7-Pyrrolidinethoxy-4′-Methoxyisoflavone Prevents Amyloid β–Induced Injury by Regulating Histamine H3 Receptor-Mediated cAMP/CREB and AKT/GSK3β Pathways

In studies on the treatment of Alzheimer’s disease (AD), in which cognition is enhanced even modestly or selectively, it has been considered that the histamine H3 receptor (H3R) may be a potential target. In this study, we aimed at evaluating the ability of 7-pyrrolidinethoxy-4′-methoxyisoflavone (indicated as LC1405), a novel potential H3R antagonist identified from our H3R antagonist screening system, to ameliorate amyloid β (Aβ)-induced cognitive deficits, and to explore the underlying mechanisms that are related to H3R-modulated signaling. Our results demonstrated that LC1405 effectively reduced the progression of Aβ-associated disorders, such as improved learning and memory capabilities, preserved tissues from suffering neurodegeneration and ultrastructural abnormalities, and ameliorated cholinergic dysfunction in an APP/PS1 double transgenic mouse model of AD. In an in vitro model, LC1405 protected neuronal cells against copper-induced Aβ toxicity, as demonstrated by the improvement in cell viability and decrease in neuronal apoptotic ratio. In addition, treatment with LC1405 resulted in the up-regulation of acetylcholine (ACh) or histamine release and provided neuroprotection through cellular signaling cascades involving H3R-mediated cAMP/CREB and AKT/GSK3β pathways. Furthermore, the beneficial effects of LC1405 on Aβ-mediated toxicity and H3R-mediated cAMP/CREB and AKT/GSK3β axes were reversed after pharmacological activation of H3R. In conclusion, our results demonstrated that LC1405 blocked Aβ-induced toxicity through H3R-modulated signaling transduction both in vitro and in vivo. The results also suggested that LC1405 might have translational potential as a complementary therapy to control disease progression in AD patients who developed cognitive deficits with H3R-related ACh neurotransmission abnormality.

To identify H3R ligands, our in-house database, including 29170 compounds, was used as the screening library. Each database was processed by removing the inorganic counter ions, adding hydrogen atoms, deprotonating strong acids, protonating strong bases, generating stereo isomers and valid single 3D conformers by means of washing and minimizing energy in the molecular operating environment (MOE).

Radioligand binding assays
This study was performed to validate the affinity of H3R of predicted H3R compounds using the cellbased H3R antagonism assay. The substituted pyridazinone analogs were tested using in vitro binding assays by displacement of [ 3 H] N α -methylhistamine in membranes isolated from HEK-293 cells that were transfected with cloned human H3R (hH3R) that was reported previously (Kottke et al., 2011). Briefly, HEK-293 cells stably expressing recombinant hH3R were harvested, centrifuged and homogenized in ice-cold binding buffer (50 mmol/L Na2HPO4/KH2PO4, pH 7.5). The homogenate was centrifuged (23,000 g, 30 min, 4°C) and the pellet was resuspended in binding buffer to constitute the membrane preparation that was used for the binding assay. Incubations of the membrane suspension were performed for 90 min at 25°C with either [ 3 H] N α -methylhistamine (5 nM) alone, or a combination with different concentrations of the test compounds (seven concentrations between 0.1 nM and 100 μM). Non-specific binding was determined in the presence of the selective H3R antagonist pitolisant (10 nM), and any bound radioligand was separated from free radioligand by filtration through glass microfiber filters (GF/B Whatman, Clifton, NJ, USA). Unbound radioligand was removed by three washing steps using ice-cold medium (50 mM Tris-HCl, 120 mM NaCl, pH 7.4). The data were analyzed by Hill transformation and Ki values were determined by the Cheng-Prusoff equation (Cheng and Prusoff, 1973). The binding analysis for mouse and rat H3Rs were performed as described above for hH3R. The binding analysis of [3 H] mepyramine, [ 125 I] iodoaminopotentidine and [ 3 H] histamine binding to H1, H2 and H4 receptors, respectively, was performed according to previous reports (Bakker et al., 2004;Łażewska et al., 2014).

Screening for H3R antagonists in a cell-based assay
The cell-based H3R antagonism assay was performed by using U2OS cells that stably contain the human H3R that was linked to a preliminarily integrated beta-arrestin/TEV protease fusion protein and the beta-lactamase (bla) reporter gene under control of an UAS response element (Invitrogen, Carlsbad, CA, USA). H3R-U2OS cells were cultured in FreeStyle™ 293 Expression Medium (Invitrogen, Carlsbad, CA, USA) and plated at 7500 cells/well in a 384-well format and incubated for 18 h at 37°C. Cells were incubated with the test compounds or thioperamide (Sigma-Aldrich, St. Louis, MO, USA), which served as a positive control for 30 min at 37°C. Then, cells were stimulated with 400 nM methylhistamine (Sigma-Aldrich, St. Louis, MO, USA) for 5 h at 37°C. Assays were performed in triplicate using seven test compound concentrations between 0.01 nM and 100 μM. Cells were loaded with LiveBLAzer™-FRET B/G substrate (Invitrogen, Carlsbad, CA, USA) for 2 h prior to the assay.
Fluorescence emission values at 460 nm and 530 nm were obtained using a Spark 20M multimode microplate reader (Tecan Group Ltd., Mannedorf, Switzerland), and the percentage of inhibition was plotted against the indicated concentrations of the test compound. IC50 values were graphically determined from log concentration-inhibition curves.

cAMP accumulation assay
Intracellular cAMP accumulation was measured using HEK293 cells expressing hH3R, co-treated with forskolin, (R)-(−)-α-methylhistamine (RAMH) and LC1405. Briefly, forskolin (10 μM), RAMH (15 nM) and LC1405 (ranging from 0.1 nM-100 μM) were added simultaneously to cell suspension. Cells stimulation was performed for 30 min at room temperature. The cells were treated with lysis buffer, and then 10 μL of the lysate was tested for measurement of cAMP concentration. cAMP accumulation levels were determined using a cAMP assay kit (R&D Systems, Minneapolis, MN, USA). The optical density at 570 nm was read on a Spark 20M multimode microplate reader (Tecan Group Ltd., Mannedorf, Switzerland). Measured OD signal was translated into actual quantities of produced cAMP on the basis of cAMP standard curve.

