Laboratory of neuroreceptors and neuroregulators

Department of Molecular Neurobiology

Head: Sergey Kozlov, D.Sc
serg@ibch.ru+7(495)336 4022

Toxinology, neurotoxins, ionotropic receptors, protein structure, recombinant polypeptides, bioactive molecules for medicine

The laboratory conducts research of biologically active components from natural sources, interacting with a variety of targets in the cell membrane, as well as developing a common approach for the identification, isolation, structural and functional analysis and use as a thin tool which modulate the activity of the nerve cells membrane receptors. Laboratory research allows to be closer to an understanding the molecular mechanisms of the natural ligand specificity action and lays the foundation for the development of fundamentally new highly specific drugs and insecticides.

The laboratory cooperates with The Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, (Magazanik L.G.), Federal Scientific Clinical Center of Physical-Chemical Medicine (Govorun V.M.), Sadykov Institute of Bioorganic Chemistry of the Academy of Sciences of Uzbekistan (Salikhov S.I.), Vavilov Institute of General Genetics RAS (Odintsova T.I., PhD), All-Russian Research Institute of Agricultural Biotechnology (prof. Babakov A.V.), University of Leuven, Belgium (Prof. Tytgat J.).

The laboratory has also performed studies in cooperation with Monsanto, Bayer, Zeneka, DuPont and other companies.

Young people are always welcome and get first class practical courses. Since its foundation, 23 PhD dissertations have been successfully defended in the Laboratory. High quality equipment, use of state-of-the-art methods, ongoing staff practical skill training, open-mindedness and ambitious scientific goals – this is what best describes the Laboratory today.

The Laboratory was founded in 1987 by Professor Eugene V. Grishin, member of the Russian Academy of Sciences. The laboratory has evolved into a world-leading scientific team studying natural neurotoxins and their neuronal receptors. The focus is on toxic components of animal venoms acting on different targets in the cell membrane. General approaches have been elaborated to active venom components identification, isolation, structure-function analyses and use as high-precision tools modulating neural membrane receptor activity.

  • Search and investigation of polypeptide toxins and lower-modulated compounds that modulate activity of membrane receptors involved in processes of generation and pain signaling (Korolkova Y.V., PhD; Andreev Ya.A., PhD; Koshelev S.G., PhD)
  • Studies of antimicrobial peptides from arthropods and plant defense peptides (Rogozhin E.A., PhD)
  • Production of recombinant polypeptide toxins (Andreev Ya.A. PhD); study of DNA complex nanostructure (Danilevich V.N.).
  • Development of agricultural crop protection methods to increase crop (Rogozhin E.)
  • Development of methods for the production of recombinant polypeptide toxins in various heterologous expression systems (Yaroslav A. Andreev, Catherine E. Maleeva)
  • Omixed technologies implementation in search and structural identification of biologically active compounds of complex multi-component mixtures (Mikov A.)
  • Creation of cell lines for the analysis of ligand-receptor interactions (Julia Korol'kova, Yaroslav A. Andreev)
  • Studying the DNA nanostructure complexes (Vasiliy Danilevich)
  • a new class of toxins such as spiders argiope – polyamine blocker of glutamate receptors (Grishin et al., 1983)
  • a family of high-selective neurotoxins from the venom of the Black Widow ("black widow") – latrotoxin (Kiyatkin et al., 1990)
  • membrane-active peptides from the venom of ants and spiders (Kozlov et al., 2006)
  • polypeptide toxins specific for sodium, potassium, calcium, proton-sensitive, TRPV1, TRPA1-channels.
  • low-molecular weight biologically active compounds from medicinal plants with analgesic effect.
  • In the study of neuronal receptors isolated and characterized for the first time all of the individual components of the mammalian sodium channel electro-excitability membranes.
NamePositionContacts
Anna Slavokhotova
Sergey Kozlov, D.Scdepart. dir.serg@ibch.ru+7(495)336 4022
Yaroslav Andreev, Ph.D.s. r. f.ay@land.ru+7(495)336-40-20
Vasilij Danilevich, Ph.D.s. r. f.dan@ibch.ru+7(495)336-40-22
Elena Eljakova, Ph.D.s. r. f.elyakova@yandex.ru+7(495)429-87-20, +7(495)995-55-57#2501
Yulija Korol'kova, Ph.D.s. r. f.july@ibch.ru+7(495)3366540
Sergej Koshelev, Ph.D.s. r. f.sknew@yandex.ru+7(495)3306683
Vera Sadykovas. r. f.
Evgenij Rogozhinr. f.rea21@list.ru+7(495)336-40-22
Dmitry Osmakovr. f.
Alexey Vasilchenkor. f.
Alexander Mikovj. r. f.mikov.alexander@gmail.com+7(495)3366540
Ekaterina Maleevaj. r. f.+7()3364022
Irina Mosharovaj. r. f.+7()3306683
Yulia Logashinaj. r. f.yulia.logashina@gmail.com+7(495)3364022
Andrey Vladimirovt. q. - lab. as.
Ljudmila Aleksandrovaeng.+7(495)336-40-22
Natalia Nigmatulinaeng.natnb@mail.ru
Kseniya Lybovaeng.lubova.ksenia@gmail.com
Anna Baranovares. eng.
Timur Bozinres. eng.
Irina Gavryushinares. eng.

