Zheng F et al. (2017), (S)-5-ethynyl-anabasine, a novel compound, is a...

XB-ART-52874

Int J Parasitol Drugs Drug Resist 2017 Apr 01;71:12-22. doi: 10.1016/j.ijpddr.2016.12.001.

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(S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu-ACR-16 receptor.

Zheng F , Du X , Chou TH , Robertson AP , Yu EW , VanVeller B , Martin RJ .

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Nematode parasites infect ∼2 billion people world-wide. Infections are treated and prevented by anthelmintic drugs, some of which act on nicotinic acetylcholine receptors (nAChRs). There is an unmet need for novel therapeutic agents because of concerns about the development of resistance. We have selected Asu-ACR-16 from a significant nematode parasite genus, Ascaris suum, as a pharmaceutical target and nicotine as our basic moiety (EC50 6.21 ± 0.56 μM, Imax 82.39 ± 2.52%) to facilitate the development of more effective anthelmintics. We expressed Asu-ACR-16 in Xenopus oocytes and used two-electrode voltage clamp electrophysiology to determine agonist concentration-current-response relationships and determine the potencies (EC50s) of the agonists. Here, we describe the synthesis of a novel agonist, (S)-5-ethynyl-anabasine, and show that it is more potent (EC50 0.14 ± 0.01 μM) than other nicotine alkaloids on Asu-ACR-16. Agonists acting on ACR-16 receptors have the potential to circumvent drug resistance to anthelmintics, like levamisole, that do not act on the ACR-16 receptors.

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Species referenced: Xenopus
Genes referenced: aopep

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	Graphical Abstract
	Fig. 1. Crystal structure of Lst-AChBP bound with nicotine (PDB code: 1UW6) and the agonist-bound model of Asu-ACR-16.(A) Ribbon diagram of the AChBP co-crystalized with nicotine, as viewed with membrane at the bottom. The principal subunit is highlighted by light pink and the complement subunit is highlighted by light purple, for clarity. Nicotine (orange) is bound in the five ligand-binding sites in the extracellular domain of AChBP.(B) Close view of the AChBP ligand-binding site. The principal subunit in light pink, the complementary subunit in light purple. Residues interacting with nicotine (orange) are represented as sticks ((+), pink; (−), purple), and water molecule is shown as red dot, view with membrane at the bottom.(C) Close view of the agonist-bound model of Asu-ACR-16 ligand-binding site. The principal subunit in light pink, the complementary subunit in light purple. The interacting residues are represented as sticks ((+), pink; (−), purple), and water molecule is shown as red dot, view with membrane at the bottom. (D) Superposition of residues in agonist-binding site, among agonist-bound form (blue), apo (no ligand) form (yellow), antagonist-bound form (green) of Asu-ACR-16 models are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 2. Ligand-binding sites of Asu-ACR-16 and its homologous proteins.(A) Surface representation in the open-up ligand-binding site of Lst-AChBP in complex with nicotine (PDB code: 1UW6). Oxygen-rich area (red), nitrogen-rich area (blue) and carbon-rich area (gray) are displayed. Empty space was observed around the 5-pyridine ring of nicotine, which suggests that the ligand-binding site is in favor of the linear functional group linking toward the 5-pyridine ring of nicotine. Little space is found around the pyrrolidine ring of nicotine.(B) Surface representation in the open-up ligand-binding site of human α7 AChR chimera in complex with epibatidine (PDB code: 3SQ6), viewed by the same angle as (A). Oxygen-rich area (red), nitrogen-rich area (blue), carbon-rich area (pink) and chloride (green) are displayed. The azabicyclic ring N1 of epibatidine was superimposed with the pyrrolidine ring N2 of nicotine, while the pyridine ring N2 of epibatidine was superimposed with the pyridine ring N1 of nicotine.(C) Surface representation in the open-up ligand-binding site of agonist-bound Asu-ACR-16 model, viewed by the same angle as (A). Oxygen-rich area (red), nitrogen-rich area (blue) and carbon-rich area (cyan) are displayed. Assuming the nicotine has the same binding pose as in (A) within the agonist-bound Asu-ACR-16, empty space around the 5-pyridine ring and pyrrolidine ring of nicotine, which allows nicotinic derivatives with modification in these positions fit into to the binding site. The black arrow indicates the likely orientation of the 5-pyridine ring moiety. (D) Surface representation in the open-up ligand-binding site of apo form Asu-ACR-16 model, viewed by the same angle as (A). Oxygen-rich area (red), nitrogen-rich area (blue) and carbon-rich area (yellow) are displayed. Assuming the nicotine has the same binding pose as in (A) within apo form Asu-ACR-16, there would be empty space around the 5-pyridine ring and pyrrolidine ring of nicotine, which would make the nicotinic derivatives with modification in these positions fit in the binding site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 3. Chemical structures of (S)-nicotine and fifteen derivatives studied. 5-substituted pyridine ring derivatives: (S)—SIB 1508Y, (S)-5-bromonicotine, (S)-nicotine-5-carboxaldehyde and 5-methylnicotine; other pyridine ring substituted derivatives: (S)-1-methylnicotinium, 6-methylnicotine and 5-(1-methyl-pyrrolidin-2-yl)-pyridin-2-ylamine (6-AN); pyrrolidine ring substituted derivatives: (S)-1’-methylnicotinium, homonicotine, nornicotine and (S)-cotinine; piperidine ring derivatives: (S)-5-ethynyl-anabasine, (S)-5-bromoanabasine, (S)-anabasine and N-methyl anabasine are shown.
	Fig. 4. Sample concentration-current recording traces for the most potent nicotine derivatives on the Asu-ACR-16 receptor. (S)-5-ethynyl-anabasine (A), (S)-5-bromoanabasine (B), (S)—SIB 1508Y (C), 5-methylnicotine (D) are depicted. For each nicotine derivative, 5 oocyte were tested as replicates. Resting membrane potential clamped at −60 mV. Downward responses to exposure of the agonists show opening of the ion-channel. Peak responses were recorded, normalized and fitted into the Hill equations.
	Fig. 5. Dose-response curves of nicotine derivatives for Asu-ACR-16. For experiment of each nicotine derivative, one oocyte was used as a group. Five replicates were performed for each group. Current responses are normalized to the first 100 μM control application (Methods).(A) ACh and (S)-nicotine as two controls.(B) Pyridine ring substituted derivatives. Responses of 30 μM 5-methylnicotine and 100 μM 6-methylnicotine are shown but were not included for fitting the Hill equation to estimate EC50, nHand Imax correspondingly due to their inhibitory effects at high concentrations.(C) Pyrrolidine ring substituted derivatives. Response of 300 μM homonicotine is shown but was not included for fitting the Hill equation to estimate EC50, nHand Imax due to its inhibitory effect at high concentration.
	Fig. 6. Correlations between binding affinities (kcal/mol) of each derivative in the apo form Asu-ACR-16 and EC50 (μM) (A), binding affinities (kcal/mol) for the selected nicotine derivatives. The correlation coefficients (r) was used for evaluating the linear regression between affinities and pharmacological parameters (r = 0.66, P < 0.05).

