Личная информация

orcid.org/0000-0002-0313-9193

Образование

Научные интересы

аллостерические взаимодействия, нейрохимия, рациональный драг-дизайн

Премии и заслуги

Премия правительства города Москвы 2016 г.

Членство в научных обществах

Европейское нейрохимическое общество (ESN) с 2015 г.

Гранты и проекты

Избранные публикации

1. Kudryavtsev D.S., Spirova E.N., Shelukhina I.V., Son L.V., Makarova Y.V., Utkina N.K., Kasheverov I.E., Tsetlin V.I. (2018). Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites. Mar Drugs 16 (4), [+]

   Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

   ID:2072
2. Shelukhina I., Spirova E., Kudryavtsev D., Ojomoko L., Werner M., Methfessel C., Hollmann M., Tsetlin V. (2017). Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants. PLoS ONE 12 (8), e0181936 [+]

   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.

   ID:1836
3. Kasheverov I.E., Chugunov A.O., Kudryavtsev D.S., Ivanov I.A., Zhmak M.N., Shelukhina I.V., Spirova E.N., Tabakmakher V.M., Zelepuga E.A., Efremov R.G., Tsetlin V.I. (2016). High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method. Sci Rep 6, 36848 [+]

   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.

   ID:1604
4. Manetti D., Bellucci C., Dei S., Teodori E., Varani K., Spirova E., Kudryavtsev D., Shelukhina I., Tsetlin V., Romanelli M.N. (2016). New quinoline derivatives as nicotinic receptor modulators. European journal of medicinal chemistry 110, 246–258 [+]

   As a continuation of previous work on quinoline derivatives, which showed some preference (2-3 times) for the α7 with respect to α4β2 acetylcholine nicotinic receptors (nAChRs), we synthesized a series of novel azabicyclic or diazabicyclic compounds carrying a quinoline or isoquinoline ring, with the aim of searching for more selective α7 nAChR compounds. Radioligand binding studies on α7* and α4β2* nAChRs (rat brain homogenate) revealed one compound (7) with a 2-fold higher affinity for the α4β2*-subtype, and four compounds (11, 13, 14 and 16) with at least 3-fold higher affinity for α7* nAChR. The most promising was 11, showing Ki∼100 nM and over 10-fold selectivity for α7* nAChR. Compounds 7, 11, 13 and 16 at 50 μM suppressed ion currents induced in the rat α4β2 nAChR and the chimeric nAChR composed of the ligand-binding domain of the chick α7 and transmembrane domain of the α1 glycine receptor, expressed in Xenopus oocytes. Calcium imaging experiments on the human α7 nAChR expressed in the Neuro2a cells and potentiated by PNU-120596 confirmed the antagonistic activity for 7; on the contrary, 11, 13 and 16 were agonists with the EC50 values in the range of 1.0-1.6 μM. Thus, the introduced modifications allowed us to enhance the selectivity of quinolines towards α7 nAChR and to get novel compounds with agonistic activity.

   ID:1356
5. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

   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.

   ID:1420
6. MalcaGarcia G.R., Hennig L., Shelukhina I.V., Kudryavtsev D.S., Bussmann R.W., Tsetlin V.I., Giannis A. (2015). Curare Alkaloids: Constituents of a Matis Dart Poison. J. Nat. Prod. , [+]

   A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of d-(-)-quinic acid, l-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. d-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

   ID:1317
7. Kudryavtsev D.S., Shelukhina I.V., Son L.V., Ojomoko L.O., Kryukova E.V., Lyukmanova E.N., Zhmak M.N., Dolgikh D.A., Ivanov I.A., Kasheverov I.E., Starkov V.G., Ramerstorfer J., Sieghart W., Tsetlin V.I., Utkin Y.N. (2015). Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic GABA Receptors. J. Biol. Chem. , [+]

   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 (TFTs) from snake venoms, specifically stained the α1β3γ2 receptor; 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 TFTs: long α-neurotoxin Ls III and non-conventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and non-competitive 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 accomodating under loop C of the receptors.