Primary neuronal cell culture and treatments
Primary cortical neuron cultures were prepared as described previously with minor modifications . Primary cultures of cortical neurons were prepared from Sprague Dawley rat embryos. In brief, the brain cortex was dissected, the cerebral vascular membrane was stripped of, digested with 0.125% trypsin, and dispersed by repeating aspirations through a Pasteur pipette following centrifugation. Cortical neurons were cultured in Neurobasal medium (Invitrogen, Carlsbad, CA, USA), supplemented with B27 (Invitrogen, Carlsbad, CA, USA) and 0.5 mM glutamine and were maintained in a humidified incubator at 37°C with 5% CO2/95% air. Half the volume of medium was removed and replaced every 3 days. Eight days after the initial plating, primary cortical neurons were used for experiments. After initiating aggregated Aβ25-35 or fibrillar β-amyloid 1-40 (fAβ1-40)-induced injury, primary cortical neurons were treated with different concentrations of LC1405 and neurons were incubated for 72 h. In Aβ25-35-and fAβ1-40-induced toxicity models, neurons were randomly divided into two groups, one group was supplemented with 10 μM Aβ25-35 or 20 μM fAβ1-40, whereas the other group was not supplemented with Aβ25-35 or fAβ1-40. Neurons in each group were further divided into subgroups based on LC1405 concentrations: 0.3 μM, 1.0 μM and 3.0 μM.

The construction of ΑPPsw cells
SH-SY5Y cells were cultured in DMEM medium supplemented with fetal bovine serum. The APP adenovirus vector was concocted by Syngentech company (Beijing, China). SH-SY5Y cell were planted in 6-well plate and the APP adenovirus vector were infected into cells. Then, after selected with puromycin, we acquired a monoclonal of cells stably expressed Swedish mutant form of human APP.

Statistical analysis
Data are represented as mean ± SEM. p < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software (SPSS, Inc., Chicago, IL, USA). Comparisons were performed using one-way ANOVA, followed by Tukey's post-hoc testing to analyze the differences between groups.

The performance of three classifiers and in silico prediction of H3R ligands
Among the three generated classifiers, the best naï ve Bayesian (NB) model, named as the M3 model, obtained the highest performance based on 16 descriptors (15 descriptors filtered and ECFP_6), which resulted in Matthews correlation coefficients of 0.990 and 0.994 for the training and test set, respectively. Moreover, the M3 classifier identified important fragments in two classifying groups (positive and negative groups) for Histamine H3, providing 20 positive fragments with favorable binding (Supplementary Fig. 1A), 20 negative fragments (Supplementary Fig. 1B), and corresponding Bayesian scores. To identify H3R lead ligands using the M3 model in drug discovery research, we performed a virtual screening using our in-house database (including 29170 compounds). Using the M3 model, 1273 compounds were identified as potential H3R ligands, among which LC1405 (7-pyrrolidinethoxy-4'-methoxyisoflavone), having a molecular weight of 381.43 Da, got a favorable score of 0.64 (Supplementary Table 2).

In vitro profile of LC1405 in binding and functional assays at the histamine receptors
We evaluated the molecular and cellular action of LC1405 on the human histamine receptor subtypes. As shown in Supplementary Table 2, LC1405 demonstrated a good affinity for hH3Rs in HEK293 cells (65.50 ± 4.48 nM), illustrated a strong activity of H3R antagonism (63.04 ±2.83 nM) and blocked the decrease of cAMP induced by RAMH (158.99 ± 7.95 nM). In addition, LC1405 had weak affinities for other histamine receptors. These results suggested that LC1405 was classified as an antagonist at H3 receptors.

LC1405 illustrated neuroprotective effects against Aβ25-35 and fAβ1-40-induced toxicity in vitro
A cytotoxicity assay was performed to evaluate the neuroprotective effects of LC1405 on rat primary cortical neurons against Aβ25-35-and fAβ1-40-induced toxicity. Cell viability was significantly decreased to 58.02 ± 2.64% and 71.01 ± 2.91% in neurons that were exposed to 10 μM Aβ25-35 and 20 μM fAβ1-40, respectively (Supplementary Fig. 2B ; p < 0.05, p < 0.001). LC1405 treatment did not have significant effects on neurons that were not exposed to Aβ25-35 or fAβ1-40 treatment at any of the concentrations tested.

Expression of Swedish mutant form of human APP were increased in monoclonal APPsw cells.
Supplementary Fig. 4A showed the information of the plasmid of Swedish mutant form of human APP(APPsw). We detected the expression of flag protein which were co-translocation with ΑPPsw and found that monoclonal APPsw cells significantly increased ΑPP695 expression compared to primary infection cells (Supplementary Fig. 4B).

Supplementary Tables
Supplementary Table 1. Molecular descriptors used in this study.