Лаборатория лиганд-рецепторных взаимодействий

Отдел молекулярных основ нейросигнализации

Руководитель: Кашеверов Игорь Евгеньевич, д. х. н.
iekash@mx.ibch.ru+7(495)330-73-74

лиганд-управляемые ионные каналы, Никотиновый холинорецептор, глициновый рецептор, компьютерное моделирование, конотоксины, синтез пептидных аналогов, радиолигандный анализ

Лаборатория организована в 2009 г. и возглавляется доктором химических наук И. Е. Кашеверовым.

Основными задачами лаборатории лиганд-рецепторных взаимодействий являются структурно-функциональные исследования некоторых Cys-петельных рецепторов (никотиновые, глициновый) с помощью пептидных лигандов природного происхождения и их искусственных аналогов, а также создание новых биологически активных пептидных соединений на основе известных холинергических лигандов.

В лаборатории проводятся работы по компьютерному дизайну новых соединений на базе структур известных холинергических лигандов (конотоксины разных классов) и моделей их комплексов с никотиновыми рецепторами или их гомологами (ацетилхолин-связывающие белки), химическому синтезу различных пептидных соединений и анализу их биологической активности с применением различных методов исследования (радиолигандный анализ, электрофизиология, плазмонный резонанс).

В лаборатории было синтезировано (в том числе и на основе дизайна с применением методов компьютерного моделирования) более 4-х десятков различных конотоксинов и их аналогов, часть из которых в дальнейшем модифицировалась радиоактивными и/или фотоактивируемыми метками. С помощью некоторых из них были структурно охарактеризованы лиганд-связывающие участки природного никотинового рецептора. С использованием другого аналога, оказавшегося более активным соединением, чем природный конотоксин, впервые была установлена кристаллическая структура ацетилхолин-связывающего белка в комплексе с пептидным антагонистом и выявлены ее принципиальные отличия от структуры комплексов с агонистами. Полученные данные стали основой для создания компьютерных моделей различных типов никотиновых холинорецепторов.

Часть представленной работы нашла отражение в недавно опубликованных обзорах: Tsetlin V., Utkin Y., Kasheverov I. (2009). Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors. Biochem Pharmacol. 78(7), 720—731 иKasheverov I.E., Utkin Y.N., Tsetlin V.I. (2009). Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors. Curr Pharm Des. 15(21), 2430—2452.

Модель комплекса аналога α-конотоксина GI с природным холинорецептором Torpedo: предложены две возможные ориентации пептида в кармане связывания между a- и γ-субъединицами на основании данных метода фотоаффинной модификации. Kasheverov IE, et al. FEBS J. 273(7), 1373—1388 (2006).

 

Избранные публикации

  1. 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
  2. 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
  3. 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
  4. 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
  5. Shelukhina I., Paddenberg R., Kummer W., Tsetlin V. (2014). Functional expression and axonal transport of α7 nAChRs by peptidergic nociceptors of rat dorsal root ganglion. Brain Struct Funct , [+]

    In recent pain studies on animal models, α7 nicotinic acetylcholine receptor (nAChR) agonists demonstrated analgesic, anti-hyperalgesic and anti-inflammatory effects, apparently acting through some peripheral receptors. Assuming possible involvement of α7 nAChRs on nociceptive sensory neurons, we investigated the morphological and neurochemical features of the α7 nAChR-expressing subpopulation of dorsal root ganglion (DRG) neurons and their ability to transport α7 nAChR axonally. In addition, α7 receptor activity and its putative role in pain signal neurotransmitter release were studied. Medium-sized α7 nAChR-expressing neurons prevailed, although the range covered all cell sizes. These cells accounted for one-fifth of total medium and large DRG neurons and <5 % of small ones. 83.2 % of α7 nAChR-expressing DRG neurons were peptidergic nociceptors (CGRP-immunopositive), one half of which had non-myelinated C-fibers and the other half had myelinated Aδ- and likely Aα/β-fibers, whereas 15.2 % were non-peptidergic C-fiber nociceptors binding isolectin B4. All non-peptidergic and a third of peptidergic α7 nAChR-bearing nociceptors expressed TRPV1, a capsaicin-sensitive noxious stimulus transducer. Nerve crush experiments demonstrated that CGRPergic DRG nociceptors axonally transported α7 nAChRs both to the spinal cord and periphery. α7 nAChRs in DRG neurons were functional as their specific agonist PNU282987 evoked calcium rise enhanced by α7-selective positive allosteric modulator PNU120596. However, α7 nAChRs do not modulate neurotransmitter CGRP and glutamate release from DRG neurons since nicotinic ligands affected neither their basal nor provoked levels, showing the necessity of further studies to elucidate the true role of α7 nAChRs in those neurons.

    ID:1081
  6. 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
  7. Vulfius C.A., Kasheverov I.E., Starkov V.G., Osipov A.V., Andreeva T.V., Filkin S.Y., Gorbacheva E.V., Astashev M.E., Tsetlin V.I., Utkin Y.N. (2014). Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2. PLoS ONE 9 (12), e115428 [+]

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

    ID:1162
  8. Utkin Y.N., Weise C., Kasheverov I.E., Andreeva T.V., Kryukova E.V., Zhmak M.N., Starkov V.G., Hoang N.A., Bertrand D., Ramerstorfer J., Sieghart W., Thompson A.J., Lummis S.C., Tsetlin V.I. (2012). Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor. J. Biol. Chem. 287 (32), 27079–86 [+]

    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 β-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 (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) 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.

