Novel Bivalent 5-HT_2A Receptor Antagonists Exhibit High Affinity and Potency in Vitro and Efficacy in Vivo

Abstract

The 5-HT receptor (5-HTR) plays an important role in various neuropsychiatric disorders, including substance use disorder and schizophrenia. Homodimerization of this receptor has been suggested, but tools that allow direct assessment of the relevance of the 5-HTR:5-HTR homodimer in these disorders are necessary. We chemically modified the selective 5-HTR antagonist M100907 to synthesize a series of homobivalent ligands connected by ethylene glycol linkers of varying lengths that may be useful tools for probing 5-HTR:5-HTR homodimer function. We tested these molecules for 5-HTR antagonist activity in a cell line stably expressing the functional 5-HTR and quantified a downstream signaling target, activation (phosphorylation) of extracellular regulated kinases 1/2 (ERK), in comparison to in vivo efficacy of altering spontaneous or cocaine-evoked locomotor activity in rats. All of the synthetic compounds inhibited 5-HT-mediated phosphorylation of ERK in the cellular signaling assay; the potency of the bivalent ligands varied as a function of linker length, with the intermediate linker lengths being the most potent. The K values for the binding of bivalent ligands to 5-HTR were only slightly lower than the values for the parent (+)-M100907 compound, but significant selectivity for 5-HTR over 5-HTR or 5-HTR binding was retained. In addition, the 11-atom-linked bivalent 5-HTR antagonist (2 mg/kg, intraperitoneally) demonstrated efficacy on par with that of (+)-M100907 in inhibiting cocaine-evoked hyperactivity. As we develop further strategies for ligand-evoked receptor assembly and analyses of diverse signaling and functional roles, these novel homobivalent 5-HTR antagonist ligands will serve as useful in vitro and in vivo probes of 5-HTR structure and function.

Synthesis and activity of functionalizable derivatives of the serotonin (5-HT) 5-HT 2A receptor (5-HT 2A R) antagonist M100907

“…Although compounds 5 and 15 displayed slightly lower potency when compared to parent (+)-M100907, these compounds retained comparable potency to a previously published series of 5-HT 2A R bivalent ligands which contain 14 and 17 ethylene glycol linkers. 3,4 These results indicate that the 1,2,3-triazole ring formed in the click reaction does not significantly affect 5-HT 2A R antagonist activity.…”

“…Intracellular calcium (Ca i 2+ ) release was monitored using FLIPR Calcium 4 Assay Kit (Molecular Devices) according to previously published protocols with minor modifications. 3,4,10–13 Cells were plated in serum-replete medium at 16–20 K cells in 150 ml in black-sided, clear bottom 96-well tissue culture plates. Cells were fed 24 h later with serum-free medium and, following 3 h incubation, medium was removed and replaced with 40 μl of fresh serum-free medium plus 40 ml of Calcium 4 dye solution supplemented with 2.5 mM of water-soluble probenicid (Invitrogen, Carlsbad CA) to inhibit extracellular transport of the dye.…”

“… a pIC 50 is presented as mean ± SEM (n = 3–5 independent experiments), except for 16 ; IC 50 values were calculated from averaged pIC 50 values. b Compound 16 was run in two independent experiments; pIC 50 reported ± SD. c Data previously published 3,4 …”

“…2 Our group reported that M100907 derivatives substituted at the methoxy group of the catechol ring retain the 5-HT 2A R antagonist activity with either the ketone ( 2 ) or racemic hydroxyl group ( 3 ) at the benzylic position. 3,4 Reported here is the installation of a polyethylene glycol (PEG) linker substituted at the methoxy group of M100907 and the chiral resolution of the molecule to provide a version of M100907 possessing an ether tether that is terminated with an azide ( 4 ) or an alkyne ( 5 ). The azide on ( 4 ) will be used for future connection to other molecules such as fluorophores, affinity tags and other receptor ligands (Fig.…”

“…Inhibition of 5-HT 2A R-mediated signaling was determined by measuring the reduction of 5-HT (1 μM) stimulated intracellular calcium (Ca 2+ ) release in CHO-K1 cells stably expressing the 5-HT 2A R. 3,4 Serotonin induces a concentration-dependent increase in Ca i 2+ release with an EC 50 of 4.2 nM (pEC 50 = 8.38 ± 0.10) and 1 μM of 5-HT exhibited maximal intracellular Ca i 2+ release (Fig. 3A).…”