References [+] :

Abongwa, Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues. 2016, Pubmed, Xenbase

Abongwa, Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues. 2016, Pubmed , Xenbase
Arias, Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors. 2000, Pubmed
Beng, Application of catalytic dynamic resolution of N-Boc-2-lithiopiperidine to the asymmetric synthesis of 2-aryl and 2-vinyl piperidines. 2011, Pubmed
Bleicher, Hit and lead generation: beyond high-throughput screening. 2003, Pubmed
Blum, Nicotinic pharmacophore: the pyridine N of nicotine and carbonyl of acetylcholine hydrogen bond across a subunit interface to a backbone NH. 2010, Pubmed , Xenbase
Celie, Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. 2004, Pubmed
Chavez-Noriega, Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes. 1997, Pubmed , Xenbase
Cosford, Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist. 2000, Pubmed
Dougherty, The cation-π interaction. 2013, Pubmed
Gárcia, First report of multiple anthelmintic resistance in nematodes of sheep in Colombia. 2016, Pubmed
Holden-Dye, Nicotinic acetylcholine receptors: a comparison of the nAChRs of Caenorhabditis elegans and parasitic nematodes. 2013, Pubmed , Xenbase
Huang, Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera. 2013, Pubmed
Li, Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist. 2011, Pubmed
Liskey, Cyanation of arenes via iridium-catalyzed borylation. 2010, Pubmed
Mongan, Novel alpha7-like nicotinic acetylcholine receptor subunits in the nematode Caenorhabditis elegans. 2002, Pubmed
Proskocil, Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells. 2004, Pubmed
Rucktooa, Insight in nAChR subtype selectivity from AChBP crystal structures. 2009, Pubmed
Rucktooa, Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein. 2012, Pubmed
Sixma, Acetylcholine binding protein (AChBP): a secreted glial protein that provides a high-resolution model for the extracellular domain of pentameric ligand-gated ion channels. 2003, Pubmed
Taly, Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. 2009, Pubmed
Taylor, Aberrant Ascaris suum Nematode Infection in Cattle, Missouri, USA. 2016, Pubmed
Trott, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. 2010, Pubmed
Van Arnam, Functional probes of drug-receptor interactions implicated by structural studies: Cys-loop receptors provide a fertile testing ground. 2014, Pubmed
Wang, Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles. 2011, Pubmed
Zheng, The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening. 2016, Pubmed , Xenbase
de Silva, Soil-transmitted helminth infections: updating the global picture. 2003, Pubmed