Development of a fluorescent bodipy probe for visualization of the

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Development of a fluorescent bodipy probe for visualization of the serotonin 5-HT1A receptor in native cells of the immune system Gloria Hernandez-Torres, Ernesto Enriquez Palacios, Miriam Mecha, Ana Feliu, Ainoa RuedaZubiaurre, Alba Angelina, Leticia Martin-Cruz, Mar Martín-Fontecha, Oscar Palomares, Carmen Guaza, Eduardo Peña-Cabrera, María L. López-Rodríguez, and Silvia Ortega-Gutierrez Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00228 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Bioconjugate Chemistry

Development of a fluorescent bodipy probe for visualization of the serotonin 5-HT1A receptor in native cells of the immune system Gloria Hernández-Torres,a Ernesto Enríquez-Palacios,b Miriam Mecha,c Ana Feliú,c Ainoa Rueda-Zubiaurre,a Alba Angelina,d Leticia Martín-Cruz,d Mar Martín-Fontecha,a Oscar Palomares,d Carmen Guaza,c Eduardo Peña-Cabrera,b María L. LópezRodríguez,a,* Silvia Ortega-Gutiérreza,*

a

Department of Organic Chemistry and dDepartment of Biochemistry and Molecular

Biology, Universidad Complutense de Madrid, Av. Complutense s/n, E-28040 Madrid, Spain b

c

Departament of Chemistry, Universidad de Guanajuato, Guanajuato 36050, Mexico

Functional and Systems Neurobiology Department, Neuroimmunology Group, Instituto Cajal, 28002 Madrid, Spain.

Corresponding author: [email protected], [email protected]

Serotonin (5-HT) modulates key aspects of the immune system. However, its precise function and the receptors involved in the observed effects have remained elusive. Among the different serotonin receptors, 5-HT1A plays an important role in the immune system given its presence in cells involved in both the innate and the adaptive immune responses, but its actual levels of expression under different conditions have not been comprehensively studied due to the lack of suitable tools. To further clarify the role of 5-HT1A receptor in the immune system, we have developed a fluorescent small molecule probe that enables the direct study of the receptor levels in native cells. This

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probe allows direct profiling of the receptor expression in immune cells using flow cytometry. Our results show that important subsets of immune cells including human monocytes and dendritic cells express functional 5-HT1A and that its activation is associated to anti-inflammatory signalling. Furthermore, application of the probe to the experimental autoimmune encephalomyelitis model of multiple sclerosis demonstrates its potential to detect the specific overexpression of the 5-HT1A receptor in CD4+ T cells. Accordingly, the probe reported herein represents a useful tool whose use can be extended to study the levels of 5-HT1A receptor in ex vivo samples of different immune system conditions.

Introduction The neurotransmitter serotonin (5-HT) regulates many essential functions in humans. Although its role and therapeutic potential in the central nervous system has been firmly established,1 the function of this biogenic amine in the immune system remains poorly understood, in spite of the facts that 5-HT receptors are widely expressed in immune system cells, high levels of 5-HT are found in the periphery, and 5-HT and its receptors have been described to affect specific immune functions.2 This dearth of data can be mainly attributable to the complexity of the serotonergic system, in which 5-HT signals through 14 different receptors (13 of which are G protein-coupled receptors, GPCRs), and to the intricacies of the immune system, composed of many different cell types involved in distinct immune responses. In this context, the lack of appropriate tools to study the role of 5-HT receptors in the immune system becomes evident, as most studies described so far rely on the quantification of receptor transcripts because the direct observation of protein levels remains challenging. This fact is due to the


