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Mar Drugs
2018 Nov 22;1612:. doi: 10.3390/md16120460.
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Orthosteric and/or Allosteric Binding of α-Conotoxins to Nicotinic Acetylcholine Receptors and Their Models.
Kryukova EV
,
Ivanov IA
,
Lebedev DS
,
Spirova EN
,
Egorova NS
,
Zouridakis M
,
Kasheverov IE
,
Tzartos SJ
,
Tsetlin VI
.
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α-Conotoxins from Conus snails are capable of distinguishing muscle and neuronal nicotinic acetylcholine receptors (nAChRs). α-Conotoxin RgIA and αO-conotoxin GeXIVA, blocking neuronal α9α10 nAChR, are potential analgesics. Typically, α-conotoxins bind to the orthosteric sites for agonists/competitive antagonists, but αO-conotoxin GeXIVA was proposed to attach allosterically, judging by electrophysiological experiments on α9α10 nAChR. We decided to verify this conclusion by radioligand analysis in competition with α-bungarotoxin (αBgt) on the ligand-binding domain of the nAChR α9 subunit (α9 LBD), where, from the X-ray analysis, αBgt binds at the orthosteric site. A competition with αBgt was registered for GeXIVA and RgIA, IC50 values being in the micromolar range. However, high nonspecific binding of conotoxins (detected with their radioiodinated derivatives) to His₆-resin attaching α9 LBD did not allow us to accurately measure IC50s. However, IC50s were measured for binding to Aplysia californica AChBP: the RgIA globular isomer, known to be active against α9α10 nAChR, was more efficient than the ribbon one, whereas all three GeXIVA isomers had similar potencies at low µM. Thus, radioligand analysis indicated that both conotoxins can attach to the orthosteric sites in these nAChR models, which should be taken into account in the design of analgesics on the basis of these conotoxins.
18-04-00844 Russian Foundation for Basic Research, 17-00-00063 komfi Russian Foundation for Basic Research, 18-04-01366 Russian Foundation for Basic Research
Figure 1. Amino acid sequences of α-conotoxin RgIA and αO-conotoxin GeXIVA with scheme of formation of their three isomers (globular, ribbon and beads) by forming disulfides between 4 cysteine residues (shaded grey and numbered).
Figure 2. HPLC profiles for synthetic peptides used in this study. (A) Co-elution of purified ribbon and globular RgIA isomers in acetonitrile gradient; retention times are shown over respective peaks. (B) Superposition of the RP HPLC profiles of analytical chromatograms for purified globular, beads and ribbon GeXIVA isomers; retention times are shown over respective peaks.
Figure 3. RP HPLC profiles in acetonitrile gradient for the products of the [127I]iodination reaction of α-conotoxin RgIA globular isomer (A) and αO-conotoxin GeXIVA beads isomer (B). The peak of oxidizer (chloramine T, peak 1), non-modified conotoxins (peaks 2), mono- (peaks 3) and di-iodinated derivatives (peak 4) are marked with bars. The numbers above the peaks indicate the corresponding measured molecular masses (Da) obtained by MALDI-TOF mass-spectrometry (A) or electrospray HPLC-MS (B).
Figure 4. Competition of conotoxins (RgIA globular and three GeXIVA isomers) with [125I]-labeled αBgt for binding to α9 LBD. The IC50 values (mean ± SEM) for GeXIVA isomers were 1.140 ± 0.012 μM (globular), 1.20 ± 0.05 μM (ribbon, dotted line) and 1.63 ± 0.02 μM (beads), as well as > 60 μM for RgIA globular isomer.
Figure 5. Inhibition of acetylcholine (10 μM)-evoked currents (A) through rat α9α10 nAChR by globular isomer of RgIA or its [127I]-derivative (5, 100, 300 nM, n = 3) and (B) through human α9α10 nAChR by ribbon isomer GeXIVA or its [127I]-derivative (1, 5, 100, 300 nM, n = 2). The bar graph data are presented as mean ± SEM. (C) and (D) Binding of the radioiodinated conotoxins RgIA globular isomer and GeXIVA beads isomer, respectively, with Ni2+-agarose resin. Binding in the presence (dense pattern bars) or absence (open bars) of α9 ECD is shown. α9 LBD binding with the radioiodinated conotoxins on the Ni2+-NTA-agarose is regarded as 100%. Each bar is the mean ± SEM value of two measurements for each concentration in two (RgIA) or three (GeXIVA) independent experiments.
Figure 6. Inhibition of [125I]-labeled αBgt binding to Aplysia californica AChBP by two RgIA isomers (globular and ribbon) and three GeXIVA isomers (globular, ribbon and beads). The IC50 values were 194 ± 14 nM and 1.00 ± 0.08 μM for the globular and ribbon RgIA analogs, respectively; and around 2.9 μM for all GeXIVA isomers. Each point is the mean ± SEM value of two measurements for each concentration in two independent experiments.
