Denis S. Kudryavtsev

Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.

Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic γ-Aminobutyric Acid (GABA) Receptors

Background: Different snake venom three-finger toxins interact with various receptors, channels, and membranes. Results: Here, we demonstrate that GABAA receptors are inhibited by α-cobratoxin, other long chain α-neurotoxins, nonconventional toxin from Naja kaouthia, and α-conotoxin ImI. Conclusion: Some toxin blockers of nicotinic acetylcholine receptors also inhibit GABAA receptors. Significance: Three-finger toxins offer new scaffolds for the design of GABAA receptor effectors. 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 α1β3γ2 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 α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 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 β/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.

Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology.

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 (Ki 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 (Ki ~ 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.

6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.

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.

Rational design of new ligands for nicotinic receptors on the basis of α-conotoxin PnIA

106 αConotoxins, comparatively short peptides of the venom of predatory marine mollusks of the genus Conus, are effective blockers of nicotinic acetylcholine receptors (nAChRs), which are actively used in studies of various nAChR types [1]. A significant advantage of conotoxins as compared to other known nAChR blockers—the polypeptide αneurotoxins of snake venom—is an initially higher specificity with respect to certain subtypes of nicotinic receptors and possibill ity to produce their various analogs by peptide synthee sis. Such analogs are actively synthesized aiming to obtain highly specific ligands for each individual nAChR subtype mainly via introducing or replacing various amino acid residues in the structure of a selected αconotoxin. For example, just a single mutation, [Ala10Leu], in αconotoxin PnIA changes its specificity from nAChR subtype α3β2 to α7 [2], which is involved in pathogeneses of several diseases (Alzheimers disease, Parkinsons disease, schizoo phrenia, etc.). Thus, the possibility to rationally design selective and potent α7 nAChR ligands is not only theoretically, but also practically important problem with a potential to become a breakthrough in molecular medicine. Numerous αconotoxin PnIA analogs have been proo duced to date, in particular, by Alaascanning mutagenesis [3] or by introduction of various charged amino acid residues to different positions in the pepp tide molecule to obtain an analog more efficient and selective with respect to the α7 receptor subtype [4]. In the last work, we selected mutations by a classical computer modeling (docking of flexible ligand to rigid receptor) using the known crystal structures of muss cleetype nAChR from the ray electric organ [5] and the complexes of acetylcholineebinding proteins (AChBPs) with several αconotoxins [6, 7] or αneurotoxin [8]. These waterrsoluble proteins, consisting of five identii cal subunits, are structural homologs of the ligandd binding Nterminal domains in all nAChRs, being the closest to the homooligomeric α7 subtype. This approach has allowed us to produce several αconoo toxin PnIA analogs with a high affinity and selectivity for the AChBPs from Lymnaea stagnalis or Aplysia call ifornica as well as an increased affinity for the human α7 nAChR. However, the best analog in the last case exhibited an affinity of approximately several hunn dreds of nanomoles per liter. This work continues the previous study and is aimed at designing a more efficient ligand for α7 nAChR using αconotoxin PnIA. Here, we applied the computational protein surface topography (PST) technique, developed at the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian …

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.

Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors

Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [125I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.

Species specificity of rat and human α7 nicotinic acetylcholine receptors towards different classes of peptide and protein antagonists

ABSTRACT Peptide and protein neurotoxins, such as &agr;‐conotoxins from Cone snails and &agr;‐neurotoxins from snake venoms, are excellent tools to identify distinct nicotinic acetylcholine receptor (nAChR) subtypes. Here we compared the rat/human species specificity of &agr;7 nAChR towards peptide and protein neurotoxins and found that &agr;‐conotoxin analogues [K11A]TxIB and [H5D]RegIIA are much more potent on the rat versus human &agr;7 receptor expressed in Xenopus oocytes. In the hope to determine the key residue responsible for the difference in &agr;‐conotoxin analogues affinities, ten single mutants of rat &agr;7 nAChR were obtained because there are 10 differences in the extracellular ligand‐binding domains of these species, and only K185R mutation decreased the affinity for &agr;‐conotoxins [K11A]TxIB and [H5D]RegIIA, down to their low affinities for human &agr;7 nAChR. On the other hand, the reverse mutation R185K in human &agr;7 nAChR resulted in the greatest increase in the affinity for both conotoxins, while a double mutation h&agr;7[S183N, R185K] made the potency of the receptor for them as high as that of rat &agr;7 nAChR. The effects of mutations at position 185 were investigated also with some other &agr;‐conotoxins and cobra venom &agr;‐cobratoxin and found to have similar but much less pronounced effects on their species specificity. Molecular modeling provided possible explanation for the high species selectivity of [K11A]TxIB and [H5D]RegIIA towards &agr;7 nAChR, opening the new way for design of their analogues with improved affinity to the human receptor. HIGHLIGHTS&agr;‐Conotoxin [K11A]TxIB and [H5D]RegIIA are more potent on rat versus human &agr;7 nAChR.Lys185 in r&agr;7 subunit mainly confers high specificity of [K11A]TxIB and [H5D]RegIIA.The residue 185 had effects on the &agr;7 species specificity towards other neurotoxins.Molecular modeling provided explanation for the high species selectivity of &agr;7 nAChR.