    ID:1083
  9. Tsetlin V., Kuzmin D., Kasheverov I. (2011). Assembly of nicotinic and other Cys-loop receptors. J. Neurochem. , [+]

    J. Neurochem. (2011) 10.1111/j.1471-4159.2010.07060.x ABSTRACT: The Cys-loop receptor family consists of nicotinic acetylcholine receptors (nAChR), glycine receptor, GABA-A and some other receptors. They fulfill a plethora of functions, whereas their malfunctioning is associated with many diseases. All three domains - extracellular ligand-binding, membrane and cytoplasmic - of these ligand-gated ion channels play important roles in the receptor assembly, delivery to the membrane surface and functional activity. In this study, we discuss the role of these domains in the assembly of the Cys-loop receptors, most comprehensively for the nAChRs. Heterologous expression and mutations of large N-terminal fragments of various subunits demonstrated their leading role in the assembly, although getting an isolated well-structured pentameric ligand-binding domain is still a problem. The long intracellular loop between transmembrane fragments M3 and M4 participates in modulating the receptor function and in clusterization of the receptor complexes because of interactions with the intracellular proteins. The transmembrane fragments play different functional roles: M2 fragments outline the channel, M4 fragments, the most remote from the channel, modulate the channel function and contact the lipid environment. The interactions of aromatic residues in the M1 and M3 fragments with those of M4 are important for the correct assembly of glycine receptor α1 subunit and for the formation of functional pentaoligomer. The role of the three receptor domains is discussed in the light of electron microscopy structure of the Torpedo nAChR, X-ray structures of agonist and antagonist complexes with the acetylcholine-binding proteins and the X-ray structures of the prokaryotic Cys-loop receptors.

    ID:406
  10. Kasheverov I.E., Zhmak M.N., Khruschov A.Y., Tsetlin V.I. (2011). Design of new α-conotoxins: from computer modeling to synthesis of potent cholinergic compounds. Mar Drugs 9 (10), 1698–714 [+]

    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.

    ID:1084
  11. Haeger S., Kuzmin D., DetroDassen S., Lang N., Kilb M., Tsetlin V., Betz H., Laube B., Schmalzing G. (2010). An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors. Nat. Struct. Mol. Biol. 17 (1), 90–8 [+]

    Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

    ID:342
  12. Kasheverov I.E., Zhmak M.N., Fish A., Rucktooa P., Khruschov A.Y., Osipov A.V., Ziganshin R.H., D'hoedt D., Bertrand D., Sixma T.K., Smit A.B., Tsetlin V.I. (2009). Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor. J. Neurochem. 111 (4), 934–44 [+]

    Изучение необычного по своим свойствам a-конотоксина ImII и его новых аналогов различными методами показало существование на природном холинорецепторе дополнительного участка связывания для этих пептидов отличного от сайта связывания «классических» агонистов и конкурентных антагонистов.

    ID:191
  13. Kasheverov I.E., Zhmak M.N., Vulfius C.A., Gorbacheva E.V., Mordvintsev D.Y., Utkin Y.N., van Elk R., Smit A.B., Tsetlin V.I. (2006). Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors. FEBS J. 273 (19), 4470–81 [+]

    В данной работе описан синтез и характеристика большой серии новых аналогов нескольких a-конотоксинов, содержащих замены ряда остатков на заряженные или противоположно заряженные. Некоторые из этих аналогов оказались значительно более эффективными лигандами соответствующих холинорецепторов, чем природные пептиды. Предложены компьютерные модели комплексов этих аналогов с рецепторами, объясняющие подобный эффект.

    ID:190
  14. Kasheverov I.E., Chiara D.C., Zhmak M.N., Maslennikov I.V., Pashkov V.S., Arseniev A.S., Utkin Y.N., Cohen J.B., Tsetlin V.I. (2006). alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor. FEBS J. 273 (7), 1373–88 [+]

     

    С использованием фотоактивируемого производного одного из конотоксинов методом фотоаффинной модификации проведено частичное картирование лиганд-связывающего участка природного холинорецептора. Исходя из полученных данных, компьютерным моделированием предположена возможность двух ориентаций молекулы пептида в этом участке.

     

    ID:189
  15. Celie P.H., Kasheverov I.E., Mordvintsev D.Y., Hogg R.C., van Nierop P., van Elk R., van Rossum-Fikkert S.E., Zhmak M.N., Bertrand D., Tsetlin V.I., Sixma T.K., Smit A.B. (2005). Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582–8 [+]

    Впервые установлена кристаллическая структура ацетилхолин-связывающего белка в комплексе с контоксином. Полученные данные позволили охарактеризовать основные структурные особенности взаимодействия конотоксинов с холинорецепторами.

    ID:104

Кашеверов Игорь Евгеньевич

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
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  • Тел.: +7(495)330-73-74
  • Эл. почта: iekash@mx.ibch.ru