α7 nAChR/α7 nicotinic acetylcholine receptor

In rat brain homogenate, PNU-282987 (compound C7) has a Ki of 27 nM and replaces the R7-selective antagonist methylaconitine (MLA) [1]. With an EC50 of 154 nM, PNU-282987 exhibits α7 nAChR agonist activity [1]. Moreover, PNU-282987 blocks 5-HT3 receptors with an IC50 of 4541nM[1].

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Several assays were used to validate that the chimera assay could be used to identify agonists of native receptors. In a binding assay, PNU-282987 displaced the α7 selective antagonist methyllycaconitine (MLA) from rat brain homogenates with a Ki of 27 nM. Also, when applied to cultured rat hippocampal neurons, PNU-282987 evoked a rapidly desensitizing inward whole-cell current that was concentration-dependent and blockable by MLA, consistent with opening of the α7 receptor [1]

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The selectivity of PNU-282987 over related receptors was also evaluated. In particular researchers were concerned with agonism of the neuromuscular junction form of the receptor (α1β1γδ) and the predominant ganglionic nAChR (α3β4). Activation of these receptors was shown to cause many of the undesirable effects of nonspecific agonists such as epibatidine and nicotine. 15 PNU-282987 showed no detectable agonist activity up to 100 μM and negligible antagonist activity (IC50 ≥ 60 μM) at both receptor subtypes. Further, PNU-282987 did not significantly displace tritiated cytisine from rat brain homogenates at 1 μM (14% inhibition), suggesting a high selectivity over the α4β2 subtype. 16 With respect to the 5-HT3 receptor, PNU-282987 displaced tritiated GR-65630 with a Ki of 1662 nM, 17 translating into a selectivity of about 62-fold for α7 compared to the high selectivity of 1 for the 5-HT3R (over 500-fold). In a cell-based FLIPR assay, PNU-282987 was found to be a functional antagonist of the 5-HT3 receptor (IC50 = 4541 nM). Broader selectivity of PNU-282987 was evaluated in a screen of 32 receptors, ion channels, and enzymes at Cerep (Rueil-Malmaison, France). At a test concentration of 1 μM, PNU-282987 produced <30% inhibition of specific binding or enzyme activity at all targets except the 5-HT3 receptor[1].

Gating deficiencies are reversed by PNU-282987 (compound C7) (iv; 1, 3 mg/kg) [1]. In rat hippocampus neurons, PNU-282987 (30 μM) stimulates currents in a concentration-dependent and MLA-blockable way [1].

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Researchers also tested PNU-282987 in a rat model of the impaired sensory gating, which had been validated with the known α7 partial agonist GTS-21. 18 Systemic administration of d-amphetamine (0.3 or 1 mg/kg, iv) significantly disrupted auditory gating in anesthetized rats because of a combination of simultaneous decreases of conditioning responses with corresponding increases in test responses. Subsequent administration of the α7 nAChR agonist PNU-282987 (iv, 1 or 3 mg/kg, n = 10) significantly reversed amphetamine-induced gating deficit (Figure 4). In contrast, application of vehicle in control rats did not normalize the amphetamine-induced gating deficit (n = 9). Furthermore, PNU-282987 (1 mg/kg) had no significant effect on normal gating (n = 4) in anesthetized rats.

Brain homogenate binding assays ([3 H]-MLA, [3 H]-cytisine, [3 H]-GR65630): [1]
Male Sprague-Dawley rats (300-350 g) were sacrificed by decapitation and the brains (whole brain minus cerebellum) were dissected quickly, weighed and homogenized in 9 volumes/g wet weight of ice-cold 0.32 M sucrose using a rotating pestle on setting 50 (10 up and down strokes). The homogenate was centrifuged at 1,000 x g for 10 minutes at 40° C. The supernatant was collected and centrifuged at 20,000 x g for 20 minutes at 40° C. The resulting pellet was resuspended to a protein concentration of 1 - 8 mg/ml. Aliquots of 5 ml homogenate were frozen at -80° C until needed for the assay. On the day of the assay, aliquots were thawed at room temperature and diluted with Kreb’s - 20 mM HEPES buffer pH 7.0 (at room temperature) containing 4.16 mM NaHCO3, 0.44 mM KH2PO4, 127 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl2, and 0.98 mM MgCl2, so that 25 - 150 mg protein are added per test tube. Protein concentration was determined by the Bradford method using bovine serum albumin as the standard. For α7, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 µM MLA, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 3 nM [3 H]-MLA (25 Ci/mmol). For α4, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 mM (-)-nicotine, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 1.0 nM [3 H]-cytisine. For 5-HT3, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 µM ICS-205930, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 0.45 nM [3 H]-GR65630. For all binding assays, 0.4 ml homogenate was added to test tubes containing buffer, test compound and radioligand, and was incubated in a final volume of 0.5 ml for 1 h at 25°. The incubations were terminated by rapid vacuum filtration through Whatman GF/B glass filter paper mounted on a 48 well Brandel cell harvester. Filters were pre-soaked in 50 mM Tris HCl pH 7.0 - 0.05 % polyethylenimine. The filters were washed two times with 5 ml aliquots of cold 0.9% saline and then counted for radioactivity by liquid scintillation spectrometry. The inhibition constant (Ki) was calculated from the concentration dependent inhibition of radioligand binding obtained by fitting the data to the Cheng-Prusoff equation. PNU-282987 had a Ki in this assay of 27 ± 1 nM (n = 48).
PNU-282987 did not significantly displace tritiated cytisine from rat brain homogenates at 1 µM (inhibition = 14 ± 4 %, n=13) With respect to the 5-HT3 receptor, PNU-282987 displaced tritiated GR-65630 with a Ki of 1,662 ± 331 nM (n=10)

