title: Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors.
abstract: We have previously identified a novel class of 5-hydroxytryptamine type 3 receptor (5-HT3R) agonists sharing little structural similarity with orthosteric 5-HT3R ligands (Jørgensen et al., 2011). In the present study we have elucidated the functional characteristics and the mechanism of action of one of these compounds, trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA). In electrophysiological recordings TMPPAA was found to be a highly-efficacious partial agonist equipotent with 5-HT at the 5-HT3A receptor (5-HT3AR) expressed in COS-7 cells and somewhat less potent at the receptor expressed in Xenopus oocytes. The desensitization kinetics of TMPPAA-evoked currents were very different from those mediated by 5-HT. Moreover, repeated TMPPAA applications resulted in progressive current run-down and persistent non-responsiveness of the receptor to TMPPAA, but not to 5-HT. In addition to its direct activation, TMPPAA potentiated 5-HT-mediated 5-HT3AR signalling, and the allosteric link between the two binding sites was corroborated by the analogous ability of 5-HT to potentiate TMPPAA-evoked responses. The agonism and potentiation exerted by TMPPAA at a chimeric α7-nACh/5-HT3A receptor suggested that the ligand acts through the transmembrane domain of 5-HT3AR, a notion further substantiated by its functional properties at chimeric and mutant human/murine 5-HT3ARs. A residue in the transmembrane helix 4 of 5-HT3A was identified as an important molecular determinant for the different agonist potencies exhibited by TMPPAA at human and murine 5-HT3ARs. In conclusion, TMPPAA is a novel allosteric agonist and positive allosteric modulator of 5-HT3Rs, and its aberrant signalling characteristics compared to 5-HT at the 5-HT3AR underline the potential in Cys-loop receptor modulation and activation through allosteric sites.
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Agnes
authors.initials: A
authors.lastname: Gasiorek
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Trattnig
authors.affiliation: Saniona A/S, Baltorpvej 154, DK-2750 Ballerup, Denmark.
authors.forename: Philip K
authors.initials: PK
authors.lastname: Ahring
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Uffe
authors.initials: U
authors.lastname: Kristiansen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Frølund
authors.affiliation: H. Lundbeck A/S, Otiliavej 9, DK-2500 Valby, Denmark.
authors.forename: Kristen
authors.initials: K
authors.lastname: Frederiksen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Electronic address: aaj@sund.ku.dk.
authors.forename: Anders A
authors.initials: AA
authors.lastname: Jensen
mesh_terms: D000178:Acrylamides / Q000138:chemical synthesis / Q000494:pharmacology*; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005071:Evoked Potentials / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression; D007700:Kinetics; D051379:Mice; D050506:Mutant Chimeric Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010647:Phenyl Ethers / Q000138:chemical synthesis / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics* / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D058827:Serotonin 5-HT3 Receptor Agonists / Q000138:chemical synthesis / Q000494:pharmacology*; D014982:Xenopus laevis
chemical_list: D000178:Acrylamides; D050506:Mutant Chimeric Proteins; D010647:Phenyl Ethers; D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists; D012701:Serotonin
keywords: 5-HT(3) receptor; Ago-PAM; Allosteric agonist; Allosteric modulation; Cys-loop receptor; TMPPAA