2.2. NMDA receptor binding studies

In vitro binding affinities (K_i) of target compounds were determined by utilizing competitive radioligand binding assays with (+)-[3-³H]-MK-801 ([³H]MK-801, PerkinElmer, 15–30 Ci/mmol), in accordance with established protocols (Reynolds and Sharma, 1999, Reynolds, 2001) similar to previous investigations (Wallach et al., 2016; Colestock et al., 2018; Kang et al., 2017). Rat forebrain homogenate was used as the source of NMDA receptors (rat brains obtained from Pel-Freez Biologicals, Rogers, AR, USA) and prepared as described by Reynolds and Sharma (1999). Suspensions of 10 mM HEPES buffer (pH 7.4) containing 100 μg/mL protein, 1.36 nM [³H]MK-801, 100 μM glutamate, 10 μM glycine and seven concentrations (10⁻⁴-10⁻¹⁰ M) of unlabeled test drugs as HCl salts dissolved in HEPEs buffer (competitors) were incubated in the dark on a mechanical rocker at room temperature for 2 h. (+)-MK-801 hydrogen maleate (30 μM; MedChem Express, USA) was used to define nonspecific binding. The reaction was terminated by vacuum filtration using a 24-well cell harvester (Brandel, Gaithersburg, MD, USA) over presoaked (15 min) GF/B glass fiber filters (Brandel, Gaithersburg, MD, USA). Filters were washed with room temperature HEPES buffer (15 mL per well). Tritium trapped on the filter was measured via liquid scintillation counting (Ultima Gold, PerkinElmer), using a Beckman LS 6500 multipurpose scintillation counter (BeckmanCoulter, USA) at 57% efficiency. IC₅₀ values were obtained by utilizing Graphpad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) using nonlinear regression with log of the concentrations plotted against percent specific binding. K_i values were calculated using the equation of Cheng and Prusoff (1973). The K_d for (+)-MK-801 hydrogen maleate was determined to be 1.747 nM in previous studies. Protein concentrations were determined via the Bradford method using Coomassie protein assay reagent and rat albumin as standard (Fraction V, Sigma Aldrich, USA) (Bradford, 1976). Experiments were performed in duplicate and repeated three times. Finally (+)-MK-801 maleate (MedChem Express, USA) was also run (range 10⁻⁶–10⁻¹¹, in duplicate, and experiments repeated three times) as a comparison control for this set of experiments.