   ID:1307
8. Kudryavtsev D., Shelukhina I., Vulfius C., Makarieva T., Stonik V., Zhmak M., Ivanov I., Kasheverov I., Utkin Y., Tsetlin V. (2015). Natural compounds interacting with nicotinic acetylcholine receptors: from low-molecular weight ones to peptides and proteins. Toxins (Basel) 7 (5), 1683–701 [+]

   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.

   ID:1306
9. Kasheverov I.E., Shelukhina I.V., Kudryavtsev D.S., Makarieva T.N., Spirova E.N., Guzii A.G., Stonik V.A., Tsetlin V.I. (2015). 6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor. Mar Drugs 13 (3), 1255–66 [+]

   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.

   ID:1308
10. Kasheverov I.E., Kudryavtsev D.S., Ivanov I.A., Zhmak M.N., Chugunov A.O., Tabakmakher V.M., Zelepuga E.A., Efremov R.G., Tsetlin V.I. (2015). Rational design of new ligands for nicotinic receptors on the basis of α-conotoxin PnIA. Dokl. Biochem. Biophys. 461, 106–9 [+]

    A variety of different subtypes of nicotinic acetylcholine receptors (nAChRs) and their involvement in a number of diseases and pathologies (Parkinson’s and Alzheimer’s diseases, schizophrenia, myasthenia, nicotine addiction) dictates the needs in potent and selective ligands for each subtype. These ligands can be used as a tool for detection and characterization of the distinct nAChR subtypes, as well as be the basis for drug design. Novel cholinergic ligands can emerge in the result of search among natural sources or design (with the use of modern computer modeling) on the basis of known molecules. The significance of the first way was confirmed in our hands by the detection of affinities of a set of marine alkaloids from sponges and ascidians towards some receptor subtypes. The most active of them — makaluvamines — showed micromolar affinity for muscle and neuronal α7 nAChRs. Application of the recently presented Protein Surface Topography method to known natural antagonist of some neuronal nAChRs — α-conotoxin PnIA — resulted in design of new potent analogs with nanomolar affinities for α7 nAChR. Radioactive derivatives of these analogs were successfully applied in radioligand tests for characterization of novel compounds and could be perspective as well for detection of α7 nAChR in the various preparations. Combining these two ways (search and design) was demonstrated in the synthesis of small peptide compounds on the basis of discovered by us in venom of Burmese Viper linear peptide azemiopsin — powerful blocker of muscle-type nAChRs. Some designed peptides have retained a certain affinity to receptor and showed high practical potential: in the absence of toxicity they contributed to the reduction of facial wrinkles (patent application for cosmetic use RU2013102410; PCT/RU2014/000032).

    ID:1393
11. Kudryavtsev D., Makarieva T., Utkina N., Santalova E., Kryukova E., Methfessel C., Tsetlin V., Stonik V., Kasheverov I. (2014). Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology. Mar Drugs 12 (4), 1859–75 [+]

    Для небольшой библиотеки низкомолекулярных соединений, выделенных сотрудниками Тихоокеанского института биоорганической химии ДВО РАН из морских губок и асцидий показана активность в отношении никотиновых ацетилхолиновых рецепторов и их гомолога - ацетилхолин-связывающего белка. В статье кратко обсуждаются структурнофункциональные взамимоотношения с привлечением методов компьютерного моделирования, а также возможность практического применения описанных в статье лигандов.

    ID:1011
12. Lyukmanova E.N., Shulepko M.A., Buldakova S.L., Kasheverov I.E., Shenkarev Z.O., Reshetnikov R.V., Filkin S.Y., Kudryavtsev D.S., Ojomoko L.O., Kryukova E.V., Dolgikh D.A., Kirpichnikov M.P., Bregestovski P.D., Tsetlin V.I. (2013). Water-soluble LYNX1 residues important for interaction with muscle-type and/or neuronal nicotinic receptors. J. Biol. Chem. 288 (22), 15888–99 [+]
    ID:929