Former members:

Tseziy Egorov, D.Scl. r. f.ego@ibch.ru
Alexander Vassilevski, Ph.D.s. r. f.avas@ibch.ru
Tat'jana Valjakina, Ph.D.s. r. f.valyakina@ibch.ru
Kirill Pluzhnikov, Ph.D.s. r. f.
Maria Simonova, Ph.D.r. f.simonova@ibch.ru
Ravilja Komalevar. f.
Ol'ga Lahtinar. f.
Elena Petrova, Ph.D.r. f.petrova@ibch.ru
Aleksandr Musoljamovj. r. f.musolyamov@ibch.ru
Larisa Samohvalovaj. r. f.
A Astafevaj. r. f.a_sanya@mail.ru
Maria Sachkova, Ph.D.PhD stud.sachkovamasha@mail.ru
Alexander Arzamasovstud.Alex_arzamasov@hotmail.com
Anna Emelianovares. eng.annaemelyan@gmail.com
Zhanna Kanaevskayalab. as.zh-kanewskaya@yandex.ru
Evgeny Grishin, member of the academy of sciencesdept. head

Selected publications

  1. Osmakov DI, Koshelev SG, Andreev YA, Dubinnyi MA, Kublitski VS, Efremov RG, Sobolevsky AI, Kozlov SA (2018). Proton-independent activation of acid-sensing ion channel 3 by an alkaloid, lindoldhamine, from Laurus nobilis. Br J Pharmacol Chemother 175 (6), 924–937
  2. Babenko VV, Mikov AN, Manuvera VA, Anikanov NA, Kovalchuk SI, Andreev YA, Logashina YA, Kornilov DA, Manolov AI, Sanamyan NP, Sanamyan KE, Kostryukova ES, Kozlov SA, Grishin EV, Govorun VM, Lazarev VN (2017). Identification of unusual peptides with new Cys frameworks in the venom of the cold-water sea anemone Cnidopus japonicus. Sci Rep 7 (1), 14534
  3. Kozlov S (2017). Animal toxins for channelopathy treatment. Neuropharmacology 132 (0), 83–97
  4. Osmakov DI, Koshelev SG, Andreev YA, Kozlov SA (2017). Endogenous isoquinoline alkaloids agonists of acid-sensing ion channel type 3. Front Mol Neurosci 10 (0), 282
  5. Nekrasova OV, Volyntseva AD, Kudryashova KS, Novoseletsky VN, Lyapina EA, Illarionova AV, Yakimov SA, Korolkova YV, Shaitan KV, Kirpichnikov MP, Feofanov AV (2017). Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol 12 (2), 260–276
  6. Logashina YA, Solstad RG, Mineev KS, Korolkova YV, Mosharova IV, Dyachenko IA, Palikov VA, Palikova YA, Murashev AN, Arseniev AS, Kozlov SA, Stensvåg K, Haug T, Andreev YA (2017). New disulfide-stabilized fold provides sea anemone peptide to exhibit both antimicrobial and TRPA1 potentiating properties. Toxins (Basel) 9 (5),
  7. Slavokhotova AA, Shelenkov AA, Korostyleva TV, Rogozhin EA, Melnikova NV, Kudryavtseva AV, Odintsova TI (2017). Defense peptide repertoire of Stellaria media predicted by high throughput next generation sequencing. B SOC CHIM BIOL 135 (0), 15–27
  8. Korolkova Y, Makarieva T, Tabakmakher K, Shubina L, Kudryashova E, Andreev Y, Mosharova I, Lee HS, Lee YJ, Kozlov S (2017). Marine cyclic guanidine alkaloids monanchomycalin B and urupocidin a act as inhibitors of TRPV1, TRPV2 and TRPV3, but not TRPA1 receptors. Mar Drugs 15 (4),
  9. Logashina YA, Mosharova IV, Korolkova YV, Shelukhina IV, Dyachenko IA, Palikov VA, Palikova YA, Murashev AN, Kozlov SA, Stensvåg K, Andreev YA (2017). Peptide from sea anemone metridium senile affects transient receptor potential ankyrin-repeat 1 (TRPA1) function and produces analgesic effect. J Biol Chem 292 (7), 2992–3004
  10. Рогожин ЕА, Кисиль ОВ, Чертаев ИВ, Завриев СК (2017). Характеристика белково- пептидного экстракта семян мари белой ( Chenopodium album L.): изучение компонентного состава, антимикробных и анальгетических свойств. 62 (0), 3–8
