands. Fluorescent Probes for Aminergic GPCRs Over the past decade, substantial advancements have been made in the development of fluorescent probes targeting aminergic G protein-coupled receptors (GPCRs), encompassing key receptor families such as the adrenergic, dopaminergic, histamine, muscarinic, and serotonin receptors. Over 30 peer-reviewed publications on the development of new aminergic GPCR fluorescent probes have been identified, predominantly concentrated on adrenergic and dopaminergic receptors, which together account for more than half of the reported publications. These studies shed light on what are likely the first confirmed target-selective fluorescent probes for the β 3 -adrenoceptor (β 3 -AR) (Li et al., 2023), dopamine D 1 receptor (D 1 R) (Rosier et al. 2023), dopamine D 2 receptor (D 2 R) (Hounsou et al., 2014), dopamine D 3 receptor (D 3 R) (Allikalt et al., 2020), histamine H 1 receptor (H 1 R) (Kok et al., 2022), muscarinic M 2 receptor (M 2 R) (She et al., 2020; Gruber et al., 2020) and serotonin 5-HT 2B receptor (Azuaje et al., 2017), which are highly valuable tool compounds especially for studying GPCR pharmacology in native tissue and receptor expression systems. These studies collectively demonstrate that fluorescent probes are powerful tools for real-time receptor visualization on live cells, pharmacological profiling and high-throughput screening for potential drug compounds, as well as in vivo studies on drug-target engagement and tumor imaging in mice, highlighting their potential to advance our understanding of GPCR signaling and support ongoing drug discovery efforts. Adrenergic Receptor Zhang et al. (2015) synthesized a series of fluorescent compounds for the α 1 -adrenoceptor (α 1 -AR) based on a prazosin-derived quinazoline core. All compounds exhibited nanomolar affinities at all three α 1 -AR subtypes (α 1A -, α 1B -, and α 1D -AR) in a radioligand binding assay and 1 was an exceptional non-selective binder ( K i <1 nM) across all three α 1 -AR subtypes studied (Table 1). It was found that the presence of the carbonyl group in the amide moiety gives approximately 100-fold more potency as seen in 1 . The authors concluded that there is space, albeit limited, around the piperazine group of the orthostere to accommodate the fluorophore, which explains why the affinity remains unchanged after fluorophore conjugation. Despite the moderate quantum yield of the compounds, 1 and 2 could label α 1A - and α 1D -AR transfected HEK293A cells at concentrations of below 100 nM with clear membrane localization, which may suggest receptor antagonism (Table 1). Their fluorescence was significantly attenuated by large excess of the unlabeled α 1 -AR antagonist tamsulosin, confirming that the observed binding was specific and reversible. Additionally, compound 2 with its superior fluorescence properties within the series was also able to effectively label PC-3 prostate cancer cells at 1 μM, with the signal displaceable by 10 μM doxazosin. 2 also had minimal signal in HepG2 cells which are known to have low α 1 -AR expression, validating its use as a labelling tool for α 1 -AR overexpressing cells. Ma et al. (2016a) reported on a series of fluorescent compounds that target the α 1 -AR, comprising of the same quinazoline core reported in Zhang et al. (2015) conjugated to the Cyanine5 fluorophore via linkers of two to four carbon lengths via copper-catalyzed azide-alkyne cycloaddition (CuAAC). All six compounds showed nanomolar binding affinities in a radioligand binding assay despite being 20-fold less potent than their parent compound prazosin. Compounds which had acylated carbon chain linkers showed higher affinities at all three α 1 -AR subtypes, further confirming the importance of the carbonyl group for α 1 -AR binding (Zhang et al., 2015). Amongst all compounds, 3 exhibited the highest binding affinity at all three α 1 -AR subtypes and was subsequently selected for cell imaging studies (Table 1). Compound 3 was found to be effective in visualizing α 1 -ARs in transfected HEK293 cells as well as selected cancer cell lines PC-3 and DU145 which are known to express α 1 -ARs at high levels (Shi et al., 2007), whilst being displaceable by excess unlabeled ligand doxazosin. The authors explored the utility of 3 in in- and ex-vivo imaging, which confirmed that 3 could serve as a probe for the selective labelling of tumors expressing the α 1 -AR as well as tissue-specific distribution of the receptor subtypes in live mice imaging. The authors reported a significant decrease in fluorescence intensity in murine organs with co-administration of excess unlabeled ligand prazosin, indicating that the observed fluorescent signal was indeed attributed to the α 1 -AR expression. Finally, the authors proceeded to use 3 to image human prostate sections, where a 24-hour incubation with 3 at 300 nM resulted in stronger fluorescence in human prostate cancer and benign prostatic hyperplasia (BPH) sections compared to normal prostate slices. Notably, a 30-minute incubation was sufficient to differentiate low-level cancer tissues from the surrounding normal tissues, suggesting favorable ligand binding kinetics. In a separate paper from the same group, Ma et al. (2016b) shed light on a new series of α 1 -AR antagonist compounds with mono- or bis-γ-aminobutyric acid (GABA) peptide linkers designed based on their previous probes (Zhang et al., 2015; Ma et al., 2016a) . The bis-GABA bearing 4 had nanomolar binding affinities in radioligand binding assay at all three α 1 -AR subtypes, a high quantum yield and was at least 1000-fold selective for α 1 -AR over α 2 - or β 1 -AR (Table 1). Compound 4 could label α 1B -AR transfected HEK293A cells at 300 nM, where the fluorescence could be diminished by 3 μM prazosin. The authors proceeded to deploy 4 in a ‘ELISA-like’ Fluorescence Polarization (FP) assay and was able to determine the binding affinities of the probe at the α 1A -, α 1B - and α 1D -AR subtypes. With this, an “ELISA-like” FP-based competitive binding assay was established for high throughput screening (HTS) for α 1 -AR antagonists which included prazosin, doxazosin, and phentolamine, and their binding affinities were relatively close to those obtained in a radioligand binding assay. This new strategy allows for the use of safer fluorescent probes and lower membrane protein consumption membrane proteins (>10-fold lower) for potential future HTS screening efforts. Qin et al. (2019) developed a series of fluorescent agonists for the α 1 -AR by conjugating the solvatochromic 4-chloro-7-nitrobenzoxadiazole (NBD) fluorophore with phenylephrine using alkyl chains of different lengths as the linker. All three compounds possessed micromolar binding affinities for the α 1A and α 1D subtype (<2 μM) determined by competitive radioligand binding assay and 5 was confirmed to be an agonist at α 1B - and α 1D -AR in a calcium mobilization assay (Table 1). After confirming compound biocompatibility, the authors were able to visualize receptor internalization for all three α 1 -AR subtypes studied separately expressed on HEK293A cells using 5, in which fluorescent signal attenuation was achieved in the presence of high concentration of unlabeled ligand tamsulosin. A BRET-based binding assay was also established using 5 (for α 1A - and α 1B -AR) and it was shown that 5 was suitable to be used as a labelled probe HTS of unlabeled α 1 -AR agonists and antagonists. In another similar work by the group, Qin et al. (2021) synthesized a series of fluorescent compounds for the α 1 -AR utilizing the same phenylephrine orthostere and alkyl linkers of varying lengths, conjugated to a different fluorophore which was the 7-(diethylamino)coumarin-3-carboxylic acid. All three compounds showed micromolar binding affinities (<5 μM) for all three α 1 -AR subtypes. In terms of spectroscopic properties, 6 showed the largest increase in fluorescence signal intensity after binding (13-fold) and could label α 1A - and α 1B -AR transfected HEK293 cells at 200 nM, fully displaceable by 3 μM tamsulosin (Table 1). 