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difficulties associated to the generation of antibodies suitable for visualization of GPCRs in native cells. It has been acknowledged that, in general, the GPCR structure, its poor immunogenicity, and a low receptor density make it difficult to generate GPCR antibodies, so the development of fluorescent antibodies is limited. Furthermore, in many occasions, the use of the antibody requires cell fixation and permeabilization, which limits their usage in fluorescent imaging in native systems.3 To overcome these drawbacks, fluorescent small molecule ligands have emerged as powerful tools to study GPCRs3-6 as they can reveal receptor expression and localization in native cells. In addition, they are particularly well suited for use in fluorescence-activated cell sorting (FACS), which is the most widely used technique for the comprehensive study of immune system cells. This is due to its suitability for the analysis of live cells, its capacity for multiplexing (simultaneous detection of different cellular markers), and its adaptation to the analysis of biological samples coming from a simple blood analysis, thus facilitating the translation of the results to human samples. Among the different 5HT receptors, in this work we have focused our attention on the 5-HT1A receptor, given its presence in cells involved in the innate immunity (such as macrophages, mast cells, and natural killer cells) and in the adaptive immune response (T and B lymphocytes),2 and its regulation of immune cell mechanisms.7-9 The role of 5-HT1A receptor in the immune response suggests that pharmacological manipulations that alter the levels of 5HT or directly modulate the 5-HT1A receptor may constitute important strategies for immunomodulation. But more interestingly, it also suggests that variations on the levels of 5-HT1A receptor may correlate with inflammatory alterations implicated in multiple diseases and hence can be used as a biomarker for certain pathologies, to evaluate their severity or the response to pharmacological treatments. However, the function of the 5HT1A receptor in the immune system remains largely elusive due to the lack of



appropriate tools to interrogate the biological systems. Previously described fluorescent ligands for the 5-HT1A receptor can be used in microscopy but have limitations for use in flow cytometers due to the specific wavelengths required for excitation and/or emission.10, 11 Alternatively, the use of biotinylated ligands requires a two-step labelling which can affect the sensitivity of the signal.12, 13 Hence, our aim is to develop a new fluorescent ligand with affinity for the 5-HT1A receptor and suitable for use in flow cytometry experiments, which enables the detection of changes in the receptor expression levels in cells of the immune system and is suitable for direct use in cells coming from a simple blood analysis.

Results and discussion Design and synthesis of the fluorescent probe In our search for the appropriate fluorescent probe we selected the high affinity 5-HT1A ligand UCM-2550 (1, Scheme 1) because we knew that this compound admits the introduction of different moieties in the 7a position of the bicyclohydantoin without important losses of affinity.10, 14 With respect to the most appropriate fluorophore, we have chosen the bodipy (borondipyrromethene) core because of its outstanding properties such as photostability, electrical neutrality, high fluorescent quantum yield, high molar extinction coefficient, tunable fluorescent characteristics, and narrow emission bandwidth.15 Taking into account synthetic accessibility, we considered the possibility of introducing the fluorophore by means of a click chemistry reaction, so a small in-house library of azide- and terminal alkyne-decorated bodipys was selected and analysed for excitation and emission wavelengths and fluorescence intensity. Among them, bodipys 2 and 3, with the best overall properties (Supporting Table S1) were


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chosen for attachment to the UCM-2550 core through a copper (I)-catalysed cycloaddition reaction to yield probes 5 and 8 (Scheme 1). Compound 5 showed better fluorescence values (Supporting Table S1 and Supporting Figure S1), both in terms of maximal wavelengths suitable for use in the flow cytometer and intensity, as well as higher affinity by the 5-HT1A receptor with an excellent Ki value of 3 nM versus 90 nM shown by compound 8 and with selectivity ranging from 50 to 330 fold against the rest of serotoninergic receptors (5-HT2A, 5-HT4e, 5-HT5a, 5-HT6, and 5-HT7). Together, these data prompted us to evaluate the potential of probe 5 for FACS experiments.

Scheme 1. Conditions and reagents: (a) i) lithium diisopropylamide, THF, 0 ºC, 30 min, ii) 6-iodo-1-hexyne, THF, 0 ºC to rt, 24 h, 16%; (b) ascorbic acid, CuSO4·5H2O, water/dichloromethane (1:1), 25 ºC, 12 h, 69% (for 5) and 58% (for 8); (c) i) lithium diisopropylamide, THF, 0 ºC, 30 min, ii) 1,4-dibromobutane, THF, 0 ºC to rt, 24 h, 48%; (d) NaN3, water/dimethylformamide (1:1), 60 ºC, 15 h, 90%.



