Development of Fluorescent Ligands for the Human 5-HT_1A Receptor

Abstract

In this work, we report the design and synthesis of a set of fluorescent probes targeting the human 5-HT_1A receptor (h5-HT_1AR). Among the synthesized compounds, derivative 4 deserves special attention as being a high-affinity ligand (K_i = 2 nM) with good fluorescent properties (I_em > 1000 au and a fluorescence quantum yield, Φ_f, of 0.26), which enables direct observation of the h5-HT_1AR in cells. Thus, it represents the first efficacious fluorescent probe for the specific labeling of h5-HT_1AR in cells. Our results provide the basis for the introduction of a variety of tags in scaffolds of G protein-coupled receptor (GPCR) ligands that enable visualization, covalent binding, or affinity pull-down of receptors. These strategies should contribute to the optimization of the therapeutic exploitation of known or new members of the GPCR superfamily by providing valuable information about their location or level of expression.

The development of compounds that enable direct observation of subfractions of the proteome has become of paramount importance in the quest to understand biological processes at the systems level. In this context, activity-based protein profiling (ABPP) can be considered as one of the major contributions. This approach has been successfully applied to the study of different enzyme families¹ and has been recently extended to the nicotinic acetylcholine receptor ion channel.² However, the superfamily of G protein-coupled receptors (GPCRs), which account for more than the 50% of the druggable genome,³ remains to be addressed.¹ The concept of ABPP probes for GPCRs is more complex than for enzymes or ion channels due to the different GPCR conformations that coexist in dynamic equilibrium and also because GPCR ligand binding can switch conformations between active and inactive state(s).^4,5 This fact might account for the lack of molecular probes able to generate knowledge on GPCRs in a high-throughput manner. Usually, information about the presence of a given GPCR is derived from detection of its cognate RNA, which might not correlate with the actual levels of protein.

Therefore, the development of probes for visualization of GPCRs can be considered as the first step in the application of ABPP principles to this field. With this broad objective in mind, we have initiated a project aimed at the search of fluorescent probes that enable direct visualization of GPCRs in complex biological systems. Although some GPCR fluorescent ligands have been recently described, their suitability for application in cell systems has proven difficult.⁶ Moreover, the introduction of alternative tags other than fluorophores to obtain additional information in a high-throughput manner comparable to ABPP approaches in the field of enzymes is still lacking. We have focused on the 5-HT_1A receptor (5-HT_1AR), one of the most important GPCRs from a therapeutic point of view. This serotonin (5-HT) receptor is involved in the regulation of key processes in neurobiology, including excitotoxicity, pain, and anxiety.⁷ Furthermore, variation of 5-HT_1AR levels has been linked to the pathogenesis of a number of diseases.⁸ Therefore, the availability of agents able to directly visualize the 5-HT_1AR would be of importance. In fact, some potent fluorescent 5-HT_1AR ligands have been recently reported,⁹ although their use in cell systems was not optimized. In this work, we report the design of fluorescent probes based on the introduction of a fluorophore in the scaffold of our previously characterized 5-HT_1AR agonists without affecting affinity. Among the synthesized compounds, derivative 4 deserves special attention as being a potent 5-HT_1AR ligand (K_i = 2 nM) with good fluorescent properties (I_em > 1000 au and a fluorescence quantum yield, Φ_f, of 0.26), which enables direct observation of human 5-HT_1AR (h5-HT_1AR) in cells. These results support the validity of our approach and represent the starting point for further optimization in terms of other fluorophores or tags, experiments that are under way in our laboratory.

The design of the new fluorescent probes for the 5-HT_1AR was based on our previously reported potent agonists 1 (UCM-310590)¹⁰ and 2 (UCM-2550)¹¹ (K_i = 5.5 and 2.4 nM, respectively; Figure 1). Using these arylpiperazines as scaffolds, we have explored different structural possibilities for the introduction of the fluorescent tag. Extensive structure−activity relationship (SAR) studies together with computational models of 5-HT_1AR−ligand interactions previously performed in our group (see ref (11) and references cited therein) have allowed us to identify the molecular determinants of the ligand−receptor interaction. On the basis of this knowledge, we envisioned that introduction of the fluorescent tag at position 7a of the bicyclohydantoin should not notably affect 5-HT_1AR affinity,¹² whereas modifications in the aryl substituent of the piperazine moiety might involve deeper changes in the affinity and selectivity of the ligands.¹³ Therefore, we have considered two possibilities: (i) attachment of the tag to position 7a of the bicyclohydantoin using an aliphatic chain as spacer (compounds 3−8) and (ii) replacement of the aromatic moiety of 1 and 2 (compounds 9−12) by the tag (Figure 1).

Figure 1.

Design of fluorescent probes for the h5-HT_1A receptor.