6 at a concentration of 300 nM could clearly show receptor internalization through the extension of fluorescence into cytoplasm, with decreased intensity after a few minutes. A BRET-based competitive binding assay for the all three subtypes of α 1 -AR was also developed using 6, which has been proven it suitable for high throughput screening of unlabeled α 1 -AR agonists and antagonists. Mitronova et al. (2017) synthesized multiple fluorescent compounds targeting the β 2 -AR, utilizing either BI-167107 (agonist) or carazolol (antagonist) as the orthostere, conjugated to various fluorophores via PEG or alkyl linkers. Results from Fluorescence Resonance Energy Transfer sensor-enabled cyclic adenosine monophosphate (FRET-cAMP) assay using β 2 -AR expressing HEK 293 cells showed that BI-167107-derived compounds (including 7 ) retained agonistic function and had potencies within the nano- to sub-nanomolar range (<10 nM) while the only carazolol-based probe ( 8 ) was an antagonist with a binding affinity ( K i app ) of 162 nM (Table 1). It was interesting to note that binding affinity improved to 29 nM for 8 if measurements were taken 30 minutes after ligand addition, which the authors attributed to complex processes involving receptor desensitization, internalization etc. as previously reported by Baker et al. (2010b). Slow on-rate binding kinetics could also be a potential explanation for this observation, but this cannot be confirmed as binding kinetic studies were not performed. Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) saturation binding assay with 7 (β 2 -AR = 7.2 nM, β 1 -AR = 63.4 nM) and 8 (β 2 -AR = 14.7 nM, β 1 -AR = 48.6 nM) also demonstrated that selectivity for β 2 -AR over β 1 -AR was nine-fold and three-fold respectively. Imaging with confocal microscopy using U2OS cells expressing β 2 -AR-YFP fusion protein showed that 7 and 8 could effectively label the cells at a concentration of 100 nM, with monitoring of receptor internalization possible for the agonist 7 through visible punctate staining inside the cells whereas 8 was fully localized at the cell membrane. Compounds 7 and 8 were fully displaceable by high concentration of their original unlabeled parent ligand, inferring minimal non-specific binding. The same results were observed in the labelling of endogenous pancreatic CAPAN-1 cells with a much lower β 2 -AR expression level, which then allowed the measurement of lateral resolution in Stimulated Emission Depletion (STED) microscopy, in which images for 7 gave a two-fold improvement in lateral optical resolution over confocal microscopy. The fluorescence properties of 7 and 8 did not significantly change with varying pH, thus are resilient probe compounds in various cellular microenvironments. Using a combination of different linkers (PEG or dipeptides) and fluorophores, Goulding et al. (2021) synthesized nine fluorescent ligands based on the β 2 AR antagonist ICI 118551, most of which demonstrated high affinity for β 2 AR ( K D <100 nM) and strong selectivity over the β 1 AR in a NanoBRET saturation binding assay performed on NLuc-β 1 and NLuc-β 2 AR transfected HEK293T cells. Compound 9, one of the most β 2 AR selective ligands, was successfully used as the labelled ligand in a NanoBRET competition binding assay to determine binding affinities of known, unlabeled β-AR ligands, with the values obtained in good agreement with literature values (Table 1). Subsequently, in a confocal imaging experiment involving β 2 AR-transfected HEK293T cells and 9, internalization of fluorescent signal was observed, to which the author proposed it could be either due to co-internalization with the receptor or independent diffusion into the cell. Membrane and cytosolic fluorescence signal were clearly reduced in the presence of high concentration of an unlabeled antagonist, indicating 9 binding specificity. Bioluminescence imaging involving NLuc-β 2 AR transfected HEK293T cells yielded similar results. Finally, the authors employed CRISPR/Cas9 genome editing to tag endogenously expressed β 2 ARs in HEK293T cells with NLuc that afforded a cell line with low endogenous expression levels of β 2 AR and repeated the saturation, binding and luminescence microscopy experiments using 9 . The results confirmed that utility of 9 remain preserved even in cells with native expression levels of β 2 AR, highlighting its potential application to study native cell lines. Li et al. (2022) reported a series of fluorescent compounds for the β-AR with a focus on compound 10, featuring mirabegron, a β 3 -AR agonist as the orthostere bound to a pyridinium (Py) fluorophore through a simple alkyl amide linker (Table 1). Fluorescent compounds based on coumarin and NBD fluorophores achieved via direct conjugation or via the same alkyl amide linker were also synthesized. Compound 10 had sub-micromolar binding affinities (100-350 nM) across all three β-AR subtypes in a radioligand binding assay with no subtype selectivity observed, in contrast to the β 3 -selective parent compound mirabegron. In cell imaging experiments, compound 10 was successful in the labelling of β-AR-transfected HEK293 cells of all β-AR subtypes at 500 nM, fully displaceable by 40 μM of unlabeled antagonists for the respective β-AR subtypes, indicating binding specificity. The fluorescent signal was mainly localized on the plasma membrane with minimal internalization observed, potentially suggesting that the probe may be an antagonist. Compound 10 was also proven to be applicable in the NanoBRET saturation binding assay, where a good BRET signal quality was obtained due to favorable fluorescence properties of the pyridinium fluorophore, with affinity values obtained comparable to those acquired from radioligand binding assays. To achieve β 3 -selective fluorescent compounds, Li et al. (2023) developed a new series of fluorescent compounds bearing a Py-5 dye based on 10, with a focus on linker optimization. Surprisingly, only the fluorescent compounds with the PEG linker displayed β 3 selectivity, providing further proof that linker SAR does matter in the pharmacological activity of the final fluorescent conjugate. Compound 11 is likely the most affine and selective β 3 -AR selective probe identified till date, displaying nanomolar affinity (10 nM) at β 3 -AR, a 45-fold and 16-fold selectivity for β 3 -AR over β 1 -AR and β 2 -AR respectively (Table 1). The K D of 11 at β 3 -AR was 47 nM in a saturation NanoBRET binding assay. In cellular imaging, 11 demonstrated selective binding to β 3 -AR-transfected HEK293 cells at 100 nM, which was fully displaceable in the presence of excess non-labelled antagonist. The authors attributed this finding to the PEG linker which minimized interaction with lipid membranes and thus reducing non-specific binding as previously proposed by Karpenko et al. (2014). The fluorescent signal was mainly located on the cell surface with limited signal in the cytoplasm, potentially indicating antagonistic activity of the probe. Further investigation on the utility of 11 to visualize β 3 -AR in melanoma cell lines A375 and B16F10 as well as NCI-H1299 and A2780 tumor xenografts indicated that 11 at 200 nM was able to selectively label β 3 -AR expressing tumor tissue slices, in which the fluorescent signal was significantly reduced in the presence of high concentration of the unlabeled antagonist. This provided convincing evidence that the binding of the fluorescent probe to β 3 -AR is specific and that it may be a useful tool in cancer diagnosis. Dopamine Receptor Rosier et al. (2023) reported the synthesis of six fluorescent compounds targeting D 1 -like receptors