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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.
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; (â), 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; (â), 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)âSIB 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)-1â-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)âSIB 1508Y (C), 5-methylnicotine (D) are depicted. For each nicotine derivative, 5 oocyte were tested as replicates. Resting membrane potential clamped at â60 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).
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