We have selected the dansyl (Ds) group [(5-dimethylamino)naphthalene-1-sulfonyl] as the fluorescent tag since it is of moderate size and it is commercially available as sulfonyl chloride; therefore, it can be readily introduced into the structures of compounds 1 and 2, and it has satisfactory fluorescent properties to be used in a cellular environment.¹⁴

The new arylpiperazine derivatives 3−12 were synthesized following standard methodologies (Schemes 1 and 2). Bicyclohydantoins 1,¹⁰2,¹¹ and 26(10) were prepared according to previously described procedures. Detailed synthetic procedures for all new compounds are described in the Supporting Information.

Scheme 1. Synthesis of Compounds 3−8.

Reagents and conditions: (a) LDA, THF, 0 °C to room temperature, 24 h, 46−89%. (b) NaN₃, H₂O/DMF, 50 °C, 24 h, 87−92%. (c) PPh₃, H₂O/THF, room temperature, 24 h, 84−92%. (d) DsCl, Et₃N, CH₂Cl₂, room temperature, 16 h, 55−82%. (e) NaI, Et₃N, DMF, 50 °C, 16 h, 40−80%.

Scheme 2. Synthesis of Compounds 9−12.

Reagents and conditions: (a) Et₃N, CH₃CN, 60 °C, 24 h, 53−99%.

Target compounds 3−12 were assessed for in vitro affinity at the h5-HT_1AR by radioligand binding assays, using [³H]-8-hydroxy-DPAT in membranes from transfected HEK-293 EBNA cells (see the Supporting Information for details). The inhibition constant K_i was calculated from the IC₅₀ value using the Cheng−Prusoff equation,¹⁵ and the values in Table 1 are the means of 2−4 independent experiments. The fluorescent properties of compounds 3−12 (Table 1) were measured as 10 μM solutions of the corresponding compound in the same buffer solution used for the binding assays.

Table 1. 5-HT_1AR Affinity and Fluorescence Values for Compounds 1−12.

^aThe values are the means ± SEMs from two to four independent experiments performed in triplicate.

^bFluorescence values were determined at 10 μM in binding assay buffer (50 mM Tris-HCl and 0.5 mM MgSO₄, pH 7.4) at 25 °C.

^cWavelengths for the maximal absorption and emission. The excitation slit was 2.5 nm in all cases.

^dThe Φ_f values were determined in binding assay buffer at 25 °C.

^eValue from ref (10).

^fn.d., not determined.

^gValue from ref (11).

High 5-HT_1AR affinity values were obtained when the Ds tag was attached by means of a spacer to the tertiary carbon of the bicyclohydantoin of 1 and 2 (compounds 3−8). These results are consistent with our previous SAR studies and receptor−ligand interaction models, which indicated that position 7a admits voluminous substituents without affecting receptor binding. In general, derivatives with R² = 1-naphthyl showed higher affinities than their corresponding 2-methoxyphenyl counterparts, as seen, for example, in compound 4 vs 3 (K_i values of 2 and 11 nM, respectively) or in compound 6 vs 5 (K_i values of 5 and 15 nM, respectively). Further elongation of the spacer did not significantly increase binding affinity, as shown by the 2-methoxyphenyl derivatives 3 vs 5 and 7 vs 8 (K_i values of 11 vs 15 and 7 vs 9 nM, respectively). In particular, the new ligands 4 and 6, with K_i values of 2 and 5 nM, respectively, showed affinities comparable to their parent compounds 1 and 2 (K_i of 5.5 and 2.4 nM, respectively). On the other hand, when the aromatic moiety of 1 or 2 was replaced by the fluorescent tag (compounds 9−12), a variety of K_i values were obtained. Direct attachment of the Ds group to the piperazine (compound 9) led to a complete lack of 5-HT_1AR affinity (K_i > 1000 nM). However, when the N,N-dimethylaminosulfonyl-substituted naphthalene ring is used as a Ds surrogate, affinity was restored. The position of the N,N-dimethylaminosulfonyl function in the naphthalene ring was found to be crucial for affinity, as illustrated by the diversity of K_i values obtained for ligands 10−12, with K_i values ranging from 2.4 to 350 nM. Among them, analogue 12 deserves special attention since it keeps the high affinity value of parent compound 2 [K_i (12) = 2.4 nM and K_i (2) = 2.4 nM].

A study of the fluorescent properties of all synthesized compounds with a constant excitation slit (2.5 nm) showed that, in general, 1-naphthyl derivatives bearing the tag in position 7a of the bicyclohydantoin exhibit the highest intensity emission I_em values (compounds 4 and 6, with I_em > 1000 au for an emission slit of 2.5 nm). With respect to the spacer between the scaffold and the tag, the results suggest that its elongation increases the fluorescence intensity as shown in methoxyphenyl derivatives 5 vs 3 (I_em > 1000 au vs 218 au, respectively, for an emission slit of 5 nm). Nevertheless, the presence of an amine in the spacer was detrimental for the fluorescent properties, since derivatives 7 and 8 were poorly fluorescent (I_em values of 169 au and 144 au for an emission slit of 5 nm, respectively). Compounds 9−12, bearing the fluorescent moiety attached to the piperazine, showed a diversity of fluorescence intensities, with I_em values ranging from low to moderate (I_em from 66 to above 1000 au for an emission slit of 5 nm). Additionally, Φ_f values were determined for those compounds with I_em > 1000 au for an emission slit of 5 nm (Table 1).