Flow cytometry experiments in cells transfected with the 5-HT1A receptor First, we assessed the capacity of compound 5 to label the 5-HT1A receptor in cells that overexpress this receptor (Figure 1). The obtained results show that probe 5 (100 nM) can be used to detect the 5-HT1A receptor with an excellent signal-to-noise ratio whereas neither the original ligand UCM-2550 (1) nor the bodipy-azide (2) exhibited any significant fluorescence when used alone (Figure 1A). In addition, probe 5 can readily differentiate between transfected and non-transfected cells (Figure 1B). Based on these results, we proceeded to the use of probe 5 in cells from the immune system with native levels of expression of the 5-HT1A receptor. For this purpose, we selected the Jurkat cell line as a widely used model of human T lymphocytes.

Figure 1. (A) Flow cytometry analysis of CHO cells stably transfected with h5-HT1AR using probe 5. Histograms display CHO cells stained with vehicle (gray), compound 1 (orange) or 2 (blue) or probe 5 (red), all of them used at 100 nM. (B) Flow cytometry analysis of wild type CHO cells and CHO cells stably transfected with h5-HT1AR cells using probe 5 (100 nM). Histograms show wild-type CHO cells stained with vehicle (blue) or with probe 5 (orange) and CHO cells stably transfected with h5-HT1AR stained with vehicle (gray) or with probe 5 (red).


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Detection of the 5-HT1A receptor in human T lymphocytes Early reports described the presence of 5-HT1A receptor mRNA in Jurkat cells,16 as well as the upregulation of 5-HT1A receptor levels following activation with proinflammatory stimuli.17 Hence, we considered this cell line as a good model to assess the capacity of probe 5 to detect 5-HT1A receptor in a direct manner and also to confirm that the probe was sensitive enough to reveal changes in the 5-HT1A levels due to immune stimulation. In this regard, probe 5 enabled the clear detection of 5-HT1A receptor in Jurkat cells (Figure 2A,B) and also to visualize the increase in the 5-HT1A expression following pro-inflammatory stimuli such as lipopolysaccharide (LPS) (Figure 2C,F). To verify that the intensification in fluorescence was actually due to the increase of the 5-HT1A receptor levels, we measured the expression of the receptor by western blot (see Supporting Figure S2). The obtained results showed a clear correlation with those obtained with the use of flow cytometry, further supporting the reliability of probe 5 to profile the levels of the receptor. To confirm the generality of these results, we also stimulated the cells with phorbol 12myristate 13-acetate (12-O-tetradecanoylphorbol-13-acetate or PMA) observing a similar effect (Figure 2D,F) and we corroborated that the use of an anti-inflammatory compound such as dexamethasone decreased the levels of 5-HT1A, change that could be detected by probe 5 (Figure 2E,F). These results suggest that 5-HT1A receptor expression is significantly related with inflammation. Hence, probe 5 could provide a real-time measurement of receptor expression as a direct read-out of inflammatory environment. To further extend the potential of probe 5 in relevant biological systems we addressed the analysis of expression and the functional implications of the 5-HT1A receptor in immune cells



involved in inflammation such as monocytes and monocyte-derived human dendritic cells as well as the profile of immune cells in multiple sclerosis, a disease driven by a pro-inflammatory T cell response.

Figure 2. Flow cytometry analysis of Jurkat cells using probe 5. Representative dot plots and histograms of cells labelled with (A) vehicle, or (B) compound 5 (250 nM). Receptor expression increases after exposure to pro-inflammatory stimuli such as (C) LPS (20 ng/mL) or (D) PMA (10 ng/mL) and (E) decreases in the presence of the antiinflammatory compound dexamethasone (10 µM). (F) Graph bar shows the quantification of fluorescence intensity in the different conditions. FSC, forward scatter indicating the size of the gated cells. **, p

Development of a fluorescent bodipy probe for visualization of the

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