In vitro binding assay for target validation

The receptor binding affinity of KR-37524 was determined by profiling services at Eurofins Cerep (Test No.: FR095-0019211, US034-0011488; I’Evescault, France) using radioligand binding assays for seven distinct human receptors and transporters; α-1A adrenergic receptor (ADRA1A), D(2) dopamine receptor (DRD2), histamine H2 receptor (HRH2), sodium-dependent noradrenaline transporter (SLC6A2), 5-hydroxytryptamine receptor 2A (HTR2A), sodium-dependent serotonin transporter (SLC6A4), and voltage-dependent L-type calcium channel subunit α-1C (CACNA1C). To evaluate the percentage (%) inhibition of specific binding, all radioligand binding assays were performed in 96 well plates at 37℃ in binding buffer (25 mM HEPES, 100 mM NaCl, 2 mM MgCl₂, and 1 mM 3-[(3-cholamidopropy) dimethyl ammonio]-1-propanesulfonate [CHAPS] at pH 7.4 [NaOH]). The human recombinant receptor membranes of the α-1A adrenergic receptor (ADRA1A), sodium-dependent noradrenaline transporter (SLC6A2), 5-hydroxytryptamine receptor 2A (HTR2A), and sodium-dependent serotonin transporter (SLC6A4) were used in the CHO cell membrane, and D(2) dopamine receptor (DRD2), histamine H2 receptor (HRH2), and voltage-dependent L-type calcium channel subunit α-1C (CACNA1C) were used in the HEK293 cell membrane overexpressed by each receptor. The specific radiolabeled ligands of α-1A adrenergic receptor (ADRA1A), D(2) dopamine receptor (DRD2), histamine H2 receptor (HRH2), sodium-dependent noradrenaline transporter (SLC6A2), 5-hydroxytryptamine receptor 2A (HTR2A), sodium-dependent serotonin transporter (SLC6A4), and voltage-dependent L-type calcium channel subunit α-1C (CACNA1C) were [³H]prazosin, [³H]methyl-spiperone, [¹²⁵I]APT, [³H]nisoxetine, [³H]ketanserin, [³H]imipramine, and [³H]PN200-110, respectively. Nonspecific binding was defined in the presence of 0.1 mM epinephrine, 10 μM butaclamol, 100 μM tiotidine, 1 μM desipramine, 1 μM ketanserin, 10 μM imipramine, and 1 μM isradipine, respectively. Ligand binding was determined by filtration of the assay mixture over GF/C Whatman filters (Cytiva, Marlborough, MA, USA). After washing the filters, liquid scintillation counting was performed to quantify the radioactivity. Compound binding was calculated as the % inhibition of the binding of a radioactively labeled ligand specific to each target. In each experiment, and when applicable, the respective reference compound was tested concurrently with KR-37524, and the data were compared with historical values determined at Eurofins. The experiment was performed in accordance with the Eurofins validation standard operating procedure. The receptor binding assays were performed in triplicate, and each result was determined in three independent experiments.