which consist of the D 1 -receptor (D 1 R) and D 5 -receptor (D 5 R). Fluorophores 5-TAMRA and DY549-P1 were linked to the D 1 R antagonist SCH-23390 via short aliphatic and PEG-based linkers, and it was found that 12 showed nanomolar binding affinities for D 1 R and D 5 R in radioligand binding studies, with K i values of 4.6 nM and 24 nM respectively and confirmed to be a neutral antagonist through a BRET-based G s heterotrimer dissociation assay (Table 2). Compound 12 demonstrated remarkable selectivity, exhibiting over a 1,000-fold and 500-fold preference for D 1 R and D 5 R respectively compared to D 2 R within the D 2 -like receptor subtypes. Experiments involving 12 at final ligand concentration of 50 nM in confocal microscopy revealed fast-on fast-off ligand kinetics at D 1 R, with minimal cellular uptake observed. Hounsou et al. (2014) synthesized six fluorescent derivatives of the D 3 R partial agonist BP897, incorporating different fluorophores and linkers of varying lengths. In a TR-FRET binding assay with Lum4-Tb as the donor performed on SNAP-tagged D 1 -D 5 R expressing HEK293 cells, all six probes retained high affinity for D 3 R ( K D = 1–30 nM) and minimal binding to D 1 R, D 4 R, and D 5 R. The authors observed that the linker and fluorophore had differing influence on the affinity of the final fluorescent compound, which was highly receptor- specific. Compound 13 was selected for use in further studies as it exhibited nanomolar affinities and excellent selectivity for the D 2 R and D 3 R over the other dopamine receptor subtypes (Table 2). This included serving as a labelled ligand in a TR-FRET competitive binding assay, which led to the successful determination of the binding affinities of known unlabeled ligands at both D 2 R and D 3 R that were largely agreeable to K i values obtained from radioligand binding assay in the literature. Using 13 in HEK293 cells co-expressing SNAP-tagged D 1 R and HALOTag-D 3 R, the authors confirmed heterodimer formation between the D 1 R and D 3 R via TR-FRET and determined that D 1 R co-expression does not significantly alter the binding affinity of compound 13 to D 3 R. This was further confirmed via TR-FRET competition binding assays with known D 1 R- and D 3 R-selective ligands as their K i values coincided with those defined for D 3 R, concluding that the receptor interaction between D 3 R and D 1 R had no impact on the affinity of the ligands tested. Tabor et al. (2016) worked on monovalent and divalent fluorescent dopamine receptor compounds to explore ligand-induced dimerization of D 2 R, which comprises both the short (D 2S R) and the long isoforms (D 2L R). Two compounds were found to be of interest: 14 that featured a 1,4-disubstituted phenylpiperazine head group and the cyanine 3B fluorophore connected by an amino acid-based linker, and 15 that was based on (S)-5-OH-DPAT, a strong D 2 R agonist orthostere (Table 2). Both compounds exhibited nanomolar binding affinities for the human D 2S -, D 2L - and D 3 R in radioligand binding studies, with 15 displaying sub-nanomolar affinity ( K i = 0.4 nM) for D 3 R and at least 16-fold selectivity over the D 2 R-subtypes. Under TIRF microscopy, compound 14 was able to label SNAP-D 2L receptors at 38 nM, which presented as individual mobile fluorescent spots on the plasma membrane. Using 14 and 15 separately in TIRF microscopy experiments, the authors found that D 2L R dimerization increased in the presence of the agonist 15, which was also the case in experiments involving the D 2S R and D 3 R subtypes. Allikalt et al. (2020a) shed light on a series of novel fluorescent compounds targeting the D 2 R and D 3 R that included 16 and 17, both of which exhibited high affinities at the D 2L R/D 2S R (<100 nM) and sub-nanomolar affinities at the D 3 R in radioligand binding studies (Table 2). Among the two compounds, 16 is the more D 3 R-selective compound (20-fold selectivity over D 2 R subtypes) whereas 17 is the more affine compound at the D 2L R and D 2S R (<10 nM). Both compounds were confirmed to be D 2S R agonists, proven through their ability to recruit β-arrestin-2 in a functional assay based on enzyme fragment complementation. In TIRF microscopy, 17 produced high-resolution images of D 2S R- and D 3 R-expressing CHO cells as discrete fluorescent spots at concentrations of 10 nM and 1 nM respectively, which was surmountable in the presence of an antagonist. Compound 16 was investigated in NanoBRET assays using both live cells and membrane preparations, where it demonstrated a sub-nanomolar affinity at D 3 R and in close agreement with its binding affinity determined from radioligand binding assays. Subsequently, the authors studied ligand association and dissociation kinetics for 16, where it displayed a concentration-independent dissociation profile with a mean residence time of 19 minutes. Compound 16 was also employed in a NanoBRET competitive binding assay to determine binding affinities of established antipsychotics at the D 3 R, though the values were found to be slightly lower than those obtained by classical radioligand binding. Allikalt et al. (2020b) conjugated the dopaminergic antagonist N-(p-aminophenethyl)spiperone (NAPS) directly to a Cy3B fluorophore, giving rise to 18, that displayed sub-nanomolar affinities for D 3 R ( K i = 0.7 nM) in a radioligand binding assay (Table 2). Compound 18 at 1 nM in fluorescence microscopy enabled real-time observation of ligand-receptor binding in HEK293-D 3 R cells, which was surmountable by an unlabeled antagonist. In a fluorescent microscopy-based saturation binding assay, 18 was determined to be highly affine at D 3 R and was further employed in competitive binding assay format against dopamine (agonist) and butaclamol (antagonist), where the K i values determined were similar to those reported in other studies. Prokop et al. (2021) synthesized a Sulfo-Cy5-tagged cariprazine analogue 19 that retained high affinity and selectivity for D 3 R ( K i = 1.3 nM) in a radioligand binding assay, which was confirmed to be a weak partial agonist at D 3 R in a BRET-based G i1 -activation assay (Table 2). In imaging studies performed using Stochastic Optical Reconstruction Microscopy (STORM), 19 was found to bind exclusively to D 3 R, with minimal off-target binding and displaceable by a selective D 3 R antagonist. Binding was significantly reduced in cells expressing D 2 R and F338A-mutated D 3 R, further confirming binding specificity. The authors proposed that the use of fluorescent ligands provides significant improvement to super-resolution imaging as they rapidly homogenise within live tissue preparations, and overcoming limitations associated with immunolabelling that often produce clusters of localisation points during imaging. Using acute brain slices as well as fresh frozen brain tissues in STORM imaging in the presence of 19, the authors subsequently identified the hilar subregion of the Islands of Calleja (IC) within the tubular striatum as the primary site of D 3 R-dependent cariprazine binding, confirmed by the absent of signal in IC of D 3 R knockout mice. Subsequently, the authors demonstrated that in vivo administration of cariprazine to live mice displaced 19 binding in the IC, specifically the granule cell axons, thus confirming that this region corresponded to real in vivo target engagement site of the drug cariprazine. Elek et al. (2022) reported five bis(BF 2 ) pyridine-based chromophore (BOPPY)-based fluorescent compounds which targeted the D 2 R and D 3 R, with 20 as the most promising as it demonstrated the best balance between fluorescence and pharmacological properties (Table 2). Compound 20 was green shifted, had reasonably high quantum yield, exhibited the highest affinity for the D 3 R ( K i = 22.5 nM) in radioligand binding assay and displayed 15-fold selectivity for D 3 R