Abolishing Dopamine D_2long/D₃ Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H₂ Receptor Agonists

Abstract

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H 2 receptor (H 2 R) agonists, but their applicability as pharmacological tools to elucidate the largely unknown H 2 R functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D 2 -like receptors (especially to the D 3 R). To improve the selectivity, a series of novel carbamoylguanidinetype ligands containing various heterocycles, spacers, and side residues were rationally designed, synthesized, and tested in binding and/or functional assays at H 1−4 and D 2long/3 receptors. This study revealed a couple of selective candidates (among others 31 and 47), and the most promising ones were screened at several off-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42, and 7.35) are responsible for the different affinities at the H 2 -and D 2long/3 -receptors. These results provide a solid base for the exploration of the H 2 R functions in the brain in further studies.

Fluorescent Tools for Imaging and Ligand Screening of Dopamine D2-Like Receptors

“…Homogenates containing the D 1 R and D 5 R were prepared and radioligand binding experiments with cell homogenates were performed as previously described with minor modifications. [36,37] For radioligand competition binding assays homogenates were incubated in BB at a final concentration of 0.3 μg (D 1 R), 0.3 μg (D 2long R), 0.7 μg (D 3 R), 0.5-1.0 μg (D 4.4 R), or 0.4 μg (D 5 R) protein/well. [ 3 H]SCH-23390 (D 1 R ( K d = 0.23 nM) and D 5 R ( K d = 0.20 nM)) was added in final concentrations of 1.0 nM (D 1 R) and 1.0 nM (D 5 R).…”

Shedding Light on the D1-Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1and D5Receptors

“…Homogenates containing the D1R and D5R were prepared and radioligand binding experiments with cell homogenates were performed as previously described with minor modifications. [66,67] For radioligand competition binding assays homogenates were incubated in BB at a final concentration of 0.3 μg (D1R), 0.3 μg (D2longR), 0.7 μg (D3R), 0.5-1.0 μg (D4.4R), or 0.4 μg (D5R) protein/well. [ 3 H]SCH-23390 (D1R ( K d = 0.23 nM) and D5R ( K d = 0.20 nM)) was added in final concentrations of 1.0 nM (D1R) and 1.0 nM (D5R).…”

“…Homogenates containing the D 1 R and D 5 R were prepared and radioligand binding experiments with cell homogenates were performed as previously described with minor modifications. [66,67] For radioligand competition binding assays homogenates were incubated in BB at a final concentration of 0. Competition binding curves were fitted using a four-parameter fit ("log(agonist) vs. response-variable slope").…”

Shedding Light on the D1‐Like Receptors: A Fluorescence‐Based Toolbox for Visualization of the D1 and D5 Receptors**

“…Homogenates containing the D 1 R and D 5 R were prepared and radioligand binding experiments with cell homogenates were performed as previously described with minor modifications. [77,78] For radioligand competition binding assays homogenates were incubated in BB at a final concentration of 0. D 5 R) at room temperature, bound radioligand was separated from free radioligand through PEI-coated GF/C filters using a Brandel harvester (Brandel Inc., Unterföhring, Germany), filters were transferred to (flexible) 1450-401 96-well sample plates (PerkinElmer, Rodgau, Germany) and after incubation with scintillation cocktail (Rotiszint eco plus, Carl Roth, Karlsruhe, Germany) for at least 3 h, radioactivity was measured using a MicroBeta2 plate counter (PerkinElmer, Waltham, MA, USA).…”