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. 2018 Jan 25;8(1):1572.
doi: 10.1038/s41598-018-19714-2.

Development of Novel Fluorescent Histamine H 1-receptor Antagonists to Study Ligand-Binding Kinetics in Living Cells

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Free PMC article

Development of Novel Fluorescent Histamine H 1-receptor Antagonists to Study Ligand-Binding Kinetics in Living Cells

Leigh A Stoddart et al. Sci Rep. .
Free PMC article

Abstract

The histamine H1-receptor (H1R) is an important mediator of allergy and inflammation. H1R antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor H1R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor. Two different pharmacophores (mepyramine or the fragment VUF13816) were used to generate fluorescent H1R antagonists conjugated via peptide linkers to the fluorophore BODIPY630/650. Kinetic properties of the probes showed wide variation, with the VUF13816 analogues having much longer H1R residence times relative to their mepyramine-based counterparts. The kinetics of these fluorescent ligands could also be monitored in membrane preparations providing new opportunities for future drug discovery applications.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Chemical structure and molecular modelling of fluorescent ligands for the H1R. (a) Chemical structures of fluorescent mepyramine (10, 11, 12) and VUF13816 (23, 24, 25) derivatives (b) Modelled binding pose of 23 in the H1R, based on the experimentally validated binding mode of VUF13816, in the H1R crystal structure (PDB code: 3RZE).
Figure 2
Figure 2
Pharmacological characterisation and live cell confocal imaging of fluorescent ligands at the H1R. (a and b) Inhibition of specific [3H]mepyramine binding to cell homogenates transiently expressing the human H1R by mepyramine (a) and VUF13816 (b) based fluorescent antagonists. Data points represent mean ± SEM of triplicate determinations. Graphs shown are representative of three experiments performed. (c and d) CHO-K1 cells stably expressing the human H1R were preincubated with 100 nM of the indicated fluorescent antagonist for 30 min at 37 °C prior to the addition of increasing concentrations of histamine and changes in intracellular calcium monitored. Data were normalized to basal (in the absence of histamine or antagonist) and 100 µM histamine for each experiment. The data shown represent the mean ± SEM of four experiments performed in triplicate. (e and f) Live cell confocal images of CHO cells expressing H1-YFP incubated with 50 nM (e) 10 or (f) 23 at 37 °C in the absence (top panels) or presence (bottom panels) of 10 µM mepyramine. Single equatorial images were taken of YFP (left hand panels) and BY630/650 (right hand panels). YFP and BY630/650 images are shown in greyscale to avoid issues with colour rendering. For each compound, images in the presence and absence of mepyramine were obtained using identical settings for laser power, detector offset and gain. Data shown are representative of images obtained in three independent experiments. Scale bars = 20 µm.
Figure 3
Figure 3
Association binding of [3H]mepyramine at HA-H1R and Nluc-H1R. Specific [3H]mepyramine binding over time to cell homogenate transiently expressing the human H1R was measured for cells expressing the HA-tagged receptor (a) or the Nluc-tagged receptor (b). Data points represent mean ± SEM of duplicate determinations. Graphs shown are representative of 12 (a) or 4 (b) experiments performed.
Figure 4
Figure 4
Saturation and competition NanoBRET studies with Fluorescent Ligands 10 and 23. (a and b) Saturation binding curves from BRET experiments in Nluc-H1R HEK cells treated with increasing concentrations of (a) 10 or (b) 23 in the absence (black circles) or presence (red squares) of 10 µM mepyramine. The data shown are representative of six independent experiments performed in triplicate. (cf) Inhibition of BRET signal in Nluc-H1R HEK293T cells treated with 25 nM 10 (c,d) or 25 nM 23 (e,f) and increasing concentrations of the unlabelled ligands; tripolidine, loratadine, mepyramine and promethazine (c,e) or levocetirizine, fexofenadine, olopatadine and doxepin (d,f). Data were normalized to maximal BRET signal in the absence of competitor. The data shown represent the mean ± SEM of at least three experiments (as detailed in Table 3) performed in triplicate.
Figure 5
Figure 5
NanoBRET association kinetic studies with mepyramine and VUF13816 based fluorescent ligands. Nluc-H1R HEK293 cells were treated with the indicated concentrations of 10 (a), 11 (b), 12 (c), 23 (d), 24 (e) and 25 (f) and BRET monitored at room temperature every min for 60 min (ac) or 90 min (df). The data shown are representative examples from four (10, 23, 24, 25), five (11) or six (12) independent experiments performed in triplicate.
Figure 6
Figure 6
Use of fluorescent ligand 10 to determine the kinetics of unlabelled ligands. Nluc-H1R HEK293 cells were treated with 10 nM 10 and the indicated concentrations of promethazine (a) and loratidine (b) and BRET monitored at room temperature every min for 60 min. The data shown are representative examples from five independent experiments performed in triplicate.
Figure 7
Figure 7
NanoBRET saturation, competition and kinetic binding studies with fluorescent ligand 10 in cell membranes. (a). Saturation BRET binding curves from membranes of Nluc-H1R HEK293 cells treated with increasing concentrations of 10 in absence (black circles) or presence (red squares) of 10 µM mepyramine. The data shown are representative of five independent experiments performed in triplicate. (b and c) Inhibition of BRET signal in membranes of Nluc-H1R HEK293T cells treated with 25 nM 10 and increasing concentrations of unlabelled ligands; tripolidine, loratadine, mepyramine and promethazine (b) or levocetirizine, fexofenadine, olopatadine and doxepin (c). Data were normalized to the maximal BRET ratio in each experiment. Data points are combined mean ± SEM from four separate experiments performed in triplicate (d). Membranes of HEK293T cells stably expressing Nluc-H1R treated with various concentrations of 10 were monitored by BRET at room temperature every min for 30 min. All experiments using membranes expressing Nluc-H1R were performed in the presence of 1 mg/ml saponin. The data shown are representative of four independent experiments performed in triplicate.

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