over D 2 R. Although the utility of 20 was not studied, its selectivity for D 3 R may be useful for use in D 3 R pharmacological research. Nagl et al. (2023) developed a series of fluorescent compounds targeting the D 2 R consisting of a 5-TAMRA or DY549-P1 fluorophore linked to a spiperone scaffold through either a γ-aminobutyric acid (GABA)-based short linker or a longer PEG-based linker. Among them, 21 featuring a GABA linker and the 5-TAMRA fluorophore demonstrated the highest binding affinity across the D 2 -like receptor subtypes (D 2 R, D 3 R and D 4 R), displaying close to 100-fold selectivity over the D 1 -like receptor subtypes (D 1 R and D 5 R) (Table 2). Compound 21 was also confirmed to be a potent antagonist in a BRET-based G o 1 heterotrimer dissociation assay at D 2 R, D 3 R and D 4 R with similar potencies. In confocal microscopy experiments, 21 at ligand concentration of 50 nM showed rapid accumulation at the cell surface of D 2L R-GFP2-transfected HEK293T cells, reflecting fast association kinetics. Histamine Receptor Stoddart et al. (2018) reported six histamine H 1 receptor (H 1 R)-targeting fluorescent compounds based on the orthostere mepyramine or VUF13816 - a small molecule H 1 R binder previously discovered via computational approach (de Graaf et al., 2011). These compounds possessed varying peptide linker composition with BODIPY TM -630/650-X fluorophores. All six compounds demonstrated similar high affinity (<100 nM) across three different assays and have been confirmed to be antagonists in a calcium mobilization assay. It was observed that the binding kinetics of the VUF13816-based compounds indicated slow-off kinetics, with 22 displaying a receptor residence time of close to 1 hour (Table 3). All compounds were used in confocal imaging at a concentration of 50 nM, with the authors reporting clear visualization of membrane localization, which diminished in the presence of high concentrations of unlabeled antagonist mepyramine. There were minimal cellular uptake and non-specific binding observed compared to prior work (Rose et al., 2012), to which the authors attributed the incorporation of hydrophilic peptide linkers to improved physicochemical properties of the final fluorescent compounds. The authors also developed and validated the first NanoBRET assay for the H 1 R to study receptor affinity and binding kinetics using 22 and was successful in determining the binding affinity and kinetics of known unlabeled ligands. Expanding the work of Stoddart et al., Kok et al. (2022) explored peptide-linker SAR to optimize ligand-receptor interaction for the discovery of more affine fluorescent compounds for the H 1 R using 22 as the lead compound. The authors first synthesized and tested several series of unlabeled peptide congeners to study peptide linker SAR, then developed three series of fluorescent compounds featuring a BODIPY TM 630/650 fluorophore. All compounds were pharmacologically tested using the NanoBRET assay previously reported by Stoddart et. al., where the authors observed that almost all fluorescent compounds exhibited high binding affinity at the H 1 R, regardless of differences in linker and orthostere. The authors concluded that linker SAR in the unlabeled congeners did not translate to the context of the full fluorescent compounds and had no significant impact on the compound potency, selectivity, or binding kinetics. The authors further demonstrated that the BODIPY TM 630/650-based fluorophore conferred high binding affinity to H 1 R fluorescent compounds, providing convincing evidence that the fluorophore is a major determinant of overall ligand binding affinity for H 1 R fluorescent compounds, which was in line with findings from a previous study (Baker et al., 2010a). The authors also proposed that there may be additional binding structures on the outer surface of the H 1 R which are specific to the BODIPY TM 630/650 fluorophore based on computational modelling studies, which however could not be verified without structural experiment confirmation. A notable outcome of this study was the discovery of 23, a potent and selective fluorescent compound for H 1 R with a receptor residence time of over an hour (Table 3). Additionally, 23 was capable of H 1 R visualization at concentrations as low as 10 nM using confocal microscopy with minimal cellular uptake and non-specific binding, deeming it a valuable tool for cell imaging. Gratz et al. (2020) synthesized three H 2 R targeting fluorescent compounds with varying fluorophores (Py-1, 5-TAMRA, BODIPY TM 630/650-X) based on the H 2 R antagonist BMY-25368 (Cavanagh et al., 1989). Two compounds demonstrated high affinity (<100 nM) across four different assays (NanoBRET, radioligand binding at Sf9 membranes, flow cytometry, β-arrestin-2 recruitment) and were confirmed to be antagonists via β-arrestin-2 recruitment assay. It was worth noting that despite the BODIPY TM 630/650-X based fluorescent compound being the most affine, it showed adsorption to plastic vessels due to its high lipophilicity, which led to a decline in signal over time, thereby highlighting the importance of physicochemical properties in fluorescent compounds for their utility in pharmacological studies. Compound 24 was identified to possess slow-off kinetics at the H 2 R, with a dissociation half-life of 5 hours and used to determine the binding affinity of known unlabeled ligands via Nano-BRET-based competitive binding experiments on live HEK293 cells expressing NLuc-H 2 R (Table 3). The results highlighted certain discrepancies in binding affinity data due to factors such as unlabeled ligand profile as agonists or antagonists, variation of receptor targets in different assays as well as assay buffer, particularly relating to sodium ions due to its allosteric effect on agonist binding to several GPCRs (Katritch et al., 2014). Bartole et al. (2020) derived six fluorescent compounds by conjugating a Py-5 fluorophore to histamine analogues with varying alkyl chain length, four of which displayed high affinity at the H 3 R in competitive radioligand binding assay. Compound 25 exhibited nanomolar affinity and selectivity for the H 3 R and H 4 R over the H 1 R and H 2 R, which was confirmed to be a partial agonist at the H 3 R and an inverse agonist at the H 4 R using luciferase reporter gene assay (Table 3). Interestingly, by extending the alkyl spacer by a single methylene group, the resulting analogue was more selective for the H3R over the H4R (37-fold), with an inversion in pharmacological profile – behaving as an antagonist at the H 3 R and a partial agonist at the H 4 R. This provides further evidence that even small changes in the linker moiety may influence the pharmacological profile of the final fluorescent conjugate. In a NanoBRET real-time kinetic binding experiment, 25 displayed fast-on fast-off kinetics at all receptor constructs studied (hH 3 R, hH 4 R, mH 4 R), with a receptor half-life ranging from 0.18 to 1.15 min. Given that 25 was the most affine fluorescent probe at the H 4 R, the authors proceeded with confocal microscopy using 25 to visualize H 4 R-expressing live cells at a concentration of 200 nM, which was successfully displaced by the addition of excess unlabeled ligand. Fluorescence was detected intracellularly despite 25 being an inverse agonist at the H 4 R, to which the authors attributed the internalization to constitutive endocytosis of the hH 4 R by β-arrestin or clathrin-independent mechanisms, which may include passive diffusion due to high fluorescent compound lipophilicity (Rose et al., 2012). The authors subsequently evaluated the utility of 25 in the characterization of unlabeled H 3 R and H 4 R ligands using NanoBRET binding assay, obtaining similar affinities to published data, though discrepancy was observed for unlabeled agonists, to which the authors adduced