title: Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor.
abstract: Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 μM) than in the resting state ([(3)H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.
authors: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.S.S., J.B.C.); Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (A.D.J., R.A.G.); and Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville,Texas (A.K.H., Z.-J.W.).
authors: Ayman K
authors: AK
authors: Hamouda
authors: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.S.S., J.B.C.); Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (A.D.J., R.A.G.); and Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville,Texas (A.K.H., Z.-J.W.).
authors: Ze-Jun
authors: ZJ
authors: Wang
authors: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.S.S., J.B.C.); Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (A.D.J., R.A.G.); and Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville,Texas (A.K.H., Z.-J.W.).
authors: Deirdre S
authors: DS
authors: Stewart
authors: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.S.S., J.B.C.); Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (A.D.J., R.A.G.); and Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville,Texas (A.K.H., Z.-J.W.).
authors: Atul D
authors: AD
authors: Jain
authors: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.S.S., J.B.C.); Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (A.D.J., R.A.G.); and Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville,Texas (A.K.H., Z.-J.W.).
authors: Richard A
authors: RA
authors: Glennon
authors: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.S.S., J.B.C.); Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (A.D.J., R.A.G.); and Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville,Texas (A.K.H., Z.-J.W.) jonathan_cohen@hms.harvard.edu.
authors: Jonathan B
authors: JB
authors: Cohen
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D006801:Humans; D006842:Hydrocarbons, Brominated / Q000737:chemistry / Q000494:pharmacology*; D026121:Indole Alkaloids / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D009132:Muscles / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014101:Torpedo / Q000033:anatomy & histology / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
chemical_list: D006842:Hydrocarbons, Brominated; D026121:Indole Alkaloids; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; C524215:desformylflustrabromine