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.

All publications (show selected)

Loading...

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

Design of a new compound highly selective to the α3β2 nicotinic acetylcholine receptor (nAChR) and elucidation of mechanism of its action

Two nicotinic acetylcholine receptor (nAChR) subtypes, α3β2 and α3β4, play an important role in various physiological processes, and are also involved in some pathologies. To date, almost no highly effective compounds are known to distinguish these subtypes. Based on the structure of the α-conotoxin LvIA from the venom of Conus lividus marine mollusk, novel analog [N9A]LvIA with an affinity for α3β2 nAChR 3 orders of magnitude greater than for α3β4 subtype was obtained by alanine scanning. The crystal structure of this compound with an acetylcholine-binding protein and computer modeling of the [N9A]LvIA in complex with α3β2 and α3β4 receptors revealed amino acid residues in the β2 subunit - Phe106, Ser108, Ser113, and Ser168, which determine the high selectivity of the new ligand to the α3β2 subtype, which was confirmed by mutations of these residues.

Publications

  1. Zhu X, Pan S, Xu M, Zhang L, Yu J, Yu J, Wu Y, Fan Y, Li H, Kasheverov IE, Kudryavtsev DS, Tsetlin VI, Xue Y, Zhangsun D, Wang X, Luo S (2020). High Selectivity of an α-Conotoxin LvIA Analogue for α3β2 Nicotinic Acetylcholine Receptors Is Mediated by β2 Functionally Important Residues. J Med Chem 63 (22), 13656–13668

An integrated approach: from toxin-receptor interactions to highly sensitive diagnostics

In collaboration with Laboratory of molecular toxinology,  Department of molecular bases of neurosignalization

The use of radioligand analysis, electrophysiology, and fluorescence in the study of toxin-receptor interactions was expanded by the use of gel chromatography of complexes in combination with mass spectrometry to determine the interaction constants, as well as the involvement of leading Russian spectroscopists, which made it possible to characterize in detail the binding of neurotoxins to peptides simulating the active site receptor [1], as well as to achieve detection in the blood plasma of troponin (an indicator of heart disease) in a uniquely low concentration of 1pg / ml [2].

Three-finger neurotoxins and new analogues of bradykinin-potentiating peptides were found in the venom of vipers for the first time, using transcriptomics and quantitative proteomics

In collaboration with Laboratory of Biological Testing,  Laboratory of molecular toxinology

From Azemiops feae viper venom, we have isolated previously azemiopsin, a new neurotoxin, that inhibited the nicotinic acetylcholine receptor. To characterize other A. feae toxins, quantitative proteomics was used and 120 unique proteins were identified. In total, identified toxins represented 14 families, among which bradykinin-potentiating peptides with unique amino acid sequences were found and manifested biological activity in vivo. Proteomic analysis also revealed non-conventional three-finger toxins belonging to the group of toxins with neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venoms. In parallel, transcriptome analysis of the venom gland was carried out by the next generation sequencing, and additionally identified 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

Phenoxazine-based scaffold for designing G4-interacting agents

In collaboration with The group of molecular tools for living system studies

G-quadruplexes (G4) represent one class of non-canonical secondary nucleic acid structures that are currently regarded as promising and attractive targets for anti-cancer, anti-viral and antibacterial therapy. We probe a new i-clamp-inspired phenoxazine scaffold for designing G4-stabilizing ligands. The length of the protonated aminoalkyl tethers (‘arms’) of the phenoxazine-based ligand was optimized in silico. Two double-armed ligands differing in the relative orientation of their arms and one singlearmed ligand were synthesized. The two-armed ligands significantly enhanced the thermal stability of the G-quadruplex structures (increasing the melting temperature by up to 20 °C) and displayed G4 selectivity over duplex DNA. The ligands look promising for biological studies and the phenoxazine scaffold could be a starting point for designing new G4-interacting compounds.