was due to the factors mentioned previously in Gratz et. al. In the following year, Rosier et al. (2021) developed a high affinity, H 3 R selective fluorescent compound by linking a 5-TAMRA dye to JNJ-5207852 (Apodaca et al., 2003), a H 3 R antagonist, via a PEG linker. Compound 26 demonstrated sub-nanomolar affinity at the H 3 R across four different assay formats, fast-off binding kinetics in NanoBRET binding experiments and was confirmed to be a neutral antagonist in a BRET G i2 sensor assay (Table 3). Compound 26 was also found to be >100,000-fold selective for H 3 R across the three other histamine receptor subtypes. Off-target screening for 14 other GPCRs was also conducted using a NanoBRET-based binding assay at ligand concentrations of 200 nM, where results revealed no significant off-target effects. Compound 26 displayed specific and displaceable binding of the fluorescent compound to cell surface H 3 R receptors at a concentration of 5 nM in confocal microscopy imaging experiments. It was also proven to serve well as a labelled ligand in a NanoBRET competitive binding assay used to determine the binding affinity of a range of known unlabeled ligands (agonists and antagonists). Furthermore, compound 26 proved suitable for total internal reflection fluorescence (TIRF) single-molecule microscopy to study receptor dynamics for the H 3 R, making it one of the best fluorescent compounds for the H 3 R ever to be reported in terms of ligand affinity, selectivity and proven utility. A fluorescent compound for each of histamine H 4 R and dopamine D 4 R has been reported by Wang et. al. (2024) and were synthesized based on the H 4 R antagonist adriforant and D 4 R antagonist rotigotine as parent compounds, linked via an extended PEG linker to the fluorophore Cy5 and MPA, respectively. The fluorescent compounds 27 and 28 were not pharmacologically characterized and information regarding ligand pharmacological profile, affinity, selectivity and binding kinetics were unknown (Table 2 and 3). Ligand binding for both compounds was only confirmed by flow cytometry experiments performed at 10 µM ligand concentration on H 4 R-expressing RAW264.7 and D 4 R-expressing HT29 (D 4 R) cell lines, as well as their ability to visualize target receptor localization on cell membrane at 10 µM ligand concentration using laser confocal scanning microscopy on the same cell lines. Results showed that only compound 27 exhibited strong fluorescence in rhomboid RAW 264.7 cells, indicating specific binding to inflammatory cells with high H 4 R expression, whereas only compound 28 exhibited strong fluorescence in round HT29 cells, indicating specific binding to colorectal cancer cells with high D 4 R expression. In in vivo imaging experiments, the authors were able to differentiate between inflammation and cancer tissues in inflammation-subcutaneous tumor model mice, which was supported by in vitro imaging studies after the mice were sacrificed and Western blot experiments. The authors also explored the combined application of the probes to distinguish between different developmental stages of colorectal mice using in vivo fluorescence monitoring. This allowed the authors to identify and differentiate the stages of inflammation, colorectal adenoma and carcinoma in an in vivo mice model, which was further confirmed by in vitro imaging of isolated colon after the mice were sacrificed and histopathological analysis of isolated tissues. Muscarinic Receptor She et al. (2020) synthesized a series of fluorescent compounds for the muscarinic M 2 receptor (M 2 R) based on an amine-functionalized dibenzodiazepinone (DIBA) derivative as previously reported by Pegoli et al. (2017), conjugated to pyrylium or various indolinium-type cyanine dyes as the fluorophore. Notably, 29 which featured a cyanine dye exhibited sub-nanomolar affinities at the M 2 R in radioligand binding studies and a 10-fold M 2 R selectivity against all other muscarinic receptor subtypes (Table 4). Compound 29 was determined to be an antagonist at the M 2 R and M 4 R by IP 1 accumulation assay using live M 2 - and M 4 R-expressing HEK293 cells. Flow cytometry saturation binding assay further confirmed the orthosteric binding nature of 29 as in the presence of the known orthosteric antagonist atropine completely prevented one-site (monophasic) specific M 2 R binding of 29 . Interestingly, 29 displayed monophasic association but incomplete monophasic dissociation in flow cytometry binding kinetics studies, to which the authors suggested that enhanced rebinding may be present due to simultaneous interactions with multiple binding sites. Based on observations in dissociation experiments and saturation binding experiments involving allosteric M 2 R modulators, the authors proposed that 29 exhibited dualsteric binding to the orthosteric and allosteric pocket at M 2 R, which through molecular dynamics simulation, the authors speculated that the allosteric vestibule was primarily occupied by the linker moieties. Confocal imaging studies using 29 at 30 nM in live CHO-hM 2 R cells demonstrated specific binding to M 2 R with majority of the observed signal being localized at the plasma membrane with no substantial increase in intracellular signal over time. The authors also employed 29 in high-content imaging binding assay using CHO-hM 2 R cells to determine the binding affinities for orthosteric and allosteric M 2 R, where correlation between fluorescent and radioligand data was high. The authors attributed this to the nearly fully reversible binding nature of 29 to M 2 R. Gruber et al. (2020) expanded on their previous work in She et al. surrounding DIBA-based M 2 R fluorescent compounds and synthesized six fluorescent compounds conjugated to various fluorescent dyes. All compounds exhibited nanomolar to sub-nanomolar affinities for M 2 R in a radioligand binding assay. Selectivity of these compounds for M 2 R over M 1 R and M 4 R was minimal, though it was more pronounced over the M 3 R and M 5 R, exceeding 10-fold across all six compounds. The TAMRA-labelled 30 was the most affine ( K i = 0.2 nM) with good selectivity (>10-fold) across all muscarinic receptor subtypes except M 4 R (Table 4). Compound 30 was also highly affine at M 2 R determined by a flow cytometric saturation binding assay, with K D values obtained comparable to data from radioligand binding assay, suggesting highly specific binding. The authors confirmed the compatibility of the fluorescence lifetime of 30 (~2.4 ns) for fluorescence anisotropy-based assays and BRET-based binding assays, though no further work to explore probe utility was reported. Yang et al. (2022) synthesized four DIBA-based fluorescent compounds targeting the M 2 R, with the conjugation of the DIBA-based congener to the Cyanine5 (Cy5) fluorophore achieved through click chemistry, linked via simple alkyl or PEG spacers. Flow cytometry saturation binding experiments on live CHO cells stably expressing M 1 R or M 2 R revealed that all four compounds bind strongly to M 2 R with nanomolar affinities. Notably, 31, the only compound to incorporate the short alkyl linker, exhibited nanomolar binding affinity for M 2 R ( K D = 1.8 nM) with a 58-fold selectivity over M 1 R, as well as slow-on slow-off binding kinetics at the M 2 R (Table 4). In confocal microscopy experiments, 31 at 2 nM ligand concentration specifically labelled CHO-M 2 R cells and could be fully displaced by in the presence of the antagonist atropine, indicating binding was specific and reversible. Compound 31 was successful in determining the binding affinities of known unlabeled orthosteric and allosteric ligands at the M 2 R in a flow-cytometry based competitive binding experiment, as K D values obtained were similar to reported K i values obtained from radioligand binding assays. The authors also highlighted the ability of the positive