On the basis of their 5-HT_1AR affinity and fluorescence profiles, compounds 4 and 6 were selected as suitable candidates for cell visualization experiments (K_i values of 2 and 5 nM and Φ_f of 0.26 and 0.24, respectively). These compounds also showed a high Stokes shift, with an excitation wavelength of 320 nm and maximal emission around 480 nm, with both parameters in the suitable range for these experiments. Although both ligands are similar in terms of affinity and fluorescence profiles, during optimization of the experimental conditions for cell labeling, we observed slightly better results with probe 4 than with probe 6; therefore, 4 was selected for further experiments. Selectivity of this derivative for 5-HT_1AR vs other metabotropic 5-HT receptors was assessed by a screening radioligand binding assay that included 5-HT_2A, 5-HT_4e, 5-HT_5a, 5-HT₆, and 5-HT₇ receptors. Compound 4 (at a concentration of 1 μM) displaces more than the 80% of the corresponding radioligand at 5-HT_2A, 5-HT_5a, and 5-HT₇ receptors, and it does not significantly bind to 5-HT_4e or 5-HT₆ receptors. These results are in agreement with the in vitro profile of the parent compound 2 and support the initial hypothesis that it is possible to introduce a tag in the 7a position of the bicyclohydantoin of these ligands without dramatically changing their in vitro profiles.

To assess the full potential of this type of probe for visualization of the 5-HT_1AR in cells, 4 was used in in vitro cell labeling experiments. The incubation of cells with probe 4 (250 nM) allowed direct observation of the h-5HT_1AR in stably transfected CHO cells using conventional confocal microscopy (λ_exc = 405 nm) (Figure 2A). To assess the specificity of the labeling, transfected cells were incubated with compound 4 in the presence of an excess of the high-affinity (K_i = 38 ± 9 nM) and nonfluorescent (I_em ≅ 0 au, λ_exc = 350 nm for excitation/emission slits of 2.5/10 nm) 5-HT_1AR ligand belonging to the same series (R¹ = H, R² = 3-benzoylphenyl; see the Supporting Information), which essentially eliminated fluorescent labeling (Figure 2B). Figure 2C shows cellular autofluorescence in the absence of probe. Considered together, all of these results confirm the suitability of 4 as a probe for in vitro visualization of the h5-HT_1AR.

Figure 2.

Labeling of CHO cells stably transfected with h5-HT_1AR with fluorescent compound 4. Cells were incubated in the presence of 4 (250 nM) for 10 min, washed, fixed, mounted, and then observed by confocal microscopy (A). To assess specificity, cells were labeled under the same conditions with 4 (250 nM) in the presence of an excess (50 μM) of a nonfluorescent 5-HT_1AR ligand of the same series (R¹ = H, R² = 3-benzoylphenyl) (B) or in the absence of probe (C). The scale bar represents 20 μm. Preparations were visualized under a SP2 Leica confocal microscope with the 63× objective with a constant laser beam and photodetector sensitivity and are representative of two or three independent experiments.

In summary, the synthesized compounds described herein represent the first efficacious fluorescent probes for the labeling of the h5-HT_1AR in cells. Moreover, most of the tagged ligands showed nanomolar affinity for the h5-HT_1AR, supporting the generality of our approach. These results are the starting point for introducing a variety of tags in these hydantoin-arylpiperazine ligands for further optimization of the probes in terms of binding selectivity and fluorescence (other fluorophores might be better in terms of wavelength emission, intensity, or both) and for incorporation of other tags that enable covalent binding or affinity pull-downs. These strategies should contribute to an optimized therapeutic exploitation of known or new members of the GPCR superfamily by providing valuable information about their location or level of expression. All of these experiments are currently under way in our laboratory and will be reported in due course.

Abbreviations

5-HT, serotonin; ABPP, activity-based protein profiling; au, arbitrary units; Ds, dansyl; Φ_f, fluorescence quantum yield; GPCR, G protein-coupled receptor; h5-HT_1AR, type 1A of the human 5-HT receptor; I, intensity; K_i, ligand−receptor equilibrium dissociation constant; SAR, structure−activity relationship.

Supporting Information Available

Full synthetic procedures, analytical and spectral characterization data of the synthesized compounds including elemental analyses, experimental details for the determination of binding affinity, and experimental protocols for spectroscopic measurements, cell labeling, and visualization. This material is available free of charge via the Internet at http://pubs.acs.org.