  11. Mikov AN, Fedorova IM, Potapieva NN, Maleeva EE, Andreev YA, Zaitsev AV, Kim KK, Bocharov EV, Bozin TN, Altukhov DA, Lipkin AV, Kozlov SA, Tikhonov DB, Grishin EV (2015). ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom. Sci Rep 5 (0), 17232
  12. Astafieva AA, Enyenihi AA, Rogozhin EA, Kozlov SA, Grishin EV, Odintsova TI, Zubarev RA, Egorov TA (2015). Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: De novo sequencing and biological activity. Plant Sci 238 (0), 323–329
  13. Dubovskii PV, Vassilevski AA, Kozlov SA, Feofanov AV, Grishin EV, Efremov RG (2015). Latarcins: Versatile spider venom peptides. Cell Mol Life Sci 72 (23), 4501–4522
  14. Rogozhin EA, Slezina MP, Slavokhotova AA, Istomina EA, Korostyleva TV, Smirnov AN, Grishin EV, Egorov TA, Odintsova TI (2015). A novel antifungal peptide from leaves of the weed Stellaria media L. B SOC CHIM BIOL 116 (0), 125–132
  15. Миков АН, Козлов СА (2015). Структурные особенности цистеин-богатых полипептидов из ядов морских анемон. 41 (5), 511–523
  16. Danilevich VN, Artemov VV, Smith SS, Gainutdinov RV, Mulyukin AL (2014). The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR. J Biomol Struct Dyn 32 (12), 1979–1992
  17. Kozlov SA, Lazarev VN, Kostryukova ES, Selezneva OV, Ospanova EA, Alexeev DG, Govorun VM, Grishin EV (2014). Comprehensive analysis of the venom gland transcriptome of the spider Dolomedes fimbriatus. Sci Data 1 (0), 140023
  18. Ryazantsev DY, Rogozhin EA, Dimitrieva TV, Drobyazina PE, Khadeeva NV, Egorov TA, Grishin EV, Zavriev SK (2014). A novel hairpin-like antimicrobial peptide from barnyard grass (Echinochloa crusgalli L.) seeds: Structure-functional and molecular-genetics characterization. B SOC CHIM BIOL 99 (1), 63–70
  19. Slavokhotova AA, Rogozhin EA, Musolyamov AK, Andreev YA, Oparin PB, Berkut AA, Vassilevski AA, Egorov TA, Grishin EV, Odintsova TI (2014). Novel antifungal α-hairpinin peptide from Stellaria media seeds: Structure, biosynthesis, gene structure and evolution. Plant Mol Biol 84 (12), 189–202
  20. Slavokhotova AA, Naumann TA, Price NPJ, Rogozhin EA, Andreev YA, Vassilevski AA, Odintsova TI (2014). Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases. FEBS J 281 (20), 4754–4764
  21. Pluzhnikov KA, Kozlov SA, Vassilevski AA, Vorontsova OV, Feofanov AV, Grishin EV (2014). Linear antimicrobial peptides from Ectatomma quadridens ant venom. B SOC CHIM BIOL 107 (0), 211–215
  22. Vassilevski AA, Sachkova MY, Ignatova AA, Kozlov SA, Feofanov AV, Grishin EV (2013). Spider toxins comprising disulfide-rich and linear amphipathic domains: A new class of molecules identified in the lynx spider Oxyopes takobius. FEBS J 280 (23), 6247–6261
  23. Andreev YA, Kozlov SA, Korolkova YV, Dyachenko IA, Bondarenko DA, Skobtsov DI, Murashev AN, Kotova PD, Rogachevskaja OA, Kabanova NV, Kolesnikov SS, Grishin EV (2013). Polypeptide modulators of TRPV1 produce analgesia without hyperthermia. Mar Drugs 11 (12), 5100–5115
  24. Osmakov DI, Kozlov SA, Andreev YA, Koshelev SG, Sanamyan NP, Sanamyan KE, Dyachenko IA, Bondarenko DA, Murashev AN, Mineev KS, Arseniev AS, Grishin EV (2013). Sea anemone peptide with uncommon β-hairpin structure inhibits acid-sensing ion channel 3 (ASIC3) and reveals analgesic activity. J Biol Chem 288 (32), 23116–23127