Azam,
Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors.
2009, Pubmed
Azam,
Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors.
2009,
Pubmed
Azam,
Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA.
2012,
Pubmed
,
Xenbase
Azam,
Molecular interaction of α-conotoxin RgIA with the rat α9α10 nicotinic acetylcholine receptor.
2015,
Pubmed
Brams,
A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.
2011,
Pubmed
Celie,
Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant.
2005,
Pubmed
,
Xenbase
Changeux,
The nicotinic acetylcholine receptor: the founding father of the pentameric ligand-gated ion channel superfamily.
2012,
Pubmed
Christensen,
RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.
2017,
Pubmed
,
Xenbase
Clark,
The three-dimensional structure of the analgesic alpha-conotoxin, RgIA.
2008,
Pubmed
Darlak,
Facile preparation of disulfide-bridged peptides using the polymer-supported oxidant CLEAR-OX.
2004,
Pubmed
Durek,
Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors.
2017,
Pubmed
Dutertre,
AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity.
2007,
Pubmed
,
Xenbase
Dutertre,
Nicotinic acetylcholine receptor inhibitors derived from snake and snail venoms.
2017,
Pubmed
Hansen,
Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations.
2005,
Pubmed
Kasheverov,
Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors.
2006,
Pubmed
Kasheverov,
Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.
2009,
Pubmed
Kasheverov,
Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor.
2009,
Pubmed
,
Xenbase
Kasheverov,
Design of new α-conotoxins: from computer modeling to synthesis of potent cholinergic compounds.
2011,
Pubmed
Kasheverov,
High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method.
2016,
Pubmed
Kawashima,
Non-neuronal cholinergic system in regulation of immune function with a focus on α7 nAChRs.
2015,
Pubmed
Lebbe,
Conotoxins targeting nicotinic acetylcholine receptors: an overview.
2014,
Pubmed
Li,
Anti-hypersensitive effect of intramuscular administration of αO-conotoxin GeXIVA[1,2] and GeXIVA[1,4] in rats of neuropathic pain.
2016,
Pubmed
Lin,
From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR.
2016,
Pubmed
Luo,
Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist.
2015,
Pubmed
,
Xenbase
Lyukmanova,
NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.
2011,
Pubmed
,
Xenbase
Lyukmanova,
Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.
2016,
Pubmed
,
Xenbase
McIntosh,
Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes.
1999,
Pubmed
McIntosh,
Alpha9 nicotinic acetylcholine receptors and the treatment of pain.
2009,
Pubmed
,
Xenbase
Mohammadi,
Conotoxin Interactions with α9α10-nAChRs: Is the α9α10-Nicotinic Acetylcholine Receptor an Important Therapeutic Target for Pain Management?
2015,
Pubmed
Nirthanan,
Three-finger alpha-neurotoxins and the nicotinic acetylcholine receptor, forty years on.
2004,
Pubmed
Romero,
Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain.
2017,
Pubmed
Rucktooa,
Insight in nAChR subtype selectivity from AChBP crystal structures.
2009,
Pubmed
Sgard,
A novel human nicotinic receptor subunit, alpha10, that confers functionality to the alpha9-subunit.
2002,
Pubmed
,
Xenbase
Smit,
A glia-derived acetylcholine-binding protein that modulates synaptic transmission.
2001,
Pubmed
Spindel,
Cholinergic Targets in Lung Cancer.
2016,
Pubmed
Tsetlin,
Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications.
2004,
Pubmed
Tsetlin,
Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors.
2009,
Pubmed
Tsetlin,
Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators.
2015,
Pubmed
Ulens,
Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP.
2006,
Pubmed
Utkin,
Three-finger toxins, a deadly weapon of elapid venom--milestones of discovery.
2013,
Pubmed
Vincler,
Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors.
2006,
Pubmed
Wang,
Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation.
2003,
Pubmed
Whiteaker,
Synthesis and characterization of 125I-alpha-conotoxin ArIB[V11L;V16A], a selective alpha7 nicotinic acetylcholine receptor antagonist.
2008,
Pubmed
,
Xenbase
Yu,
Determination of the α-conotoxin Vc1.1 binding site on the α9α10 nicotinic acetylcholine receptor.
2013,
Pubmed
Zhangsun,
αO-Conotoxin GeXIVA disulfide bond isomers exhibit differential sensitivity for various nicotinic acetylcholine receptors but retain potency and selectivity for the human α9α10 subtype.
2017,
Pubmed
,
Xenbase
Zouridakis,
Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain.
2014,
Pubmed
,
Xenbase