M 2 R allosteric modulator LY2119620 in displacing compound 31 from the M 2 R, further confirming the dualsteric binding mode of the DIBA-type antagonists at M 2 R. Lastly, 31 was capable of visualizing the M 2 R in sinoatrial nodes (SAN) isolated from mice at ligand concentration of 50 nM, confirmed by the high colocalization of fluorescence between 31 and the staining of Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) antibodies originally expressed in pacemaker channels in the SAN. Kockenburger et al. (2022) reported on the development of ten fluorescent compounds for the M 3 R, achieved by linking M 3 R-binding biphenylcarbamate analogues to cyanine-5 or TAMRA-based fluorescent dyes using alkyl linkers of varying lengths. Radioligand competition binding assays performed on membrane preparation of HEK293T cells transiently and separately expressing M 1 -M 5 R indicated that all ten fluorescent compounds bind considerably well to all receptor subtypes ( K i mostly <10 nM). Fluorescent compounds with the highly lipophilic Cyanine5 dye were not pursued further in this study as they were highlighted to have high levels of unspecific binding due to poor physicochemical properties, despite being some of the most affine compounds in this series. Compound 32 was not M 3 R-selective, but in fact was more affine at the M 1 R and M 4 R with sub-nanomolar affinities, suggesting possible utility in M 1 R and M 4 R studies (Table 4). Compound 33, on the other hand, displayed comparable affinities across all muscarinic receptor subtypes ( K i <10 nM) and lacked selectivity for M 3 R over other muscarinic receptor subtypes (Table 4). Both compounds were confirmed to be antagonists in two functional assay formats, namely IP 1 accumulation and β-arrestin recruitment assays. Additionally, Compound 33 was more photostable than 32 as it possessed the TAMRA dye which was known for its high photostability along with a decent quantum yield. In flow cytometry binding experiments using CHO-hM 3 R and CHO-hM 1 R cells, 32 and 33 displayed specific and saturable binding, though were much less affine at the M 3 R when compared to data obtained from radioligand binding assays. In flow cytometry kinetics experiments, both compounds exhibited monophasic association and dissociation curves, possessing favorable binding kinetics for application as molecular compounds for M 3 R studies. This was subsequently confirmed in a flow cytometry competition binding experiment using both compounds to determine binding affinities of known unlabeled ligands at the M 3 R, from which the results were in close agreement with data reported in literature. Compounds 32 and 33 were determined to have no cytotoxic effects at up to 1 µM concentration and were subsequently used in confocal microscopy at 100 nM and 60 nM respectively to image CHO-hM 3 R cells. Results showed a strong preference of both compounds to localize at the cell membrane along with highly specific and reversible binding, confirmed by the complete loss of fluorescence in presence of the antagonist atropine. Serotonin Receptor Azuaje et. al. (2017) reported on a preliminary series of fluorescent compounds targeting the serotonin 5-HT 2 receptor (5-HT 2 ) by conjugating 1-(2,5-Dimethoxy-4-iodophenyl)-propan-2-amine (DOI) to a dansyl dye with alkyl or ether linkers of varying lengths. From initial radioligand binding tests, the authors discovered that a propyl linker was optimal for 5-HT 2B R selectivity, and this was incorporated during fluorescent compound synthesis involving a wide range of fluorophores. The rhodamine-based 34 possessed moderate affinity ( K i = 262 nM) and potency (EC 50 = 225 nM) for the 5-HT 2B receptor in radioligand binding and IP accumulation assays respectively, along with favorable fluorescence properties (Table 5). This allowed for a concentration-dependent specific labelling of CHO-K1-5-HT 2B cells, whereby 34 under confocal microscopy demonstrated fluorescence signal internalization, which was expected from the agonistic nature of 34 . Hernandes et al. (2018) developed two fluorescent compounds by conjugating the 5-HT 1A receptor ligand UCM-2550 to two distinct BODIPY TM -based fluorophores separately through CuAAC reaction. The authors shared that in a radioligand binding assay, 35 demonstrated high affinity ( K i = 3 nM) and selectivity (up to 330-fold over most 5-HT receptor subtypes) for the 5-HT 1A receptor, though the actual affinity values for the other 5-HT receptor subtypes were not reported (Table 5). In flow cytometry experiments, 35 was capable of visualizing 5-HT 1A receptor in various cell lines with native levels of expression, including human T lymphocytes, monocytes, and dendritic cells, as well as changes of 5-HT 1A receptor expression in the presence of pro-inflammatory stimuli in these different cells. The authors revealed the significance of the 5-HT 1A receptor activation in promoting anti-inflammatory responses in monocytes and dendritic cells. In an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, it was found that 5-HT 1A receptor expression was significantly increased in CD4+ cells in diseased mice compared to healthy mice, with 35 capable of detecting the activated T cells in the model. These findings highlight the potential of 35 as a tool compound to study pathological inflammation and related diseases such as multiple sclerosis. Sarkar et al. (2021) conjugated the NBD fluorophore to serotonin via single amino acid spacer, affording three different fluorescent compounds. Amongst them, 36 showed the highest affinity ( K i = 1.4 µM) in a radioligand competition binding assay at 5-HT 1A receptor, in which the author attributed the low micromolar affinity to the loss of the serotonin amine moiety, an essential feature for aminergic GPCR binding, post-fluorophore conjugation (Table 5). Confocal microscopy experiments confirmed that 36 at a ligand concentration of 7 µM could label 5-HT 1A receptors in live CHO-K1 cells with signal internalization observed thus potentially suggesting agonist behavior, as well as minimal nonspecific binding and displaceable in the presence of unlabeled serotonin. Spectral imaging revealed a red edge excitation shift (REES) of 29 nm when the ligand was bound to the receptor, indicating localization in a restricted, hydrophobic microenvironment and consistent with the receptor’s membrane-embedded binding site. Garvey et. Al. (2022) shed light on two novel fluorescent compounds targeting the 5-HT 1A receptor by conjugating analogues of the 5-HT 1A receptor agonist 8-OH-DPAT separately to dansyl and 7-nitrobenzofurazan fluorophores linked via a hexyl spacer. Notably, 37 displayed high affinity ( K i = 1.8 nM) for the 5-HT 1A receptor in a radioligand binding assay (Table 5). Fluorescent microscopy experiments demonstrated that 37 was able to selectively label the 5-HT 1A receptor in live pancreatic islet cell monolayered cultures, which presented as vesicles within the cell cytoplasm, and displaceable in the presence of a 5-HT 1A receptor antagonist, validating ligand binding specificity. The authors also demonstrated that 5-HT 1A receptor expression was shared by human islet α and β cells, whereby subcellular localization of 5-HT 1A receptor coincided with insulin-containing vesicles and to some extent the D 2 R in β-cells.