Publications

  1. Tsvetkov VB, Varizhuk AM, Lizunova SA, Nikolenko TA, Ivanov IA, Severov VV, Belyaev ES, Shitikov EA, Pozmogova GE, Aralov AV (2020). Phenoxazine-based scaffold for designing G4-interacting agents. Org Biomol Chem 18 (31), 6147–6154

New endogenous modulators of the acetylcholine system and selective agonists acting on cancer and immune cells

In collaboration with Laboratory of molecular toxinology,  Laboratory of oxylipins,  Department of molecular bases of neurosignalization

It was established for the first time that endogenous analogs of acetylcholine, in which unsaturated fatty acids replace the acetyl group, are modulators of the acetylcholine system capable of inhibiting muscle and neuronal alpha7 nicotinic acetylcholine receptors (nAChR), as well as acetylcholinesterase. Arachidonoylcholine has been found to exhibit antioxidant activity in in vitro and ex vivo models. In A549 cancer cells, whose growth is enhanced by the activation of alpha7 nAChR, these compounds dose-dependently inhibited their growth by 50%. Using PNU 282,987, a selective alpha7 nAChR agonist, we investigated the effect of activation of these receptors on cytokine expression and, for the first time, on the expression of membrane macrophage markers. It was found that the expression of HLA-DR, CD54, and CD11b increases, while the expression of the CD14 receptor decreases. At the same time, along with the previously known decrease in the expression of TNF-α, a significant decrease in the expression of the cytokine IL-10 was noted, which may be of great importance for the fight against sepsis and immunosuppression in the "cytokine storm".

 

1. Akimov MG, Kudryavtsev DS, Kryukova EV, Fomina-Ageeva EV, Zakharov SS, Gretskaya NM, Zinchenko GN, Serkov IV, Makhaeva GF, Boltneva NP, Kovaleva NV, Serebryakova OG, Lushchekina SV, Palikov VA, Palikova Y, Dyhenko IA, Kasheverov IE, Tsetlin VI, Bezuglov VV. Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System. Biomolecules. 2020 Feb 12; 10 (2): 283.
2. Akimov MG, Dudina PV, Fomina-Ageeva EV, Gretskaya NM, Bosaya AA, Rudakova EV, Makhaeva GF, Kagarlitsky GO, Eremin SA, Tsetlin VI, Bezuglov VV. Neuroprotective and Antioxidant Activity of Arachidonoyl Choline, Its Bis-Quaternized Analogues and Other Acylcholines. Dokl Biochem Biophys. 2020 Mar; 491 (1): 93-97.
3. Siniavin AE, Streltsova MA, Kudryavtsev DS, Shelukhina IV, Utkin YN, Tsetlin VI. Activation of α7 Nicotinic Acetylcholine Receptor Upregulates HLA-DR and Macrophage Receptors: Potential Role in Adaptive Immunity and in Preventing Immunosuppression. Biomolecules. 2020 Mar 27; 10 (4): 507.

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

In collaboration with Laboratory of molecular toxinology

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

In collaboration with Laboratory of molecular toxinology

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

Elucidation of molecular basis of Odontosyllis bioluminescence

In collaboration with Laboratory of biomolecular NMR-spectroscopy,  Laboratory of Chemistry of Metabolic Pathways

The researchers from Yampolsky lab have successfully characterized three key low-molecular-weight components of Odontosyllis undecimdonta bioluminescence system: luciferin,  oxyluciferin (Green) and a nonspecific luciferin oxidation product (Pink). These compounds were revealed to be highly unusual tricyclic heterocycles containing three sulfur atoms in different electronic states. Together the structures of these low-molecular-weight components of Odontosyllis bioluminescent system have enabled us to propose chemical transformation pathways for the enzymatic (luminescent) and non-enzymatic (dark) oxidation of luciferin. Moreover Odontosyllis oxyluciferin was established to be the only green primary emitter described for any known bioluminescent marine organism.

