Zheng F et al. (2016), The Ascaris suum nicotinic receptor, ACR-16, as...

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Int J Parasitol Drugs Drug Resist 2016 Feb 10;61:60-73. doi: 10.1016/j.ijpddr.2016.02.001.

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The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.

Zheng F , Robertson AP , Abongwa M , Yu EW , Martin RJ .

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Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.

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Species referenced: Xenopus
Genes referenced: ecd

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	Graphical Abstract
	Fig. 1. (A) Sequence and numbering of the ECD-Asu-ACR-16 and its alignment with the template, human α7 nAChR chimera subunit. Completely conserved residues (red background) and partially conserved residues (yellow background) are indicated. Secondary structures are shown schematically above the sequences. α1 represents α helix. β1-7 represent β sheet. η1 represents α helix. The Cysteine loop and loops A - F are labeled by dark green bars. Residues in the orthosteric binding site are indicated by arrows (principal subunit, pink; complementary subunit, orange). Residues in three allosteric binding pockets are highlighted by arrows (principal subunit of agonist sub-pocket, turquoise; complementary subunit of agonist sub-pocket, green; principal subunit of vestibule pocket, dark green; complementary subunit of vestibule pocket, gold; principal subunit of top pocket, purple). (B) Sequence and numbering of the TID-Asu-ACR-16 and its alignment with the template TID-Tma-AChR subunit A. Completely conserved residues (red background) and partially conserved residues (yellow background) are indicated. Four transmembrane α helixes (M1, M2, M3 and M4) are shown schematically above the sequences. Residues in the allosteric binding pocket are indicated by arrows (principal subunit, pink; complementary subunit, orange). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 2. (A) Ribbon diagram of the antagonist-bound model of ECD-Asu-ACR-16 viewed from the synaptic cleft, showing the location of the orthosteric binding site and agonist sub-pocket. For clarity, only the front two subunits are highlighted (principal subunit, light pink; complementary subunit, yellow). The residues that contribute to the orthosteric binding site (principal side, pink; complementary side, orange) and the agonist sub-pocket (principal side, turquoise; complementary side, green) are represented by sticks and highlighted inside the red dotted circle. (B) Ribbon diagram of the antagonist-bound model of TID-Asu-ACR-16 viewed above the membrane, showing the location of two transmembrane allosteric binding sites. For clarity, only the front two subunits are highlighted (principal subunit, light pink; complementary subunit, yellow). The residues that contribute to the intersubunit site (principal side, pink; complementary side, orange) and intrasubunit site (principal side, purpleblue) are represented by sticks and highlighted inside the red dotted circle. (C) Ribbon diagram of the antagonist-bound model of full-length Asu-ACR-16 viewed parallel to the membrane plane, showing the location of the orthosteric binding site and the agonist sub-pocket in the extracellular domain, the intersubunit and intrasubunit binding sites in the transmembrane domain. For clarity, only the front two subunits are highlighted (principal subunit, light pink; complementary subunit, yellow). The residues that contribute to the ligand binding sites are represented by sticks (orthosteric site: (+), pink; (−), orange; agonist sub-pocket: (+), turquoise; (−), green; intersubunit transmembrane site: (+), pink; (−), orange; intrasubunit transmembrane site: purpleblue) and highlighted inside the red dotted circle. (D) Detailed view of the orthosteric binding site and agonist sub-pocket in the antagonist-bound model of ECD-Asu-ACR-16. The principal subunit is colored light pink, whereas the complementary subunit is colored yellow. The residues that contribute to the orthosteric binding site (principal side, pink; complementary side, orange) and the agonist sub-pocket (principal side, turquoise; complementary side, green) are represented by sticks and highlighted inside the red dotted circle. Carbon is in either turquoise or green. Nitrogen is in blue. Oxygen is in red. (E) Detailed view of the transmembrane allosteric binding sites in the antagonist-bound model of TID-Asu-ACR-16. The principal subunit is colored light pink, whereas the complementary subunit is colored yellow. The residues that contribute to intersubunit site (principal side, pink; complementary side, orange) and intrasubunit site (principal side, purpleblue) are represented by sticks and highlighted inside the red dotted circle. Carbon is in either pink or orange or purpleblue. Nitrogen is in blue. Oxygen is in red. Sulfur is in yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 3. Binding modes of four virtual screening hits in the orthosteric binding site, the agonist sub-pocket, the intersubunit and intrasubunit transmembrane allosteric binding pockets of the antagonist-bound model of Asu-ACR-16: (A), (B), (C), (D) fomc-2; (E), (F), (G), (H) SB-277011-A; (I), (J), (K) fomc-1; (L), (M), (N), (O) (+)-butaclamol Cl. Hits docked into the binding pockets are represented by sticks (carbon in yellow; ring in white; nitrogen in blue; oxygen in red). (A), (E), (I) and (L) show the four hits bound in the orthosteric binding site of the antagonist-bound model of ECD-Asu-ACR-16. The front two subunits are highlighted (principal subunit, light pink; complementary subunit, yellow). The residues in the orthosteric binding site are labeled (principal side, pink; complementary side, orange) to show the location of the orthosteric binding site. (B), (F), (J) and (M) show the four hits bound in the agonist sub-pocket of the antagonist-bound model of ECD-Asu-ACR-16. The front two subunits are highlighted. The residues in the agonist sub-pocket are labeled (principal side, turquoise; complementary side, green) to show the location of the agonist sub-pocket. (C), (G) and (N) show the four hits bound in the intersubunit transmembrane site of the antagonist-bound model of TID-Asu-ACR-16. The front two subunits are highlighted. The residues in the intersubunit transmembrane site are labeled (principal side, pink; complementary side, orange) to show the location of the intersubunit transmembrane binding site. (D), (H), (K) and (O) show the four hits bound in the intrasubunit transmembrane site of the antagonist-bound model of TID-Asu-ACR-16. The residues in the intrasubunit transmembrane site are labeled (purpleblue) to show the location of the intersubunit transmembrane 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. 4. Effects of four virtual screening hits on Asu-ACR-16 mediated ach responses. Sample traces for: (A) fmoc-2, (B) SB-277011, (C) (+)-butaclamol Cl, (D) fmoc-1 concentration-inhibition relationships on Asu-ACR-16. Mla in (D), which stands for methyllycaconitine citrate salt, was used as an antagonist control of Asu-ACR-16. All four hits did not induce the current response by themselves, while produced the concentration-depended inhibition of ach current response. (E) is the magnified figure of part of (D) as an example to show the four parameters needed to measure the inhibition percentage. Imax control was the peak current of the control 30 s application of 100 μM ach. Imax was the peak current of the 100 μM ach that preceded the 10 s co-application of ach and antagonist. Iant was the minimal current during the co-application of 100 μM ach and antagonist. Iant control was the current at the same time point from the beginning of the 30 s application as Iant during the control 30 s application of 100 μM ach.
	Fig. 5. (A) Effects of four virtual screening hits on Asu-ACR-16 mediated ach responses. Fmoc-2, fmoc-1, (+)-butaclamol Cl and SB-277011-A concentration-inhibition curves for Asu-ACR-16. Results were expressed as mean % inhibition of currents elicited by 100 μM ach ± S.E.M. (B) Bar chart representing the IC50 (mean ± S.E.M, μM) of each plots in (A). The rank order series of inhibition based on IC50 for four hits is: SB-277011-A (3.12 ± 1.29 μM, n = 4) < (+)-butaclamol Cl (9.85 ± 2.37 μM, n = 4) ≈ fmoc-1 (10.00 ± 1.38 μM, n = 4) < fmoc-2 (16.67 ± 1.95 μM, n = 4). * represents p < 0.05 (unpaired t-test). (C) Ach concentration-response plots for Asu-ACR-16 in the absence of hits as a control (ach) and in the continual presence of four hits identified in (A). Ach concentration-response curves for Asu-ACR-16 in the presence of 3 μM of four hits: fmoc-1, fmoc-2, SB-27011-A and (+)-butaclamol Cl. (D) Bar chart (mean ± S.E.M, %) representing the reduced maximum current response of ach concentration-response curves in (C). The series of reduced maximum response of each hits compared to that of ach by unpaired t-test is: 10 μM SB-27011-A (5.51 ± 1.38%, n = 4), 3 μM (+)-butaclamol Cl (17.22 ± 1.94%, n = 4), 3 μM SB-27011-A (23.25 ± 1.80%, n = 5), 3 μM fmoc-2 (49.92 ± 3.27%, n = 4), 3 μM fmoc-1 (61.25 ± 3.08%, n = 4) and ach (97.45 ± 1.19%, n = 4). * represents p < 0.05, represents p < 0.01, * represents p < 0.001. All four hits significantly inhibited the maximum current response induced by ach. (E) Bar chart (mean ± S.E.M, μM) displaying the EC50 of ach concentration-response curves in (C). The series of variable EC50 of each hits compared to that of ach by unpaired t-test is: 10 μM SB-27011-A (29.40 ± 2.27 μM, n = 4), 3 μM fmoc-1 (8.62 ± 1.04 μM, n = 4), 3 μM fmoc-2 (8.01 ± 0.18 μM, n = 4), 3 μM SB-27011-A (7.17 ± 0.33 μM, n = 5), ach (5.92 ± 0.29 μM, n = 4) and 3 μM (+)-butaclamol Cl (3.94 ± 0.66 μM, n = 4). The EC50 of all four hits obviously shift away from the control when applied.

References [+] :

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