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Mar Drugs
2014 Mar 28;124:1859-75. doi: 10.3390/md12041859.
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Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology.
Kudryavtsev D
,
Makarieva T
,
Utkina N
,
Santalova E
,
Kryukova E
,
Methfessel C
,
Tsetlin V
,
Stonik V
,
Kasheverov I
.
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For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([¹²⁵I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (K(i) 0.5-1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [¹²⁵I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (K(i) ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.
Figure 1. Chemical structures of compounds from marine sponges and ascidians (1–13), for which putative cholinergic activities were examined by computational and experimental methods.
Figure 2. Inhibition of [125I]-αBgt binding to L. stagnalis AChBP with the most active compounds studied. Numbering the compounds corresponds to the numbering in Figure 1. The corresponding curves are marked with symbols: 1—filled circles; 2—open circles; 3—filled squares; 4—open squares; 5—filled diamonds; 6—open diamonds; 9—open triangles; 12—filled triangles; 13—stars. Each point is a mean ± s.e.m value of two or three measurements for each concentration. The curves were calculated from the means ± s.e.m. using ORIGIN 7.5 program (see Experimental Section). The respective Ki values are listed in Table 1.
Figure 3. Schematic planar image of the model of makaluvamine G (6) docked to HEPES-bound form of L. stagnalis AChBP binding site (a) in comparison with the same presentation of the X-ray structure of the complex of the same protein with clothianidin (PDB ID—2ZJV) (b); intermolecular bonds in ligands are colored in magenta; the same links in the AChBP amino acid residues involved in the formation of hydrogen bonds (green lines) are shown in orange. The AChBP amino acid residues forming hydrophobic contacts with ligands are presented as red combs. Those of them that are the same for both ligands are encircled by red lines. Atoms of carbon, oxygen, nitrogen, and sulfur are colored in black, red, blue and yellow, respectively. Water molecule involved in the formation of hydrogen bonds is shown in cyan; (c) superposition of 3D structures of makaluvamine G (6), docked to AChBP and crystal structure of clothianidine AChBP complex. Carbons of makaluvamine G (6), clothianidine and AChBP are shown in light blue, pink and grey respectively; oxygens are shown red, nitrogens are shown blue; hydrogen bonds are brown; (d) superposition of α-cobratoxin structure (blue) (PDB ID—1YI5) and the best solution for debromohymenialdesine (7) (magenta) in silico docked to L. stagnalis AChBP (gray).
Figure 4. Inhibition of initial rate for [125I]-αBgt binding to T. californica nAChR (a) or human α7 nAChR (b) with the most active compounds studied. Numbering the compounds and their symbols correspond to the numbering in Figure 1 and symbols in Figure 2, respectively. Each point is a mean ± s.e.m value of two or three measurements for each concentration. The curves were calculated from the means ± s.e.m. using the ORIGIN 7.5 program (see Experimental Section). The respective Ki values are listed in Table 2.
Figure 5. Relative inhibition of agonist-evoked current by 10 μM compounds on murine muscle-type nAChR (a) and on human α7 nAChR (b). Numbering the compounds in x axis corresponds to the numbering in Figure 1. Asterisks indicate significant (p < 0.05, according to Student’s test) differences between inhibition effects on murine muscle- and human α7 nAChRs for compounds (4), (6) and (11).
Figure 6. Electrophysiological measurements of 10 μM rhizochalin (1) (a) and makaluvamine G (6) (b) activity on muscle nAChR. Black and grey rectangles represent application of acetylcholine and tested compound, respectively. From left to right: control acetylcholine-evoked current, acetylcholine-evoked current in the presence of tested compound and acetylcholine-evoked current after 15 min of wash out. Current inhibition dose-response curves (c): concentrations of tested compounds from 1 to 100 μM were used to evaluate IC50 (values placed on figure according to numbering from Figure 1). The symbols used for the respective compounds are the same as in Figure 2 and Figure 4.
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