3h spiperone

UCL-2190^{41 (link)}, Ciproxifan⁴² and Pitolisant^{27 (link)} were from own laboratory stocks of which synthesis and analytics have been described previously. G9a-inhibitors A-366 and UNC-0642 as well as G9a enzyme, S-adenosylmethionine (SAM, (2S)-2-amino-4-[[(2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl-methylsulfonio]butanoate), biotinylated histone H3 (1–21) fragment and Dulbecco´s modified eagle medium (DMEM, article no. D5671) were purchased from Sigma-Aldrich, Taufkirchen, Germany. Fetal bovine serum albumin (FBS Good-Forte) and Dulbecco´s Phosphate Buffered Saline (DPBS) were provided by PAN biotech (Aidenbach, Germany). The radioligands [³H]N^α-methylhistamine, [³H]histamine, [³H]spiperone and [³H]SCH23390 were purchased from PerkinElmer (Rodgau, Germany) as well as AlphaLISA materials such as AlphaLISA H3K9me2 acceptor beads, streptavidin-coated donor beads, detection buffer (5x) and white 384-well microplates (OptiPlate). Human or animal blood/tissue/cell samples have not been used in this study.

Rat striatum was homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM ice-cold Tris-HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.1% ascorbic acid and 5 µM pargyline. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.5 nM [³H]spiperone (16.2 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris-HCl buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.1% ascorbic acid and 5 µM pargyline. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [³H]spiperone, 50 µL of 5 µM (+)-butaclamol. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [³H]spiperone, 50 µL of 50 µM new compounds. The tubes were incubated at 37 °C for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.

Receptor binding studies were carried out as described previously27 (link)50 (link)52 (link). The radioligand [³H]spiperone (specific activity 80.6 Ci mmol⁻¹) (Perkin Elmer) was used in saturation experiments to determine the K_d and B_max values for the membrane preparations of stably transfected CHO cells expressing the human D_2L, D_2S48 (link), D₃49 (link) and SNAP-D_2L receptors, respectively,. The K_i values for the compounds were obtained by competition experiments. In brief, the assay were carried out in the 96-well plates at protein concentration of 1–8 μg assay⁻¹ tube in a final volume of 200 μL and [³H]spiperone at final concentrations of 0.125–0.200 nM for D_2L, D_2S, D₃ and SNAP‐D_2L receptors. The K_D values of [³H]spiperone for D_2L, D_2S, D₃ and SNAP-D_2L receptors, were 0.053–0.085, 0.067–0.120, 0.095–0.180 and 0.110–0.130 nM respectively, and the corresponding B_max values were in the range of 610–640, 1500–4800, 4200–6450 and 2000–2100 fmol mg⁻¹.

HEK293T cells were grown to a confluence of 70–80% and transfected with the Nluc-D₃R plasmids using polyethyleneimine (PEI) as the transfection reagent (PEI/DNA ratio 3:1). After incubation in DMEM/F12 with 10% FBS at 37 °C, 5% CO₂ for 48 h, cells were harvested and cell membranes were prepared as described previously^{34 (link)}. The protein concentration was determined using the method of Lowry and bovine serum albumin as standard^{63 (link)}. Membranes (protein concentration 5–20 µg·mL⁻¹) were incubated with the radioligand [³H]spiperone (0.05 nM–2.00 nM, Perkin Elmer) for 1 h at 37 °C in binding buffer (50 mM Tris, 1 mM EDTA, 5 mM MgCl₂, 100 µg·mL⁻¹ bacitracin, 5 µg·mL⁻¹ soybean trypsin inhibitor, pH 7.4). Non-specific binding was determined in the presence of 10 µM haloperidol. Reactions were terminated by filtration through GF/B filters soaked with 0.3% PEI solution. Dried filters were sealed with scintillation wax and bound radioactivity was determined with a Microbeta Counter (Perkin Elmer). Data were analyzed with the one-site saturation binding model implemented in PRISM8.0 (GraphPad Software Inc., San Diego, CA) to determine the equilibrium dissociation constant (K_D) and the receptor expression level (B_max).