  25. Utkina LL, Andreev YA, Rogozhin EA, Korostyleva TV, Slavokhotova AA, Oparin PB, Vassilevski AA, Grishin EV, Egorov TA, Odintsova TI (2013). Genes encoding 4-Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: Multimodular structural organization, instraspecific variability, distribution and role in defence. FEBS J 280 (15), 3594–3608
  26. Kudryashova KS, Nekrasova OV, Kuzmenkov AI, Vassilevski AA, Ignatova AA, Korolkova YV, Grishin EV, Kirpichnikov MP, Feofanov AV (2013). Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study. Anal Bioanal Chem 405 (7), 2379–2389
  27. Philyppov IB, Paduraru ON, Andreev YA, Grishin EV, Shuba YM (2012). Modulation of TRPV1-dependent contractility of normal and diabetic bladder smooth muscle by analgesic toxins from sea anemone Heteractis crispa. Life Sci II 91 (1920), 912–920
  28. Kabanova NV, Vassilevski AA, Rogachevskaja OA, Bystrova MF, Korolkova YV, Pluzhnikov KA, Romanov RA, Grishin EV, Kolesnikov SS (2012). Modulation of P2X3 receptors by spider toxins. BIOCHIM BIOPHYS ACTA 1818 (11), 2868–2875
  29. Dubinnyi MA, Osmakov DI, Koshelev SG, Kozlov SA, Andreev YA, Zakaryan NA, Dyachenko IA, Bondarenko DA, Arseniev AS, Grishin EV (2012). Lignan from thyme possesses inhibitory effect on ASIC3 channel current. J Biol Chem 287 (39), 32993–33000
  30. Gladkikh I, Monastyrnaya M, Leychenko E, Zelepuga E, Chausova V, Isaeva M, Anastyuk S, Andreev Y, Peigneur S, Tytgat J, Kozlovkaya E (2012). Atypical reactive center Kunitz-type inhibitor from the sea anemone Heteractis crispa. Mar Drugs 10 (7), 1545–1565
  31. Andreev YA, Korostyleva TV, Slavokhotova AA, Rogozhin EA, Utkina LL, Vassilevski AA, Grishin EV, Egorov TA, Odintsova TI (2012). Genes encoding hevein-like defense peptides in wheat: Distribution, evolution, and role in stress response. B SOC CHIM BIOL 94 (4), 1009–1016
  32. Dubovskii PV, Vassilevski AA, Samsonova OV, Egorova NS, Kozlov SA, Feofanov AV, Arseniev AS, Grishin EV (2011). Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin. FEBS J 278 (22), 4382–4393
  33. Lazarev VN, Polina NF, Shkarupeta MM, Kostrjukova ES, Vassilevski AA, Kozlov SA, Grishin EV, Govorun VM (2011). Spider venom peptides for gene therapy of Chlamydia infection. Antimicrob Agents Chemother 55 (11), 5367–5369
  34. Nolde SB, Vassilevski AA, Rogozhin EA, Barinov NA, Balashova TA, Samsonova OV, Baranov YV, Feofanov AV, Egorov TA, Arseniev AS, Grishin EV (2011). Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli). J Biol Chem 286 (28), 25145–25153
  35. Slavokhotova AA, Odintsova TI, Rogozhin EA, Musolyamov AK, Andreev YA, Grishin EV, Egorov TA (2011). Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds. B SOC CHIM BIOL 93 (3), 450–456
  36. Kozlov S, Grishin E (2011). The mining of toxin-like polypeptides from EST database by single residue distribution analysis. BMC Genomics 12 (0), 88
  37. Vassilevski AA, Fedorova IM, Maleeva EE, Korolkova YV, Efimova SS, Samsonova OV, Schagina LV, Feofanov AV, Magazanik LG, Grishin EV (2010). Novel class of spider toxin: Active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide. J Biol Chem 285 (42), 32293–32302
  38. Grishin EV, Savchenko GA, Vassilevski AA, Korolkova YV, Boychuk YA, Viatchenko-Karpinski VY, Nadezhdin KD, Arseniev AS, Pluzhnikov KA, Kulyk VB, Voitenko NV, Krishtal OO (2010). Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain. Ann Neurol 67 (5), 680–683