Functional High-Throughput Screens for α7/5-HT3 chimera, 5-HT3, neuromuscular junction (α1β1γδ) and ganglionic (α3β4) nAChRs: [1]
The α 7/5-HT3 chimera and the 5-HT3 receptor were stably expressed in SH-EP1 cells. TE671 and SH-SY5Y cells were used as an endogenous source for neuromuscular junction and ganglionic nAChRs, respectively. All functional high throughput screens were conducted as calcium flux assays using the Fluorescence Imaging Plate Reader. Transfected SH-EP1 cells were grown in minimal essential medium (MEM) containing nonessential amino acids supplemented with 10% fetal bovine serum, L-glutamine, 100 units/ml penicillin/streptomycin, 250 ng/ml fungizone, 400 µg/ml Hygromycin-B, and 800 µg/ml Geneticin. TE671 and SH-SY5Y cells were grown according to published methods. All cells were grown in a 37° C incubator with 6% CO2 . The cells were trypsinized and plated in 96 well plates with dark side walls and clear bottoms at density of 26×104 cells per well two days before analysis. Cells were loaded in a 1:1 mixture of 2 mM Calcium Green-1, AM prepared in anhydrous dimethylsulfoxide and 20% pluronic F-127. This reagent was added directly to the growth medium of each well to achieve a final concentration of 2 µM Calcium Green-1, AM. Cells were then incubated in the dye for one hour at 37° C and then washed with 4 cycles of Bio-Tek plate washer. Each cycle was programmed to wash each well four times with Mark’s Modified Earle's Balanced Salt Solution (MMEBSS) composed of (in mM): CaCl2 (4), MgSO4 (0.8), NaCl (20), KCl (5.3), D-Glucose (5.6), Tris-HEPES (20), N- methyl-D-glucamine (120), pH 7.4. After the third cycle, the cells were allowed to incubate at 37° C for at least ten min. The final volume of MMEBSS in each well was 100 µl. FLIPR was set up to excite Calcium Green with at 488 nm using 500 mW of power and reading fluorescence emission above 525 nm. A 0.5 s exposure was used to illuminate each well. Fluorescence was detected using a F-stop set of either 2.0 or 1.2. Specifically, after 30 s of baseline recording, test compounds were added to each well of a 96 well plate using 50 µL from a 3× stock. In each experiment, 4 wells were used as solvent controls. Data from cell-based FLIPR assays for PNU-282987: α7/5-HT3 receptor chimera (EC50 = 178 ± 5 nM (n=70)). 5-HT3 receptor functional antagonist (IC50 = 4,541 ± 413 nM, n=46)). α3: no detectable agonist activity at concentrations up to 100 µM (n = 69), for antagonist activity, IC50 ≥ 60µM (n = 70) α1 no detectable agonist activity at concentrations up to 100 µM (n = 69), for antagonist activity, IC50 ≥ 60µM (n = 10).

Animal/Disease Models: Rat[1]
Doses: 1, 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Dramatically reversed amphetamine-induced gating defects.

