Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

Hamouda, Ayman K.; Wang, Zejun; Stewart, Deirdre S.; Jain, Atul D.; Glennon, Richard A.; Cohen, Jonathan B.

doi:10.1124/mol.115.098913

Cited by 19 publications

(17 citation statements)

References 63 publications

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“…Mutagenesis studies using an EC 75 concentration of ACh suggested that dFBr binds at extracellular β2/β2 and β2/α4 sites (Weltzin and Schulte, ). These sites are also supported by photoaffinity labelling of Torpedo nAChRs (Hamouda et al , ). Another study using mutagenesis, SCAM and docking indicates that dFBr binds at the interface between the extracellular and transmembrane domains when testing its PAM effect at an EC 10 concentration of ACh (Alcania et al , ).…”

Section: Introductionmentioning

confidence: 84%

“…(R)‐7‐bromo‐N‐(piperidin‐3‐yl)benzo[b]thiophene‐2‐carboxamide (Br‐PBTC) acts from the extracellular C‐terminus of α2 or α4 in association with nearby parts of the α subunit (Wang et al , ). Desformylflustrabromine (dFBr) was initially reported to act at the extracellular domain (Weltzin and Schulte, ; Hamouda et al , ; Weltzin and Schulte, ), but has recently been reported to act in the transmembrane domain near the C‐tail of α4 near where Br‐PBTC and estradiol act (Alcania et al , ). Type II PAMs for heteromeric nAChRs that act directly at or close to the desensitization gate may be found.…”

Section: Introductionmentioning

confidence: 99%

“…The antihelminthic cholinergic agonist morantel enhances channel gating through β2/α3 non‐canonical sites (Wu et al , ; Seo et al , ). dFBr selectively potentiates α4β2 nAChRs (Hamouda et al , ; Weltzin and Schulte, ). At concentrations more than 10 μM, dFBr inhibits activation as a channel blocker (Weltzin and Schulte, ).…”

Section: Introductionmentioning

confidence: 99%

“…At concentrations more than 10 μM, dFBr inhibits activation as a channel blocker (Weltzin and Schulte, ). It also inhibits α7 and muscle type nAChRs (Kim et al , ; German et al , ; Hamouda et al , ).…”

Section: Introductionmentioning

confidence: 99%

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Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors

Wang

Lindstrom

2017

British J Pharmacology

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Section: Introductionmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors

Wang

Lindstrom

2017

British J Pharmacology

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“…Photoaffinity labeling identifies amino acid residues within the protein structure that are in direct contact with a bound drug and differentiates them from amino acids not contributing to the binding site but important for drug action, which can be identified by mutational analyses. Even though CMPI bound with .30-fold higher affinity in the Torpedo nAChR ion channel than in the transmitter binding sites, 55 in the g subunit, suggests that CMPI binds preferentially to the ACh binding site at the a-g interface, as is seen for d-tubocurarine, the classic muscle-type nAChR competitive antagonist, which photolabels the same amino acids in the ACh binding site as CMPI (Chiara and Cohen, 1997;Chiara et al, 1999 (Chiara and Cohen, 1997;Nirthanan et al, 2005;Srivastava et al, 2009 (Hamouda et al, 2013(Hamouda et al, , 2015 , which indicates that CMPI does not bind to the dFBr/ physostigmine/galanthamine allosteric sites in the ECD at the a/g subunit interface near the entry of the ACh binding site or in the vestibule of the ion channel.…”

Section: Discussionmentioning

confidence: 99%

Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator

Hamouda

Deba

Wang

et al. 2016

Molecular Pharmacology

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Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing the a4 subunit. In this report, we compare CMPI interactions with low-sensitivity (a4)3(b2)2 and high-sensitivity (a4)2(b2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [ 3 H]CMPI upon photolysis at 312 nm to identify its binding sites in Torpedo nAChRs. Recording from Xenopus oocytes, we found that CMPI potentiated maximally the responses of (a4) 3 (b2) 2 nAChR to 10 mM ACh (EC 10 ) by 400% and with an EC 50 of ∼1 mM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10 mM) ACh responses of (a4) 2 (b2)

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Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites

Jayakar

Ang

Chiara

et al. 2017

Methods in Molecular Biology

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Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.

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Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

Cited by 19 publications

References 63 publications

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors

Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator

Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites

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