  39. Odintsova TI, Vassilevski AA, Slavokhotova AA, Musolyamov AK, Finkina EI, Khadeeva NV, Rogozhin EA, Korostyleva TV, Pukhalsky VA, Grishin EV, Egorov TA (2009). A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif. FEBS J 276 (15), 4266–4275
  40. Nekrasova OV, Ignatova AA, Nazarova AI, Feofanov AV, Korolkova YV, Boldyreva EF, Tagvei AI, Grishin EV, Arseniev AS, Kirpichnikov MP (2009). Recombinant Kv channels at the membrane of escherichia coli bind specifically agitoxin2. J Neuroimmune Pharmacol 4 (1), 83–91
  41. Kozlov SA, Vassilevski AA, Grishin EV (2009). Secreted protein and peptide biosynthesis: Precursor structures and processing mechanisms.  (0), 225–248
  42. Odintsova TI, Rogozhin EA, Baranov Y, Musolyamov AK, Yalpani N, Egorov TA, Grishin EV (2008). Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv. B SOC CHIM BIOL 90 (1112), 1667–1673
  43. Andreev YA, Kozlov SA, Koshelev SG, Ivanova EA, Monastyrnaya MM, Kozlovskaya EP, Grishin EV (2008). Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1). J Biol Chem 283 (35), 23914–23921
  44. Vassilevski AA, Kozlov SA, Samsonova OV, Egorova NS, Karpunin DV, Pluzhnikov KA, Feofanov AV, Grishin EV (2008). Cyto-insectotoxins, a novel class of cytolytic and insecticidal peptides from spider venom. Biochem J 411 (3), 687–696
  45. Kozlov SA, Vassilevski AA, Grishin EV (2007). Peptidomics of Short Linear Cytolytic Peptides from Spider Venom.  (0), 55–70
  46. Tseng GN, Sonawane KD, Korolkova YV, Zhang M, Liu J, Grishin EV, Guy HR (2007). Probing the outer mouth structure of the hERG channel with peptide toxin footprinting and molecular modeling. Biophys J 92 (10), 3524–3540
  47. Kozlov SA, Vassilevski AA, Feofanov AV, Surovoy AY, Karpunin DV, Grishin EV (2006). Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J Biol Chem 281 (30), 20983–20992
  48. Jiang M, Zhang M, Maslennikov IV, Liu J, Wu DM, Korolkova YV, Arseniev AS, Grishin EV, Tseng GN (2005). Dynamic conformational changes of extracellular S5-P linkers in the hERG channel. J Physiol 569 (1), 75–89
  49. Botos I, Melnikov EE, Cherry S, Kozlov S, Makhovskaya OV, Tropea JE, Gustchina A, Rotanova TV, Wlodawer A (2005). Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus Lon reveals the conformational variability in the active sites of Lon proteases. J Mol Biol 351 (1), 144–157
  50. Krukovskaja LL, Baranova A, Tyezelova T, Polev D, Kozlov AP (2005). Experimental study of human expressed sequences newly identified in silico as tumor specific. Tumour Biol 26 (1), 17–24
  51. Zhang M, Korolkova YV, Liu J, Jiang M, Grishin EV, Tseng GN (2003). BeKm-1 is a HERG-specific toxin that shares the structure with ChTx but the mechanism of action with ErgTx1. Biophys J 84 (5), 3022–3036
  52. Korolkova YV, Bocharov EV, Angelo K, Maslennikov IV, Grinenko OV, Lipkin AV, Nosyreva ED, Pluzhnikov KA, Olesen SP, Arseniev AS, Grishin EV (2002). New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1. J Biol Chem 277 (45), 43104–43109
  53. Korolkova YV, Kozlov SA, Lipkin AV, Pluzhnikov KA, Hadley JK, Filippov AK, Brown DA, Angelo K, Strøbæek D, Jespersen T, Olesen SP, Jenser BS, Grishin EV (2001). An ERG Channel Inhibitor from the Scorpion Buthus eupeus. J Biol Chem 276 (13), 9868–9876
  54. Grishin EV, Korolkova YV, Kozlov SA, Lipkin AV, Nosyreva ED, Pluzhnikov KA, Sukhanov SV, Volkova TM (1996). Structure and function of the potassium channel inhibitor from black scorpion venom. Pure Appl Chem 68 (11), 2105–2109

