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, 7 (1), 10765

Development of Fluorescent Probes That Target Serotonin 5-HT 2B Receptors

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Development of Fluorescent Probes That Target Serotonin 5-HT 2B Receptors

Jhonny Azuaje et al. Sci Rep.

Abstract

Some 5-HT2B fluorescent probes were obtained by tagging 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-amine (DOI) with a subset of fluorescent amines. Some of the resulting fluorescent ligands showed excellent affinity and selectivity profiles at the 5-HT2B receptors (e.g. 12b), while retain the agonistic functional behaviour of the model ligand (DOI). The study highlighted the most salient features of the structure-activity relationship in this series and these were substantiated by a molecular modelling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human 5-HT2B receptor. One of the fluorescent ligands developed in this work, compound 12i, specifically labelled CHO-K1 cells expressing 5-HT2B receptors and not parental CHO-K1 cells in a concentration-dependent manner. 12i enables imaging and quantification of specific 5-HT2B receptor labelling in live cells by automated fluorescence microscopy as well as quantification by measurements of fluorescence intensity using a fluorescence plate reader.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Synthesis of the target fluorescent DOI analogues. Reagents and conditions: (a) K2CO3, R–X, MeCN, 80 °C, 8 h. (b) I2, THF, rt, 24 h. (c) Me-NO2, AcOH, 100 °C, 6 h. (d) Fe, AcOH, 100 °C, 12 h. (e) NH4OAc, NaCNBH4, THF, 2 h. (f) (Boc)2O, THF, 0 °C, 3 h. (g) DCC, DCM, rt, 12 h (70–93%). (h) HCl/Dioxane, DCM, 0 °C, 2 h (50–78%).
Figure 2
Figure 2
Structural and pharmacological data at 5-HT2 receptors of model dansyl probes bearing different linkers (10ad).
Figure 3
Figure 3
Three-dimensional complexes of the fluorescent probes 10a (left) and 10b (right) bound to the 5-HT2B receptor obtained from extended molecular dynamics simulation with a total time of 1.6 μs (2 times × 800 ns). The upper panel highlights ligand-receptor interactions of a representative structure. The lower panel includes information about the dynamic properties and stability of the ligand binding by depicting frames each 50 ns along a total simulation time of 800 ns. The RMSD values are calculated for DOI or the fluorescent tag with respect to the average structure at 800 ns.
Figure 4
Figure 4
Synthesis of the target fluorescent DOI analogues 12ai and structures of the fluorescent amine precursors (8ai). Reagents and conditions: (a) DCC, DCM, rt, 12 h (70–93%). (b) HCl/Dioxane, DCM, 0 °C, 2 h (50–78%).
Figure 5
Figure 5
Photophysical properties of fluorescent DOI probes 12ai.
Figure 6
Figure 6
Structures and pharmacological data at 5-HT2 receptors for the DOI-based fluorescent probes 12a–i.
Figure 7
Figure 7
Concentration-response curves obtained for representative ligands and DOI in binding experiments (A) and in functional studies (B).
Figure 8
Figure 8
(A) Superimposition of docking poses of compounds 12ai for the 5-HT2B receptor (PDB ID: 4IB4) with following color code: 12a - red, 12b - pink, 12c - green, 12d - yellow, 12e - cyan, 12f - dark blue, 12g - purple, 12h - orange and 12i - silver. (B) Individual docking poses for the probes 12ai bound to the 5-HT2B receptor (PDB ID: 4IB4).
Figure 9
Figure 9
Labelling of 5-HT2B receptors by compound 12i in cells. (A) Living parental untransfected CHO-K1 cells (CHO-K1) and CHO-K1 cells stably expressing 5-HT2B receptors (CHO-K1-5-HT2B) were incubated in the absence or presence of the indicated concentrations of compound 12i and, after compound removal, fluorescence images (excitation wavelength 520–550 nm, emission wavelength 560–630 nm, standard filter set for 5-TAMRA) were acquired using an automated high content imaging instrument at 20x magnification. (B) Sample images from CHO-K1 and CHO-K1-5-HT2B cells labelled with 1 µg/mL Hoechst 33342 (for nuclear staining) and compound 12i at a concentration of 3 μM, sampled from those quantified in (C,D). Minimum and maximum intensity and gamma correction of the images are shown in the colour scale in the panel. (C,D) Quantification of the fluorescence emission of compound 12i in images from CHO-K1 and CHO-K1-5-HT2B cells labelled with 1 µg/mL Hoechst 33342 (for nuclear staining) and compound 12i at a concentration of 3 μM, both by image analysis (C) and by direct fluorescence measurement using a plate reader (D). The graphs show mean ± SEM of 4 wells, 5 fields/well (image analysis) and mean ± SEM of the same wells (plate reader). ***p < 0.001, two-way ANOVA and Bonferroni post tests.

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References

    1. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu. Rev. Med. 2009;60:355–366. doi: 10.1146/annurev.med.60.042307.110802. - DOI - PMC - PubMed
    1. Hoyer D, et al. International union of pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin) Pharmacol Rev. 1994;46:157–203. - PubMed
    1. Gerhardt CC, van Heerikhuizen H. Functional characteristics of heterologously expressed 5-HT receptors. Eur. J. Pharmacol. 1997;334:1–23. doi: 10.1016/S0014-2999(97)01115-1. - DOI - PubMed
    1. Roth BL, Sheffler DJ, Kroeze WK. Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nat. Rev. Drug Discov. 2004;3:353–359. doi: 10.1038/nrd1346. - DOI - PubMed
    1. McCorvy JD, Roth BL. Structure and function of serotonin G protein-coupled receptors. Pharmacol. Ther. 2015;150:129–142. doi: 10.1016/j.pharmthera.2015.01.009. - DOI - PMC - PubMed

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