Laboratory of ligand-receptor interactions

Department of molecular bases of neurosignalization

Head: Igor' Kasheverov, D.Sc
iekash@ibch.ru+7(495)995-55-57#2118

ligand-gated ion channels, nicotinic receptors, glycine receptor, peptide synthesis, radioligand assay, electrophysiology

Laboratory was organised in 2009 and is headed by Dr. Igor Kasheverov

The main scope of the Ligand-receptor interactions lab is structure-function relationships of natural and artificial peptide ligands of several Cys-loop receptors (nAChR, GlyR and GABAA). Construction of completely new active compounds based on known anticholinergic ligands is also in the field of our interest.

The lab conducts computer-aided design of new compounds based on diverse conotoxins of different structural classes and modelling of their complexes with nicotinic receptors (nAChR) and acetylcholine-binding proteins (AChBP). We have at our disposal eqipment for peptide synthesis, chromatography and mass-spectrometry, which is used for synthesys and characterization of active compounds. We also have electrophysiology and radioligand assay eqipment for ligans activities research.

Our lab contacts has lots of international collaborations in Germany, France, Finland, Greece, China and Vietnam. And we are open to new contacts!

Previously, more than forty diverse conotoxins analogs were synthetized and studied in the lab. Partly these studies could be represented by following reviews:

Tsetlin V., Utkin Y., Kasheverov I. (2009). Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptorsBiochem Pharmacol. 78(7), 720—731

Kasheverov I.E., Utkin Y.N., Tsetlin V.I. (2009). Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptorsCurr Pharm Des. 15(21), 2430—2452.

Selected publications (show all)

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Scientific projects

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Igor' Kasheverov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 33, office. 236
  • Phone: +7(495)995-55-57#2118
  • E-mail: iekash@ibch.ru

The first three-finger snake neurotoxin, which distinguishes two binding sites in the muscle nicotinic receptor by affinity and dissociation kinetics (2019-12-13)

A new three-finger α-neurotoxin - αδ-bungarotoxin - was isolated and characterized from the venom of Bungarus candidus krait. It differs from α-bungarotoxin, the well-known antagonist of muscle, α7 and α9α10 neuronal subtypes of nicotinic receptors, by 11 substitutions of 74 amino acid residues. The new toxin also effectively interacted with the α7 receptor with nanomolar affinity, but it demonstrated a unique (for three-finger toxins) difference in affinity towards two different binding sites on the muscle receptor (affinity to the α-δ site was higher than to the α-γ site by 20 times). In addition, the new toxin showed kinetics of dissociation from one of the sites of muscle-type receptor much faster than α-bungarotoxin with its almost irreversible binding. Thus, a new tool for the study of muscle nicotinic receptors with a unique set of properties has been discovered.

Publications

  1. Utkin YN, Kuch U, Kasheverov IE, Lebedev DS, Cederlund E, Molles BE, Polyak IL, Ivanov IA, Prokopev NA, Ziganshin RH, Jornvall H, Alvelius G, Chanhome L, Warrell DA, Mebs D, Bergman T, Tsetlin VI (2019). Novel Long-chain Neurotoxins from Distinguish the Two Binding Sites in Muscle-type Nicotinic Acetylcholine Receptors. Biochem J 476 (8), 1285–1302

A new class of nicotinic acetylcholine receptor inhibitors has been proposed and synthesized (2019-12-13)

Based on our own and published data showing that positively charged amino acid residues play an important role in the activity of α-conotoxins, a series of peptides consisting exclusively of arginine (R) residues was synthesized, and oligoarginines (R = 6-18) were shown to be inhibitors of various subtypes of nicotinic acetylcholine receptors (nAChR), with R8 having pronounced neuronal selectivity against α9/α10 nAChR, and R16 possessing the highest affinity for α7 nAChR. Inhibitory activity was also found for a number of positively charged polymers, which, along with oligoarginines, are used for intracellular delivery of potential drug compounds, which should be taken into account as possible positive and negative effects.

Publications

  1. Lebedev D, Kryukova E, Ivanov I, Egorova N, Timofeev N, Spirova E, Tufanova E, Siniavin A, Kudryavtsev D, Kasheverov I, Zouridakis M, Katsarava R, Zavradashvili N, Iagorshvili I, Tzartos S, Tsetlin V (2019). Oligoarginine Peptides, a New Family of nAChR Inhibitors. Mol Pharmacol 96 (5), 664–673

Characterization of the "analgesic" conotoxins' binding sites on the nicotinic receptor and its models (2018-12-03)

A set of Cys-Cys isomers of "analgesic" conotoxins RgIA and GeXIVA acting through ortho- or allosteric binding sites of α9/α10 of nicotinic acetylcholine receptor, respectively, was synthesized. The study of them by direct or competitive radioligand analysis with water-soluble models of this receptor - acetylcholine-binding protein and recombinant extracellular domain of the α9 receptor subunit, showed that they effectively interact with the micromolar affinity only with the orthosteric binding site of these models.

Development of a new technique based on calcium imaging and functional characterization of mutant α7/α9 nAChRs with the use of this technique. (2017-12-15)

On the basis of the calcium imaging method, we developed a new technique that allows to effectively express functionally active "problematic" subtypes of nicotinic receptors (nAChRs) in cell lines. It involves co-expressing with the appropriate receptor subtype a chaperone and a fluorescent calcium sensor Case12. This technique allowed us to obtain 6 mutant forms of α7 nAChR with selected single substitutions of amino acid residues from α9 nAChR subtype. All the mutants together with the wild-type receptors were analyzed for affinity to acetylcholine and epibatidine using the developed technique. This helped to identify two key mutations - L119D and F187S which are responsible for selectivity of these nAChR subtypes to above-mentioned ligands. Computer simulations showed a significant change in the arrangement of ligands’ molecules in binding sites of these two mutant forms of the receptor, explaining the data obtained.

Publications

  1. 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

SLURP-1 (81 amino-acid residues, 5 disulfides), identical in the amino-acid sequence to the endogenous human protein, has been synthesized and shown to differ from all known recombinant (2017-12-15)

SLURP-1 (81 amino acid residues, 5 disulfides) has been synthesized with the amino acid sequence identical to that of the endogenous human toxin-like protein. 1H-NMR revealed the same structure as in the recombinant rSLURP-1 bearing additional N-terminal Met0. These proteins  have some differences in molecular dynamics, but differ greatly in their activity towards distinct subtypes of nicotinic receptors. Our work in general stresses the necessity of maximal approach to the structure of naturally-occurring proteins to solve the mechanisms of their endogenous activities and choosing appropriate medical applications.  

Publications

  1. Durek T, Shelukhina IV, Tae HS, Thongyoo P, Spirova EN, Kudryavtsev DS, Kasheverov IE, Faure G, Corringer PJ, Craik DJ, Adams DJ, Tsetlin VI (2018). Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors. Sci Rep 7 (1), 16606