title: Molecular determinants of subtype-selective efficacies of cytisine and the novel compound NS3861 at heteromeric nicotinic acetylcholine receptors.
abstract: Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric α3β4 and α4β2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of α3- and α4-, with β2- and β4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing β4- but not β2-subunits, NS3861 displays the opposite β-subunit preference and a complete lack of activation at α4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the β-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the β-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at α4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (α) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (β) affect agonist efficacy.
authors.affiliation: Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Harpsøe
authors.forename: Helle
authors.initials: H
authors.lastname: Hald
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Timmermann
authors.forename: Marianne L
authors.initials: ML
authors.lastname: Jensen
authors.forename: Tino
authors.initials: T
authors.lastname: Dyhring
authors.forename: Elsebet Ø
authors.initials: EØ
authors.lastname: Nielsen
authors.forename: Dan
authors.initials: D
authors.lastname: Peters
authors.forename: Thomas
authors.initials: T
authors.lastname: Balle
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
authors.forename: Jette S
authors.initials: JS
authors.lastname: Kastrup
authors.forename: Philip K
authors.initials: PK
authors.lastname: Ahring
mesh_terms: D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D001392:Azocines / Q000494:pharmacology; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004594:Electrophysiology / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008956:Models, Chemical; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011807:Quinolizines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013876:Thiophenes / Q000494:pharmacology*; D014982:Xenopus laevis; D000093843:Quinolizidine Alkaloids
chemical_list: D000470:Alkaloids; D053961:Azabicyclo Compounds; D001392:Azocines; D008024:Ligands; D011807:Quinolizines; D011978:Receptors, Nicotinic; D013876:Thiophenes; C579339:3-(3-bromothiophen-2-yl)-8-methyl-8-azabicyclo(3.2.1)oct-2-ene; C403530:nicotinic receptor alpha3beta4; C403531:nicotinic receptor alpha4beta2; C004712:cytisine; D000093843:Quinolizidine Alkaloids