Figure 1.
Chemical structure and synthetic routes for ZEL-H16.
Reaction of indole-3-propionic acid with piperidine provided 2, which was alkylated with 1-bromo-3-chloropropane in the presence of NaH in anhydrous DMF to get 3. Reaction of 3 with piperdine in refluxing acetonitrile afforded 4, followed by reduction with LiAlH4 to yield ZEL-H16.
Figure 2.
Identify ZEL-H16 as a partial agonist in CRE-driven luciferase activity assay in HEK-293 cells stably expressing hH3R.
A, Concentration inhibition curve of luciferase activity induced by ZEL-H16 with forskolin stimulation. B, Luciferase activity with Forskolin stimulation in response to ZEL-H16 and histamine in the absence and presence of PTX or Thioperamide. C. Luciferase activity with Forskolin stimulation in response to 10 µM Histamine in the presence of 2 µM ZEL-H16. D and E. Luciferase activity with Forskolin stimulation in response to ZEL-H16 (D) and histamine (E) in the absence and presence of 30 nM, 300 nM or 1 µM Thioperamide. The presented data points are the mean ±SE of triplicate values from a single experiment and are representative of three to six separate experiments (*p<0.05; **p<0.01).
Figure 3.
The selectivity of ZEL-H16 for hH3R in intracellular Ca2+ flux assay and CRE-luciferase transcription assay.
A–C, Intracellular Ca2+ flux or CRE-driven luciferase activity induced by ZEL-H16 in HEK-293 cells stably expressing hH1R (A), hH2R (B), or hH4R (C). D–F, The influence of ZEL-H16 on intracellular Ca2+ flux or CRE-driven luciferase activity induced by histamine in HEK-293 cells stably expressing hH1R(D), hH2R(E), or hH4R(F). The presented data points are the mean ±SE of triplicate values from a single experiment and are representative of three separate experiments. Statistical analysis between ZEL-H16 added or not added for each concentration point in the kinetic graph was done using a t-test (PRISM software). G, Relative expression of hH1R, hH2R, hH3R and hH4R on transfected HEK-293 cells by ELISA quantification for Flag-tagged cell-surface receptors (***p<0.001, compared to non-transfected HEK 293 cells).
Table 1.
The selectivity of ZEL-H16 on other biogenic amine GPCRs in CRE-luciferase transcription assaysa.
Figure 4.
Competition binding experiments with [3H]-N-α-methylhistamine.
A. H3R/HEK-293 cell membranes with 10 nM [3H]-N-α-methylhistamine; B. Rat cerebral cortex membranes with 2 nM [3H]-N-α-methylhistamine. The presented data are the means ±SE of values from a single experiment performed in duplicate and are representative of three to six separate experiments.
Figure 5.
Internalization of H3R-EGFP stably expressed in HEK-293 cells induced by ZEL-H16.
A, HEK-293 cells stably expressing H3R-EGFP were stimulated with histamine, ZEL-H16, or imetit for 45 min respectively. (a) control; (b) 100 µM Histamine; (c) 100 µM ZEL-H16; (d) 100 µM imetit. B, ELISA quantification for Flag-tagged cell-surface receptors showed a concentration -dependent internalization of H3R induced by ZEL-H16. C, Internalization of H3R induced by 5 µM ZEL-H16 in the absence and presence of thioperamide (*p<0.05). ELISA data are expressed as the percentage of receptors detected on the surface of agonist-untreated cells expressing H3R. Error bars represent the SEM for four replicates.
Figure 6.
Localization of internalized H3R-EGFP stably expressed in HEK-293 cells and recycling of internalized H3R to the cell surface.
A. HEK-293 cells stably expressing H3R-EGFP were incubated with or without 20 µM ZEL-H16 and 500 µM histamine in the presence of 100 g/ml Alexa Fluor546-labeled transferrin for 45 min at 37°C. B. H3R-EGFP expressing cells were treated with 100 µg/ml cycloheximide and 20 µM ZEL-H16 or 500 µM histamine at 37°C for 30 min, followed by the removal of residual agonists by washing, and further incubation in the presence of cycloheximide for the indicated time periods. The internalized receptors were recycled to the plasma membrane within 1 h after histamine removal and 3 h after ZEL-H16 removal. All pictures shown are representative of at least three independent experiments.
Figure 7.
Phosphorylation of ERK1/2 induced by ZEL-H16 in H3R-expressing HEK-293 cells.
A, time-dependent phosphorylation of ERK1/2 induced by histamine or ZEL-H16. Following 3 h of serum starvation, cells were treated with 1 µM histamine or ZEL-H16 for the indicated time period and cell lysates were analyzed for phosphorylated ERK (p-ERK) and total ERK (ERK) protein levels. Signals were quantified by densitometric image analysis and p-ERK was normalized to a loading control (ERK). The signal at each point is expressed as the percentage of the maximal p-ERK signal induced by histamine. A statistical analysis between ZEL-H16 and histamine for each time point in the kinetic graph was done using a t-test (PRISM software) (*p<0.05; **p<0.01). B, Concentration-dependent phosphorylation of ERK1/2 induced by histamine or ZEL-H16. After serum starvation, cells were incubated with increasing concentrations of ZEL-H16 or histamine (10 nM to 100 µM) and cell lysates were analyzed for p-ERK and ERK levels. Concentration -dependent phosphorylation signals were quantified by densitometric analysis and p-ERK levels were normalized to total ERK levels. The signal at each point is expressed as the percentage of the maximal p-ERK signal induced by histamine. C, The phosphorylation of ERK1/2 induced by ZEL-H16 could be entirely abolished by co-incubation with H3R antagonist thioperamide (***p<0.001). Data represent the mean ±SE of three independent experiments.
Figure 8.
Phosphorylation of ERK1/2 induced by ZEL-H16 in mouse cortical neurons.
A, Time-dependent phosphorylation of ERK1/2 induced by 5 µM ZEL-H16 in neonatal mouse cortical neuron cultures. B, The phosphorylation of ERK1/2 induced by 50 µM ZEL-H16 for 20 min was mostly abolished by 100 ng/mL PTX or 10 µM thioperamide. Signals were quantified by densitometric image analysis and p-ERK was normalized to a loading control (ERK). The signal at each point is expressed as the percentage of the maximal p-ERK signal induced by ZEL-H16. Data represent the mean ±SE of three independent experiments.
Figure 9.
Effect of ZEL-H16-induced and histamine-induced inhibition of the electrically induced contraction of the guinea-pig ileum.
Each point is the mean ± s.e.m. of three to six separate experiments.