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Patch-clamp electrophysiology: Cultured neurons were prepared according to Brewer . Briefly, Sprague- Dawley rats (postnatal day 3) were killed by decapitation and their brains were removed and placed in ice cold Hibernate-A medium. Hippocampal regions were gently removed, cut into small pieces and placed in Hibernate-A medium with 1 mg/ml papain for 60 min at 35°C. After digestion, the tissues were washed several times in Hibernate-A media and transferred to a 50 ml conical tube containing 6 ml Hibernate-A medium with 2% B-27 supplement. Neurons were dissociated by gentle trituration and plated onto poly-D-lysine/laminin coated coverslips at a density of 300 – 700 cells/mm2 , and transferred to 24-well tissue culture plates containing warmed culture medium composed of Neurobasal-A medium, B-27 supplement (2%), L-glutamine (0.5 mM), 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml Fungizone. Cells were maintained in a humidified incubator at 37°C and 6% CO2 for 1 – 2 weeks. The medium was changed after 24 h and then approximately every three days thereafter. Patch pipettes were pulled from borosilicate capillary glass using a Flaming/Brown micropipette puller and filled with an internal pipette solution composed of (in mM): CsCH3SO3 (126), CsCl (10), NaCl (4), MgCl2 (1), CaCl2 (0.5), EGTA (5), HEPES (10), ATP-Mg (3), GTP-Na (0.3), phosphocreatin (4), pH 7.2. The resistances of the patch pipettes when filled with internal solution ranged between 3 – 6 M•. All experiments were conducted at room temperature. Cultured cells were continuously superfused with an external bath solution containing (in mM): NaCl (140), KCl (5), CaCl2 (2), MgCl2 (1), HEPES (10), glucose (10), bicuculline (0.01), CNQX (0.005), D-AP-5 (0.005) tetrodotoxin (0.0005), pH 7.4. Compounds were dissolved in water or DMSO and diluted into the external bath solution containing a final DMSO concentration of 0.1% and delivered via a multibarrel fast perfusion system. Whole-cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Analog signals were filtered at 1/5 the sampling frequency, digitized, stored, and measured using pCLAMP software (Axon Instruments). All data are reported as mean ± SEM. [1]

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Auditory gating assay. Experiments were performed on Male Sprague-Dawley rats (weighing 250 to 300 gm) under chloral hydrate anesthesia (400 mg/kg, IP). The femoral artery and vein were cannulated for monitoring arterial blood pressure and administration of drugs or additional doses of anaesthetic, respectively. Unilateral hippocampal field potential (EEG) was recorded by a metal monopolar macroelectrode placed into the CA3 region (co-ordinates: 3.0 – 3.5 mm posterior from the bregma, 2.6 –3.0 mm lateral and 3.8 – 4.0 mm ventral; Paxinos and Watson, 19863 ). Field potentials were amplified, filtered (0.1 – 100 Hz), displayed and recorded for on-line and off-line analysis (Spike3). Quantitative EEG analysis was performed by means of Fast Fourier Transformation (Spike3). The auditory stimulus consisted of a pair of 10 ms, 5 KHz tone bursts with a 0.5 s delay between the first “conditioning” stimulus and second “test” stimulus. Auditory evoked responses were computed by averaging of responses to 50 pairs of stimuli presented with an interstimulus interval of 10 s. Percentage of gating was determined by the formula: (1 - test amplitude/conditioning amplitude) x 100. Amphetamine (D-amphetamine sulfate, 0.3-1 mg/kg, IV) was administered in order to disrupt sensory gating. Recordings of evoke potentials commenced 5 min after amphetamine administration, and only rats showing gating deficit exceeding 20 % were used for subsequent evaluation of α7 nAChR agonists or vehicle. Statistical significance was determined by means of two-tailed paired Student’s t-test. [1]

[1]. Discovery and structure-activity relationship of quinuclidine benzamides as agonists of alpha7 nicotinic acetylcholine receptors. J Med Chem.

PNU-282987 is a potent alpha7 nicotinic acetylcholine receptor (nAChR) agonist. PNU-282987 is also a functional antagonist of the 5-HT3 receptor. PNU-282987 can be used for the research of central and peripheral nervous systems.

C14H18CL2N2O

301.21

300.079

C, 55.83; H, 6.02; Cl, 23.54; N, 9.30; O, 5.31

123464-89-1

737727-12-7 (S enantiomer); 128311-08-0 (S enantiomer hydrochloride); PNU-282987 free base;711085-63-1

11243536

White to off-white solid powder

1.3±0.1 g/cm3

431.5±30.0 °C at 760 mmHg

214.8±24.6 °C

0.0±1.0 mmHg at 25°C

1.612

2.49

2

2

2

19

307

1

O=C(N[C@H]1CN2CCC1CC2)C3=CC=C(Cl)C=C3.[H]Cl

HSEQUIRZHDYOIX-ZOWNYOTGSA-N

InChI=1S/C14H17ClN2O.ClH/c15-12-3-1-11(2-4-12)14(18)16-13-9-17-7-5-10(13)6-8-17;/h1-4,10,13H,5-9H2,(H,16,18);1H/t13-;/m0./s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 1 mg/mL (3.32 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (3.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 50 mg/mL (166.00 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)