Aminergic GPCRs are fundamental components of the body’s signaling network, orchestrating a wide array of physiological functions and their involvement in numerous diseases has made them prominent drug targets. Ongoing advances in molecular pharmacology continue to fuel the development of more selective and efficacious therapeutic agents, i.e. biased and allosteric ligands, offering promising avenues for improved treatment strategies. For over four decades, fluorescent ligands have proven themselves to be powerful pharmacological tools in the study of aminergic GPCRs, offering versatile applications across various assay formats, including as alternatives to radioligands in receptor binding and kinetics studies, and for visualizing receptors both in vitro and in vivo, in artificial and native cell lines. The recent rise of machine learning and artificial intelligence has significantly enhanced image processing and recognition capabilities, enabling deeper and more accessible insights from cellular and tissue imaging data. This technological progress underscores the urgent need for high-affinity, target-selective fluorescent tool compounds, ideally tailored to each individual receptor subtype, to fully leverage these advanced techniques. This review summarizes a decade of dedicated efforts to develop selective fluorescent probes for the aminergic GPCRs, across multiple receptor subtypes. While notable progress has been made in terms of expanding the pharmacological toolkit, gaps remain, and continued research is essential. The next exciting step forward for fluorescent ligand research would be to explore its utility in the clinical setting, i.e. the study of relevant receptor expression and distribution patterns in individual patient samples and their implication on a particular disease onset, progression and treatment prognosis. We hope that this ‘catalogue’ of reported, non-commercially available aminergic GPCR fluorescent ligands will inspire greater collaboration between scientific and clinical researchers. Such partnerships are vital to unraveling the complexities of GPCR signaling in native tissues and receptor expression, ultimately enhancing translational outcomes from bench to bedside, for the benefit of human health.

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Small-molecule fluorescent tool compounds reported between 2014-24 for the adrenergic GPCRs. a - Radioligand binding assay a, FP assay b, calcium ion mobilization assay c, FRET-cAMP assay d, TR-FRET assay e, NanoBRET binding assay f . | Alpha-Adrenoceptor | |||| | 1 2 | Green Green | Affinity: α 1A = 0.3 nM a α 1B = 0.1 nM a α 1D = 0.4 nM a Affinity: α 1A = 4.7 nM a α 1B = 7.2 nM a α 1D = 21 nM a | Fluorescence microscopy to visualize α 1A - and α 1D -AR at [ligand] = 10 nM ( 1 ) and 50 nM ( 2 ) with observed cellular intake, and minimal non-specific binding Compound 2 only Ex vivo imaging of PC-3 prostate cancer cells at [ligand] = 1 μM with minimal non-specific binding | Zhang et al. (2015) | | 3 | Red | Affinity: α 1A = 8.2 nM a α 1B = 4.8 nM a α 1D = 14 nM a | Fluorescence microscopy to visualize α 1A -, α 1B - and α 1D -AR at [ligand] = 300 nM with minimal cellular uptake and non-specific binding In vivo and ex vivo imaging of tumor xenografts in mice at [ligand] = 50 µM Ex vivo imaging of human prostate tissue at [ligand] = 300 nM | Ma et al. (2016a) | | 4 | Green | Affinity: α 1A = 4 nM a, 0.6 nM b α 1B = 2.7 nM a, 1.2 nM b α 1D = 24 nM a, 0.7 nM b | ELISA-based FP assay - As labelled ligand to determine the binding affinity of unlabeled antagonist at α 1 -AR | Ma et al. (2016b) | | 5 | Green | α 1B - and α 1D -AR Agonist c Affinity: α 1A = 580 nM a α 1B > 10 µM a α 1D = 200 nM a Potency (EC 50 ): α 1B = 1.8 µM c α 1D = 2.8 µM c | Fluorescence microscopy to visualize α 1A -AR at [ligand] = 250 nM with observed cellular uptake and minimal non-specific binding NanoBRET assay - As labelled ligand in the determination of binding affinity of unlabeled ligand (agonist and antagonist) at α 1A - and α 1B -AR | Qin et al. (2019) | | 6 | Green | α 1 Agonist c Affinity: α 1A = 1.6 µM a, 830 nM f α 1B = 4.8 µM a, 950 nM f α 1D = 1.7 µM a,230 nM f Potency (EC 50 ): α 1A, α 1B, α 1D > 10 µM c | Fluorescence microscopy to visualize α 1A - and α 1B -AR at [ligand] = 200 nM with observed cellular uptake and minimal non-specific binding NanoBRET assay - As labelled ligand in the determination of binding affinity of unlabeled ligand (agonist and antagonist) at α 1 -AR | Qin et al. (2021) | | Beta-Adrenoceptor | |||| | 7 8 | Red Red | β 2 Agonist d Affinity: β 1 = 63 nM e β 2 = 7.2 nM e Potency (EC 50 ): β 2 = 5.7 nM d β 2 Antagonist d Affinity: β 1 = 49 nM e β 2 = 15 nM e | Fluorescence microscopy and Stimulated Emission Depletion (STED) nanoscopy to visualize β 2 -AR at [ligand] = 100 nM with observed ( 7 ) or minimal ( 8 ) cellular uptake and minimal non-specific binding, in both artificial and endogenous cell lines (pancreatic CAPAN-1 cells) | Mitronova et al. (2017) | | 9 | Red | Affinity: β 1 > 500 nM f β 2 = 28 nM f | Fluorescence microscopy and bioluminescence imaging to visualize β 2 -AR at [ligand] = 100 nM with observed cellular uptake and non-specific binding NanoBRET assay - As labelled ligand in the determination of binding affinity of unlabeled ligand (agonist and antagonist) at β 2 -AR | Goulding et al. (2021) | | 10 | Red | Affinity: β 1 = 334 nM a, 216 nM f β 2 = 107 nM a, 276 nM f β 3 = 151 nM a, 316 nM f | Fluorescence microscopy to visualize β 1 -, β 2 - and β 3 -AR at [ligand] = 200 nM with minimal cellular uptake and non-specific binding | Li et al. (2022) | | 11 | Red | Affinity: β 1 = 434 M a β 2 = 156 nM a β 3 = 9.6 nM a, 43 nM f | Fluorescence microscopy to visualize β 3 -AR at [ligand] = 100 nM with minimal cellular uptake and non-specific binding Ex vivo imaging of tumour tissue at [ligand] = 200 nM with minimal cellular uptake and non-specific binding | Li et al. (2023) | Table 2. Small-molecule fluorescent tool compounds reported between 2014-24 for the dopaminergic GPCRs. G s heterotrimer dissociation assay a, radioligand binding assay b, TR-FRET assay c, IP accumulation assay d, β-arrestin-2 recruitment assay e, NanoBRET binding assay f, fluorescence microscopy assay g, BRET G i activation assay h, BRET Gα o1 sensory assay i | Dopamine 1-like (D1, D5) Receptor | |||| | 12 | Yellow | D 1 R antagonist a D 5 R antagonist a Affinity: D 1 R = 30 nM a, 5 nM b D 2 R > 10 µM b D 3 R = 2 µM b D 4 R > 10 µM b D 5 R = 0.3 nM a, 24 nM b | Fluorescence microscopy to visualize D 1 R at [ligand] = 50 nM with minimal cellular uptake and non-specific binding | Rosier et al. (2023) | | Dopamine 2-like (D2, D3, D4) Receptor | |||| | 13 | Red | Affinity: D 1 R > 1 µM c D 2 R = 8.4 nM c D 3 R = 1.9 nM c D 4 R > 1 µM c D 5 R > 1 µM c | TR-FRET assay - As labelled ligand to determine the binding affinity of unlabeled ligands separately at D 2 R and D 3 R, as well as to study cell surface dopamine receptor heteromerization | Hounsou et al. (2014) | | 14 15 | Yellow Yellow | Affinity: hD 2L R = 3.8 nM b hD 2S R = 7.8 nM b hD 3 R = 5.3 nM b D 2L R agonist d Affinity: hD 2L R = 8.8 nM b hD 2S R = 7.1 nM b hD 3 R = 0.4 nM b Potency (EC 50 ): D 2L R = 0.1 nM d | TIRF Microscopy to study receptor dimerization at D 2S R, D 2L R and D 3 R | Tabor et al. (2016) | | 16 17 | Green Red | D 2S R agonist e Affinity: hD 2L R = 46 nM b hD 2S R = 21 nM b hD 3 R = 1 nM b, 0.7 nM f hD 4 R = 56 nM b Potency (EC 50 ): D 2S R = 410 nM e D 2S R agonist e Affinity: hD 2L R = 10 nM b hD 2S R = 4.8 nM b hD 3 R = 0.9 nM b hD 4 R = 50 nM b Potency (EC 50 ): D 2S R = 410 nM e | Compound 16 only NanoBRET assay - As labelled ligand to determine the binding affinity and kinetics of unlabeled ligands at D 3 R Compound 17 only TIRF Microscopy to visualize D 2S R and D 3 R at [ligand] = 10 nM (D 2S R) and 1 nM (D 3 R) with minimal cellular uptake and non-specific binding at D 2S R and D 3 R. | Allikalt et al. (2020a) | | 18 | Yellow | Affinity: D 3 R = 0.7 nM b, 0.5 nM g | Fluorescence microscopy to visualize D 3 R at [ligand] = 1 nM with minimal cellular uptake and non-specific binding Fluorescence microscopy binding assay - As labelled ligand to determine the binding affinity and kinetics of unlabeled ligands at D 3 R | Allikalt et al. (2020b) | | 19 | Red | D 2S R partial agonist h Affinity: D 2L R = 7.1 nM b D 3 R = 1.3 nM b 5-HT 1A = 27 nM b 5-HT 2A = 2660 nM b 5-HT 6 = 530 nM b 5-HT 7 = 5.1 nM b | Stochastic Optical Reconstruction Microscopy (STORM) to visualize D 3 R at [ligand] = 100 nM with minimal cellular uptake and non-specific binding, the ex vivo and in vivo study of receptor localization and drug target engagement in live tissue preparations | Prokop et al. (2021) | | 20 | Green | Affinity: D 2S R = 328 nM b D 3 R = 23 nM b | No reported utility | Elek et al. (2022) | | 21 | Yellow | D 2L R antagonist i D 3 R antagonist i D 4 R antagonist i Affinity: D 1 R = 68 nM b D 2L R = 11 nM a, 5.8 nM b D 3 R = 12 nM a, 2.6 nM b D 4 R = 8.8 nM a, 17 nM b D 5 R = 331 nM b | Fluorescence microscopy to visualize D 2L R at [ligand] = 50 nM with minimal cellular uptake | Nagl et al. (2023) | | 28 | Near Infrared (IR) | Pharmacological profile, affinity, selectivity and binding kinetics not determined. Ligand binding to D 4 R confirmed by flow cytometry at concentration of 10 µM. | Fluorescence microscopy to visualize D 4 R at [ligand] = 10 µM with minimal non-specific binding In vivo imaging in inflammation subcutaneous tumor model mice In vivo studies of cancer developmental stages in CRC mice | Wang et al. (2024) | Table 3. Small-molecule fluorescent tool compounds reported between 2014-24 for the histaminergic GPCRs. Calcium ion mobilization assay a, radioligand binding assay b, NanoBRET binding assay c, β-arrestin recruitment assay d, flow cytometry assay e, reporter gene assay f, BRET Gi2 sensor assay g | 22 | Red | H 1 R Antagonist a Affinity: hH 1 R = 3 nM a, 4 nM b, 8 nM c | NanoBRET assay - As labelled ligand to determine the binding affinity and kinetics of unlabeled antagonist at H 1 R Fluorescence microscopy to visualize H 1 R at [ligand] = 50 nM with minimal cellular uptake and non-specific binding | Stoddart et al. (2018) | | 23 | Red | H 1 R Antagonist a Affinity: hH 1 R = 3 nM a, 4 nM c hH 3 R > 1 µM c hH 4 R > 1 µM c | Fluorescence microscopy to visualize H 1 R at [ligand] = 10 nM with minimal cellular uptake and non-specific binding | Kok et al. (2022) | | 24 | Red | H 2 R Antagonist d Affinity: hH 2 R = 24 nM b, 45 nM c, 17 nM d, 74 nM e | NanoBRET assay - As labelled ligand in the determination of binding affinity of unlabeled ligand (agonist and antagonist) at H 2 R | Gratz et al. (2020) | | 25 | Red | H 3 R Partial agonist f H 4 R Inverse agonist d,f Affinity: hH 3 R = 3 nM b, 2 nM c hH 4 R = 14 nM b, 2 nM c, 39 nM e hH 1 R > 1 µM b hH 2 R > 1 µM b Potency: H 3 R (EC 50 ) < 10 nM f H 4 R (IC 50 ) = 15 nM d, 2 nM f | Fluorescence microscopy to visualize H 4 R at [ligand] = 200 nM with observed cellular uptake and minimal non-specific binding NanoBRET assay - As labelled ligand in the determination of binding affinity of unlabeled ligand (agonist and antagonist) at H 3 R and H 4 R | Bartole et al. (2020) | | 26 | Yellow | H 3 R Antagonist g Affinity: hH 3 R = 1 nM b, 0.2 nM c, 0.3 nM e, 1 nM g hH 1 R > 100 µM b hH 2 R > 100 µM b hH 4 R > 100 µM b | NanoBRET assay - As labelled ligand in the determination of binding affinity of unlabeled ligand (agonist and antagonist) at H 3 R Fluorescence microscopy to visualize H 3 R at [ligand] = 5 nM with minimal cellular uptake and non-specific binding TIRF-single molecule microscopy to visualize H 3 R at [ligand] = 3 nM to study receptor dynamics | Rosier et al. (2021) | | 27 | Near-IR | Pharmacological profile, affinity, selectivity and binding kinetics not determined. Ligand binding to H 4 R confirmed by flow cytometry at concentration of 10 µM. | Fluorescence microscopy to visualize H 4 R at [ligand] = 10 µM with minimal non-specific binding In vivo imaging in inflammation subcutaneous tumor model mice In vivo studies of cancer developmental stages in CRC mice | Wang et al. (2024) | Table 4. Small-molecule fluorescent tool compounds reported between 2014-24 for the muscarinic GPCRs. IP 1 accumulation assay a, radioligand binding assay b, flow cytometry assay c, high-content imaging assay d, β-arrestin recruitment assay e | 29 | Red | M 2 R antagonist a Affinity: M 1 R = 5.2 nM b, 26 nM d M 2 R = 0.5 nM b, 4.5 nM c, 12 nM d M 3 R = 155 nM b M 4 R = 4.5 nM b M 5 R =166 nM b Potency (IC 50 ): M 2 R = 58 nM a M 4 R = 138 nM a | Fluorescence microscopy to visualize M 2 R at [ligand] = 30 nM with minimal cellular uptake and non-specific binding Fluorescence microscopy assay - As labelled ligand to determine the binding affinity of unlabelled orthosteric and allosteric ligands at hM 2 R Flow cytometry assay - As labelled ligand to determine the binding affinity of unlabelled orthosteric and allosteric ligands at hM 2 R | She et al. (2020) | | 30 | Yellow | Affinity: M 1 R = 2.6 nM b M 2 R = 0.2 nM b, 1.4 nM c M 3 R = 81.3 nM b M 4 R = 1 nM b M 5 R = 178 nM b | No reported utility | Gruber et al. (2020) | | 31 | Red | Affinity: M 1 R = 105 nM c M 2 R = 1.8 nM c | Fluorescence microscopy to visualize M 2 R at [ligand] = 2 nM with minimal cellular uptake and non-specific binding Flow cytometry assay - As labelled ligand to determine the binding affinity of unlabelled orthosteric and allosteric ligands at hM 2 R Ex vivo imaging of isolated sinoatrial node sections | Yang et al. (2022) | | 32 (OFH611) 33 (OFH5503) | Red Yellow | M 3 R antagonist a,e Affinity: M 1 R = 0.7 nM b M 2 R = 9.6 nM b M 3 R = 14 nM b, 54 nM c M 4 R = 0.9 nM b M 5 R = 5.5 nM b Potency (IC 50 ): M 3 R = 300 nM a, 450 nM e M 3 R antagonist a,e Affinity: M 1 R = 1.5 nM b M 2 R = 11 nM b M 3 R = 23 nM b, 23 nM c M 4 R = 1.4 nM b M 5 R = 7.4 nM b Potency (IC 50 ): M 3 R = 130 nM a, 430 nM e | Fluorescence microscopy to visualize M 3 R at [ligand] = 100 nM ( 32 ) or 60 nM ( 33 ) with minimal cellular uptake and non-specific binding Flow cytometry assay - As labelled ligand to determine the binding affinity of unlabelled orthosteric ligands at hM 3 R | Kockenburger et al. (2022) | Table 5. Small-molecule fluorescent tool compounds reported between 2014-24 for the serotonergic GPCRs. IP accumulation assay a, radioligand binding assay b | 34 | Yellow | 5-HT 2B agonist a Affinity: 5-HT 2A = 1.2 µM b 5-HT 2B = 262 nM b 5-HT 2C = 994 nM b Potency (EC 50 ): 5-HT 2B = 225 nM a | Fluorescence microscopy to visualize 5-HT 2B receptor at [ligand] = 3 µM with observed cellular uptake and minimal non-specific binding at 5-HT 2B . | Azuaje et al. (2017) | | 35 | Green | Affinity: 5-HT 1A = 3 nM b The authors reported that affinity for 5-HT 2A, 5-HT 4e, 5-HT 5a, 5-HT 6, and 5-HT 7 was determined at CEREP-Eurofins. These values were not available in the publication. | Flow cytometry – detect 5-HT 1A receptor expression in immune cells, which included ex vivo studies involving experimental autoimmune encephalomyelitis (EAE) mice model | Hernandez-Torres et al. (2018) | | 36 | Green | Affinity: 5-HT 1A = 1.4 µM b | Fluorescence microscopy to visualize 5-HT 1A receptor at [ligand] = 7 µM with observed cellular uptake and minimal non-specific binding at 5-HT 1A . | Sarkar et al. (2021) | | 37 | Green | Affinity: 5-HT 1A = 1.8 nM b | Fluorescence microscopy to visualize 5-HT 1A receptor at [ligand] = 10 µM with observed cellular uptake and minimal non-specific binding in live human islet cells as well as to study receptor distribution and subcellular localisation within live human islet cells. | Garvey et al. (2022) | Information & Authors Information Version history Peer review timeline Published European Journal of Medicinal Chemistry Version of Record1 Mar 2026Published Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Metrics & Citations Metrics Article Usage 286views 119downloads Citations Download citation Christopher Zi Qian Choo, Joey Yun Xuan Ching, Myra Mazhar Ud Deen, et al. Small-molecule fluorescent probes for the aminergic GPCRs -- What new tool compounds do we have post-2014 and what can they do?. Authorea. 21 August 2025. 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