title: Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4β2 nicotinic acetylcholine receptors.
abstract: Modulation of Cys loop receptor ion channels is a proven drug discovery strategy, but many underlying mechanisms of the mode of action are poorly understood. We report the x-ray structure of the acetylcholine-binding protein from Lymnaea stagnalis with NS9283, a stoichiometry selective positive modulator that targets the α4-α4 interface of α4β2 nicotinic acetylcholine receptors (nAChRs). Together with homology modeling, mutational data, quantum mechanical calculations, and pharmacological studies on α4β2 nAChRs, the structure reveals a modulator binding mode that overlaps the α4-α4 interface agonist (acetylcholine)-binding site. Analysis of contacts to residues known to govern agonist binding and function suggests that modulation occurs by an agonist-like mechanism. Selectivity for α4-α4 over α4-β2 interfaces is determined mainly by steric restrictions from Val-136 on the β2-subunit and favorable interactions between NS9283 and His-142 at the complementary side of α4. In the concentration ranges where modulation is observed, its selectivity prevents NS9283 from directly activating nAChRs because activation requires coordinated action from more than one interface. However, we demonstrate that in a mutant receptor with one natural and two engineered α4-α4 interfaces, NS9283 is an agonist. Modulation via extracellular binding sites is well known for benzodiazepines acting at γ-aminobutyric acid type A receptors. Like NS9283, benzodiazepines increase the apparent agonist potency with a minimal effect on efficacy. The shared modulatory profile along with a binding site located in an extracellular subunit interface suggest that modulation via an agonist-like mechanism may be a common mechanism of action that potentially could apply to Cys loop receptors beyond the α4β2 nAChRs.
authors: From NeuroSearch A/S, Pederstrupvej 93, 2750 Ballerup, Denmark, the Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark, the Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales 2006, Australia, and.
authors: Jeppe A
authors: JA
authors: Olsen
authors: the Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales 2006, Australia, and Saniona AB, Baltorpvej 54, 2750 Ballerup, Denmark.
authors: Philip K
authors: PK
authors: Ahring
authors: the Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors: Jette S
authors: JS
authors: Kastrup
authors: the Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors: Michael
authors: M
authors: Gajhede
authors: the Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales 2006, Australia, and thomas.balle@sydney.edu.au.
authors: Thomas
authors: T
authors: Balle
mesh_terms: D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D018722:Nicotinic Agonists / Q000737:chemistry / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D010069:Oxadiazoles / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D014982:Xenopus laevis
chemical_list: C561896:3-(3-(pyridine-3-yl)-1,2,4-oxadiazol-5-yl)benzonitrile; D002352:Carrier Proteins; D018722:Nicotinic Agonists; D010069:Oxadiazoles; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D006639:Histidine; D000109:Acetylcholine
keywords: Allosteric Regulation; Benzodiazepines; Crystal Structure; Electrophysiology; Nicotinic Acetylcholine Receptors (nAChR); Protein Drug Interaction