Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4-iodoamphetamine elicited head-twitch response model

Abstract

There are seemingly conflicting data in the literature regarding the role of serotonin (5-HT) 5-HT2C receptors in the mouse head-twitch response (HTR) elicited by the hallucinogenic 5-HT2A/2B/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Namely, both 5-HT2C receptor agonists and antagonists, regarding 5-HT2C receptor-mediated Gq-phospholipase C (PLC) signaling, reportedly attenuate the HTR response. The present experiments tested the hypothesis that both classes of 5-HT2C receptor compounds could attenuate the DOI-elicited-HTR in a single strain of mice, C57Bl/6J. The expected results were considered in accordance with ligand functional selectivity. Commercially-available 5-HT2C agonists (CP 809101, Ro 60-0175, WAY 161503, mCPP, and 1-methylpsilocin), novel 4-phenyl-2-N,N-dimethyl-aminotetralin (PAT)-type 5-HT2C agonists (with 5-HT2A/2B antagonist activity), and antagonists selective for 5-HT2A (M100907), 5-HT2C (SB-242084), and 5-HT2B/2C (SB-206553) receptors attenuated the DOI-elicited-HTR. In contrast, there were differential effects on locomotion across classes of compounds. The 5-HT2C agonists and M100907 decreased locomotion, SB-242084 increased locomotion, SB-206553 resulted in dose-dependent biphasic effects on locomotion, and the PATs did not alter locomotion. In vitro molecular pharmacology studies showed that 5-HT2C agonists potent for attenuating the DOI-elicited-HTR also reduced the efficacy of DOI to activate mouse 5-HT2C receptor-mediated PLC signaling in HEK cells. Although there were differences in affinities of a few compounds at mouse compared to human 5-HT2A or 5-HT2C receptors, all compounds tested retained their selectivity for either receptor, regardless of receptor species. Results indicate that 5-HT2C receptor agonists and antagonists attenuate the DOI-elicited-HTR in C57Bl/6J mice, and suggest that structurally diverse 5-HT2C ligands result in different 5-HT2C receptor signaling outcomes compared to DOI.

Molecular Pharmacology and Ligand Docking Studies Reveal a Single Amino Acid Difference between Mouse and Human Serotonin 5-HT2A Receptors That Impacts Behavioral Translation of Novel 4-Phenyl-2-dimethylaminotetralin Ligands

“…A description of the wild-type (WT) m5-HT2 and h5-HT2 receptor cDNA constructs used was reported recently (Canal et al, 2013). Point mutations of residue 5.46 of both m5-HT2A and h5-HT2A receptors were made by polymerase chain reactions using the QuikChange II SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol.…”

“…Radioligand saturation isotherm and competitive displacement binding assays were performed in 96-well plates using 5-HT2A, 5-HT2B, or 5-HT2C receptors transiently expressed in HEK293 cells as previously described (Canal et al, 2011(Canal et al, , 2013 …”

“…Receptor-mediated phosphoinositide hydrolysis by phospholipase C (PLC) was measured as previously described (Canal et al, 2013 H-Induced scintillations were counted with a scintillation analyzer. Experiments were performed with duplicates for 5-HT and triplicates for all other drugs, and each independent experiment was performed a minimum of three times.…”

“…Drug discovery programs targeting 5-HT2 receptors are focused on treating psychosis (5-HT2A antagonists), sleep disorders (5-HT2A antagonists), cluster headaches (5-HT2 agonists), anxiety (5-HT2B/2C antagonists), and obesity (5-HT2C agonists) (Sewell et al, 2006;Abbas and Roth, 2008;Smith et al, 2010). In addition, compounds with combined 5-HT2C agonist plus 5-HT2A/2B antagonist activity, similar to certain trans-4-phenyl-2-dimethylaminotetralins (PATs), may be useful for treating substance abuse and other compulsive behavioral disorders Canal et al, 2013;Cunningham et al, 2013;Higgins et al, 2013;Morgan et al, 2013).…”

“…Using a structure-based drug design approach, novel PATs targeting 5-HT2 receptors are being developed to treat compulsive behavioral and other neuropsychiatric disorders (Rowland et al, 2008;Booth et al, 2009;Canal et al, 2013;Morgan et al, 2013). Reported here are results from in silico m5-HT2A and h5-HT2A molecular modeling/ligand docking studies, in vitro molecular pharmacology studies, and in vivo neurobehavioral studies using the DOI-elicited HTR model for the trans-(2)-and trans-(1)-enantiomers of two PAT-type drug candidates, 6-OH-7-Cl-PAT and 6-OMe-7-Cl-PAT (Fig.…”