Sergey Kozlov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 51, office. 363
  • Phone: +7(495)336 4022
  • E-mail: serg@ibch.ru

Fax: +7 (495) 330 5892

First peptide ligands potentiating the TRPA1 response to agonists and producing the analgesic and anti-inflammatory effects. (2017-11-24)

Two analgesic peptides Мs9а-1 and Ueq 12-1 were isolated from sea anemones Metridium senile and Urticina eques and characterized. Peptide Мs9а-1 contains 35 amino acid residues, and its spatial structure is stabilized by two disulfide bridges. The spatial structure of Мs9а-1 is similar to the sea anemones peptides structures described previously. Ueq 12-1 consists from 45 amino acid residues including 10 cysteine residues with an unusual distribution among sea anemone peptides. Its uncommon spatial structure resolved by NMR is partially similar to the structure of mammal’s alpha defensins. This similarity can explain a weak antimicrobial activity of Ueq 12-1 against gram-positive bacteria. Structurally different peptides Мs9а-1 and Ueq 12-1 have a similar mechanism of action onto the same biological target. Experiments in vitro on TRPA1 receptor expressed in oocytes of Xenopus laevis or in mammalian cells shown an increase of receptors’ respond to direct agonists, such as AITC and diclofenac. The intravenously peptides application in tests on mice in vivo resulted in significant analgesic and anti-inflammatory effects, while peptides’ administration did not cause pain or thermal hypersensitivity. We assume that observed effects are connected with the fact that peptides make the receptor more sensitive to their agonists (potentiating effect). So a release of endogenous inflammatory mediators leads to the desensitization of TRPA1-expressing neurons and a nociception decrease. Such enhance of the TRPA1 activity by peptides give novel opportunity for basic research and analgesic drug development.

Novel endogenous ligands of ASIC3 receptor can activate the channel independent from protons, and release human ASIC3 from the steady state desensitization at physiological pH. (2017-11-24)

First activators of acid-sensing ion channels (ASIC) with unique mechanism of action were found. The milimolar concentration of isoquinoline alkaloids (THP, reticuline), which are synthesized by mammalian cells as intermediates in the cycle of endogenous morphine synthesis from tyrosine, can decrease ASIC3 proton-mediated desensitization and at the same time effectively activates hASIC3 and rASIC3 at physiological pH 7.4 and slightly alkaline conditions. To date, a very limited pool of ASICs activators is known. All these molecules shift the channels activation limit to the higher pH values, that making the receptor to be more responsible to a small pH spikes. But still the protons contribution in the process of the channels opening considered as primary. The isoquinoline alkaloids are the first described "non-proton" activators of ASIC3 receptors.