Publications

  1. Kotlobay AA, Dubinnyi MA, Purtov KV, Guglya EB, Rodionova NS, Petushkov VN, Bolt YV, Kublitski VS, Kaskova ZM, Ziganshin RH, Nelyubina YV, Dorovatovskii PV, Eliseev IE, Branchini BR, Bourenkov G, Ivanov IA, Oba Y, Yampolsky IV, Tsarkova AS (2019). Bioluminescence chemistry of fireworm Odontosyllis. Proc Natl Acad Sci U S A 116 (38), 18911–18916

The molecular target for mammalian orphan neuropeptide - nocystatin associated with pain sensing.

In collaboration with Laboratory of neuroreceptors and neuroregulators

Nocystatin is an endogenous neuropeptide produced by neurons of the central and peripheral nervous system. It was belonged so far to the class of orphan proteins for which the molecular target has not been defined. In the Laboratory of Neuroreceptors and Neuroregulators of the IBCh RAS was discovered the peptides’ ability to evoked currents in X. laevis oocytes expressing rat ASIC1a, ASIC1b, ASIC2a, and ASIC3 that were very similar in kinetic parameters to the proton-gated response. The peptide was able to activate all subtypes of acid-sensitive channels, and its effect was concentration-dependent. General ASIC antagonists inhibited the current induced by the nocystatin application. Moreover, the channels activated by nocystatin desensitized and lose their ability to further activation by acid, therefore, low concentration of the peptide inhibits while a higher concentration stimulates the activity of ASIC channels. This can to explain its dual effect on pain sensitivity, where it acts both as a generator of pain signals and as an analgesic compound. No previously known ASICs’ ligands shown such biological effect. Thus, nocistatin is the first endogenous direct agonist of ASICs except protons which should be associated with a media property rather as the molecule.

Novel data could give a key to understanding ASICs activation regulation in the nervous system and also could be used to develop new drugs to treat pathological processes associated with ASICs activation, such as neurodegeneration, inflammation, and pain.

An article with the results of the first experiments was published in the journal Biomolecules.

Characterization of the "analgesic" conotoxins' binding sites on the nicotinic receptor and its models

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.

Motif-based design of bioactive peptides

In collaboration with Laboratory of optical microscopy and spectroscopy of biomolecules,  Laboratory of biomolecular modeling

Engineering antimicrobial peptide with a low hemolytic activity via combination of motifs of spider venom peptides. Using coarse-grained Molecular Dynamics, the depth of penetration of parent spider venom peptides (Ltc1, Oxt 4a) in model erythrocyte membrane was estimated. The artificial peptide (P5) is formed of fragments with the low depth of penetration (encircled - see Fig.), or penetrating deeply, and thus hemolytic (enclosed in ellipse). Hydrophobicity of the latter peptide was decreased via L/K mutation.

The first recombinant viper three finger toxins - antagonists of the nicotinic acetylcholine receptors of muscle and neuronal types

In collaboration with Laboratory of Molecular Diagnostics,  Science-Educational center,  Laboratory of molecular toxinology

One of the main components of the venom of the snakes from the Elapidae family are three finger toxins, which possess various types of biological activity, including inhibition of synaptic transmission by blocking the nicotinic acetylcholine receptors. So far, three finger toxins have not been found in the venom of the Viperidae snakes, although the mRNA encoding these toxins has been found in the venom glands of snakes from this family. Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. We have shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors both of neuronal and muscle type.

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

In collaboration with Laboratory of molecular toxinology

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

Development of a new technique based on calcium imaging and functional characterization of mutant α7/α9 nAChRs with the use of this technique.

In collaboration with Laboratory of molecular toxinology

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

First peptide ligands potentiating the TRPA1 response to agonists and producing the analgesic and anti-inflammatory effects.

In collaboration with Laboratory of biomolecular NMR-spectroscopy,  Laboratory of neuroreceptors and neuroregulators

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.