α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells.

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, ß2 and ß4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

α-Conotoxins Enhance both the In Vivo Suppression of Ehrlich carcinoma Growth and In Vitro Reduction in Cell Viability Elicited by Cyclooxygenase and Lipoxygenase Inhibitors.

Several biochemical mechanisms, including the arachidonic acid cascade and activation of nicotinic acetylcholine receptors (nAChRs), are involved in increased tumor survival. Combined application of inhibitors acting on these two pathways may result in a more pronounced antitumor effect. Here, we show that baicalein (selective 12-lipoxygenase inhibitor), nordihydroguaiaretic acid (non-selective lipoxygenase inhibitor), and indomethacin (non-selective cyclooxygenase inhibitor) are cytotoxic to Ehrlich carcinoma cells in vitro. Marine snail α-conotoxins PnIA, RgIA and ArIB11L16D, blockers of α3ß2/α6ß2, α9α10 and α7 nAChR subtypes, respectively, as well as α-cobratoxin, a blocker of α7 and muscle subtype nAChRs, exhibit low cytotoxicity, but enhance the antitumor effect of baicalein 1.4-fold after 24 h and that of nordihydroguaiaretic acid 1.8-3.9-fold after 48 h of cell cultivation. α-Conotoxin MII, a blocker of α6-containing and α3ß2 nAChR subtypes, increases the cytotoxic effect of indomethacin 1.9-fold after 48 h of cultivation. In vivo, baicalein, α-conotoxins MII and PnIA inhibit Ehrlich carcinoma growth and increase mouse survival; these effects are greatly enhanced by the combined application of α-conotoxin MII with indomethacin or conotoxin PnIA with baicalein. Thus, we show, for the first time, antitumor synergism of α-conotoxins and arachidonic acid cascade inhibitors.

Neurotoxins from snake venoms and α-conotoxin ImI inhibit functionally active ionotropic γ-aminobutyric acid (GABA) receptors.

Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1ß3γ2 receptor; and at 10 µm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1ß2γ2 ≈ α2ß2γ2 > α5ß2γ2 > α2ß3γ2 and α1ß3δ GABAARs. The α1ß3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the ß/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors.

Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor.

Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a ß-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 µm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 µm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1ß1εδ) than the fetal form (α1ß1γδ), EC(50) being 0.44 ± 0.1 µm and 1.56 ± 0.37 µm, respectively. The peptide had no effect on GABA(A) (α1ß3γ2 or α2ß3γ2) receptors at a concentration up to 100 µm or on 5-HT(3) receptors at a concentration up to 10 µm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

Structure and alignment of the membrane-associated antimicrobial peptide arenicin by oriented solid-state NMR spectroscopy.

The antimicrobial arenicin peptides are cationic amphipathic sequences that strongly interact with membranes. Through a cystine ring closure a cyclic ß-sheet structure is formed in aqueous solution, which persists when interacting with model membranes. In order to investigate the conformation, interactions, dynamics, and topology of their bilayer-associated states, arenicin 1 and 2 were prepared by chemical solid-phase peptide synthesis or by bacterial overexpression, labeled selectively or uniformly with (15)N, reconstituted into oriented membranes, and investigated by proton-decoupled (31)P and (15)N solid-state NMR spectroscopy. Whereas the (31)P NMR spectra indicate that the peptide induces orientational disorder at the level of the phospholipid head groups, the (15)N chemical shift spectra agree well with a regular ß-sheet conformation such as the one observed in micellar environments. In contrast, the data do not fit the twisted ß-sheet structure found in aqueous buffer. Furthermore, the chemical shift distribution is indicative of considerable conformational and/or topological heterogeneity when at the same time the (15)N NMR spectra exclude alignments of the peptide where the ß-sheet lies side ways on the membrane surface. The ensemble of experimental constraints, the amphipathic character of the peptide, and in particular the distribution of the six arginine residues are in agreement with a boatlike dimer structure, similar or related to the one observed in micellar solution, that floats on the membrane surface with the possibility to oligomerize into higher order structures and/or to insert in a transmembrane fashion.

Formation of arenicin-1 microdomains in bilayers and their specific lipid interaction revealed by Z-scan FCS.

Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.

Design of new α-conotoxins: from computer modeling to synthesis of potent cholinergic compounds.

A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog, single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes.

α9α10 nicotinic acetylcholine receptors regulate murine bone marrow granulocyte functions.

Polymorphonuclear neutrophilic granulocytes (PMNs) are extremely important in defense of the organism against infections and in inflammatory processes including neuroinflammation and pain sensation. Different subtypes of nicotinic acetylcholine receptors (nAChRs) are involved in modulation of PMN activities. Earlier we determined expression of α2-7, α9, ß3, ß4 subunits and regulatory role of α7 and α3ß2 nAChR subtypes in functions of inflammatory PMNs. Other authors detected mRNA of α9 subunit in bone marrow neutrophils (BM-PMNs). Murine BM-PMNs coming out from the bone marrow, where they develop, to blood were characterized as mature. There was no data for α10 and for the presence of functionally active α9α10 nAChRs in BM-PMNs. Here we detected for the first time mRNA expression of the α10 nAChR subunit in BM-PMNs and confirmed the expression of mRNA for α9 nAChR. With the help of α-conotoxins RgIA and Vc1.1, highly selective antagonists of α9α10 nAChRs, we have revealed participation of α9 and/or α9α10 nAChRs in regulation of cytosolic Ca2+ concentration, cell adhesion, and in generation of reactive oxygen species (ROS). Nicotine, choline, RgIA, and Vc1.1 induced Ca2+ transients in BM-PMNs, enhanced cell adhesiveness and decreased production of ROS indicating involvement of α9, possibly co-assembled with α10, nAChRs in the BM-PMN activity for recruitment and cytotoxicity.

Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant.

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three ß-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor.

alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha7 nAChR-targeting alpha-conotoxin ImI, blocked alpha7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [(125)I]-alpha-bungarotoxin from human alpha7 nAChRs in the cell line GH(4)C(1) (IC(50) 17 and 23 microM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC(50) 2.0-9.0 microM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K(d) and IC(50) 2.5-8.2 microM). On Torpedo nAChR, alpha-conotoxin [(125)I]-ImII(W10Y) revealed specific binding (K(d) 1.5-6.1 microM) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC(50) 2.7, 2.2 and 3.1 microM, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [(125)I]-ImII(W10Y) only at higher concentrations (IC(50)> or = 90 microM), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.

From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs.

The proteins of the Ly6 family have a three-finger folding as snake venom α-neurotoxins, targeting nicotinic acetylcholine receptors (nAChRs), and some of them, like mammalian secreted Ly6/uPAR protein (SLURP1) and membrane-attached Ly-6/neurotoxin (Lynx1), also interact with distinct nAChR subtypes. We believed that synthetic fragments of these endogenous proteins might open new ways for drug design because nAChRs are well-known targets for developing analgesics and drugs against neurodegenerative diseases. Since interaction with nAChRs was earlier shown for synthetic fragments of the α-neurotoxin central loop II, we synthesized a 15-membered fragment of human Lynx1, its form with two Cys residues added at the N- and C-termini and forming a disulfide, as well as similar forms of human SLURP1, SLURP2, and of Drosophila sleepless protein (SSS). The IC50 values measured in competition with radioiodinated α-bungarotoxin for binding to the membrane-bound Torpedo californica nAChR were 4.9 and 7.4 µM for Lynx1 and SSS fragments, but over 300 µM for SLURP1 or SLURP2 fragments. The affinity of these compounds for the α7 nAChR in the rat pituitary tumor-derived cell line GH4C1 was different: 13.1 and 147 µM for SSS and Lynx1 fragments, respectively. In competition for the ligand-binding domain of the α9 nAChR subunit, SSS and Lynx1 fragments had IC50 values of about 40 µM, which correlates with the value found for the latter with the rat α9α10 nAChR expressed in the Xenopus oocytes. Thus, the activity of these synthetic peptides against muscle-type and α9α10 nAChRs indicates that they may be useful in design of novel myorelaxants and analgesics.

Scorpion toxins interact with nicotinic acetylcholine receptors.

Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage-gated ion channels, while most snake neurotoxins target ligand-gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α-bungarotoxin binding to both muscle-type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK-1 (α-KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ-KTx family) from Heterometrus laoticus, both being blockers of voltage-gated potassium channels. With an IC50 of 1.6 µm, OSK1 inhibits acetylcholine-induced current through mouse muscle-type nAChR heterologously expressed in Xenopus oocytes. Other well-characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity.

Improving therapeutic potential of antibacterial spider venom peptides: coarse-grain molecular dynamics guided approach.

AIM: Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive. METHODOLOGY: How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions. CONCLUSION: The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.

Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant.

Azemiopsin (Az), a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR) of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 µM (EC100) acetylcholine but was less potent (IC50 ~ 3 µM) at α7 nAChR activated by 10 µM (EC50) acetylcholine and had a low affinity to α4ß2 and α3-containing nAChR, as well as to GABAA or 5HT3 receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30-300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg-the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20-40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 µg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.

Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors.

Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.

alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor.

alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.

Alpha-conotoxin analogs with enhanced affinity for nicotinic receptors and acetylcholine-binding proteins.

Alpha-conotoxins, neurotoxic peptides from poisonous Conus marine snails, can be subdivided into several groups targeting distinct subtypes of nicotinic acetylcholine receptors (nAChRs). Such alpha-conotoxins as, for example, GI, MI, or SIA potently block muscle-type nAChRs from muscles and from the electric organ of Torpedo ray, whereas others target distinct neuronal nAChRs: alpha-conotoxins ImI and PnIB block pentaoligomeric alpha7 nAChRs, and alpha-conotoxins MII or PnIA inhibit heteromeric nAChRs made of combinations of alpha3 or alpha6 subunits with beta2 subunit. alpha-Conotoxins interact with N-terminal extracellular ligand-binding domains of nAChRs and are indispensable tools for distinguishing various subtypes of AChRs at normal and pathological states. Although many alpha-conotoxins have been isolated from Conus venoms, there is still a great need in more potent and selective tools, which in principle can be obtained by design and synthesis of novel alpha-conotoxin analogs.

High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method.

Despite some success for small molecules, elucidating structure-function relationships for biologically active peptides - the ligands for various targets in the organism - remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules' surface as projection maps, which enables the exposure of the structure-function dependencies. Here, we use PST to uncover the "activity pattern" in α-conotoxins - neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by "standard" structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.