A pharmacological characterization of "non-proton" activators at human and rodents ASIC3 isoforms revealed a strong functional difference of the effect between the rat channels (minor effect) and human (strong potentiate response to acidification) that questioned the effectiveness of ligands proved in animal tests by their transfer to clinical study. In addition, we suggested a new approach for the new analgesics development. Bu our opinion, two systems of nociception (ASIC3) and anti-nociception (opioid receptors) shared common biosynthetic pathway of ligands and may be in mutually balanced state. The inhibition or activation of enzymes involved in the endogenous morphine biosynthesis cascade may shift the equilibrium in some direction that results in analgesia or hyperalgesia.

ASICs play an important role not only in nociception but in the signal transduction, synaptic plasticity, learning, and neuronal cells death. The discovery of endogenous "non-proton" activators proves the existence of several regulatory ways for these channels. It is not clear which of them can more be implemented in the CNS, it is possible that acidification only accompanied the ligand gated regulation. The answer to this question will be obtained in further study.

The multidirectional mechanism of action of peptide modulators of TRPV1 receptor in different conditions of receptor activation has been established. (2017-11-24)

The effect of polypeptides APHC1, APHC2 and APHC3 isolated from sea anemones Heteractis crispa was studied on rTRPV1 expressed in CHO cells. Method of electrophysiology, fluorescence spectroscopy and molecular modeling was utilized. It was established that APHCs potentiate the response of TRPV1 to low (3-300 nM) capsaicin concentrations and inhibit the response to high (>3.0 µM) concentrations of the activator. Also the dependence of peptides action onto TRPV1 was investigated for varying concentrations of activators, such as 2APB and protons, and for the activation by combined stimuli. As a result, the bimodal mechanism of APHCs action onto the receptor is depended to the stimulus power then peptides potentiate low signals but inhibit / neutral to high-amplitude responses. The proposed dual "Gating" model of TRPV1 activation suggests that APHC-polypeptides can stabilize an intermediate state during the receptor activation. By molecular modeling the expected binding site of peptides located between a pair of P-loops on TRPV1 top.

Modulators like APHCs with a dualistic effect have a definite advantage for practical medical applications, since such compounds do not inhibit the normal functioning of the receptor, but produce the desired therapeutic effect at pathologically strong activation.

Publications

  1. Nikolaev MV, Dorofeeva NA, Komarova MS, Korolkova YV, Andreev YA, Mosharova IV, Grishin EV, Tikhonov DB, Kozlov SA (2017). TRPV1 activation power can switch an action mode for its polypeptide ligands. PLoS One 12 (5), e0177077

ω-Tbo-IT1 — selective inhibitor of insect channels isolated from Tibellus oblongus spider venom (2016-03-28)

Novel disulfide-containing polypeptide toxin was found in venom Tibellus oblongus spider from Central Asia region. Here, we report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin called ω-Tbo-IT1 It has insect toxic effect with LD50 14.3 μg/g in experiments on Musca domestica larvae. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in Calliphora vicina neuromuscular junction while parameters of spontaneous ones were not affected. Inhibition was concentration dependent with IC50 value 40±10 nM and Hill coefficient 3.4±0.3. The toxin did not affect frog neuro-muscular junction, glutamatergic and GABAergic transmission in rat brain. Ca2+ currents in Calliphora vicina muscle were not inhibited, whereas in cockroach neurons at least one type of voltage gated Ca2+ currents were inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as selective inhibitor of presynaptic insect Ca2+ channels. Spatial structure analysis of recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed the set of 20 structures. The toxin comprises the conventional ICK (knottin) fold containing extended β-hairpin loop and γ-turn that are capable of doing — as we called it — “scissors-like mutual motions”. Alongside with alternative twisting of the β-sheet observed in the major β-hairpin loop such plasticity of the molecule (dynamic epitope) may play a crucial role for the receptor binding/recognition.

Publications

  1. Mikov AN, Fedorova IM, Potapieva NN, Maleeva EE, Andreev YA, Zaitsev AV, Kim KK, Bocharov EV, Bozin TN, Altukhov DA, Lipkin AV, Kozlov SA, Tikhonov DB, Grishin EV (2015). ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom. Sci Rep 5 (0), 17232

A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers (2016-03-28)

A novel peptide named ToAMP 4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, an d RP-H PLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids , and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 doe s not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4 , it was success fully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4 -mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and function al diversity of AMPs in plants.