Department of Molecular Neurobiology

All publications (show selected)

Alexey Semyanov

Patterns of Calcium Dynamics in Brain Astrocytic Networks

Laboratory of Extrasynaptic Signalling

Astrocytes encode the state of local neural environment in spatiotemporal patterns of Ca2+ activity. In turn, Ca2+ events in astrocytes trigger release of gliotransmitters, modulate synaptic plasticity and local blood flow via various mechanisms. The use of genetically encoded Ca2+ indicators has allowed to visualize astrocytic Ca2+ activity on multiple scales from thin distal processes to whole cell domains to cell networks. Interpretation of imaging data requires adequate techniques of data processing, including motion correction, denoising, signal separation to slow and fast components, segmentation of separate Ca2+ events, and revealing possible recurrent patterns in their initiation. Here we demonstrated that spontaneous Ca2+ activity in single astrocyte takes form of separate events, which primarily located in the periphery of astrocytic spatial domains. The Ca2+ events (yellow tinctures at the processed image) spanned across wide range of sizes and durations. The fluorescence amplitude, the rates of rise and decay within such Ca2+ events were linked to the local cell morphology (e.g. thickness of the process). At the network level, the Ca2+ activity was fluctuating, most of the population activity peaks was explained by appearance of large size events, engulfing whole astrocyte domains, rather than an increase in events frequency. The proposed techniques and experimental data can shed light on the principles that define the formation of spatiotemporal patterns of astrocytic Ca2+ activity and possible involvement of these patterns in cognitive tasks.

Publications

  1. Браже АР, Доронин МС, Попов АВ, Денисов Александро, Семьянов АВ (2019). Patterns of Calcium Dynamics in Brain Astrocytic Networks. Ross Fiziol Zh Im I M Sechenova 105 (11), 1436–1451

Previously, for the design of peptides with a given function, we have proposed using a convenient structural framework, namely, the α-hairpinin fold, characteristic of toxins from scorpion venom and plant defense peptides. Now, the use of the Protein Surface Topography method that we developed, has significantly improved the properties of an artificial α-hairpinin, which blocks Kv1.3 potassium channels, an important pharmacological target. The joint application of two approaches, namely, scaffold engineering and protein surface topography, can be used to obtain optimized ion channel ligands.

The physiological effect of two bisbenzylisoquinoline alkaloids having activity on ASIC1a

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Biological Testing,  Laboratory of neuroreceptors and neuroregulators

The ASIC1a is the most sensitive subtype of acid-sensing ion channel in the cell membrane, and it plays an important role in the excitation of neurons of CNS. Long time the ligands to this ASIC subtype are under intense attention for the development of drugs for pain relief, as well as protectors from strokes and neurodegenerative diseases. In in vitro experiments on heterologically expressed ASIC1a channels, the action of two bisbenzylisoquinoline alkaloids from plants was studied by electrophysiological method of two-electrode potential fixing on oocyte cells.

The alkaloid lindoldhamine extracted from the leaves of Laurus nobilis L. significantly inhibited the ASIC1a channel’s response to physiologically-relevant stimuli of pH 6.5–6.85 with IC50 range 150–9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund’s adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid. Thus, it was shown not only a prospective of plant alkaloids using for a pain relief, but was indirectly confirmed the involvement of the ASIC1a channels of the peripheral nervous system in the generation of a pain response to mild acidification.

The structural analogue named daurisoline, unlike lindoldamine, did not inhibit the activation of the ASIC1a channel by protons, but produced the second peak component of the ASIC1a current. This second peak manifested with a 2.5 seconds delay after the first fast respond followed by completely desensitization with the same kinetics as the main peak. The presence of second current components was specific characteristic of ASIC2 and ASIC3 subtypes early, but this component is sustained, that last all time while the acid stimulus presented. The discovery of the second component of ASIC1a current allows us to declare the common mechanism of opening and desensitization for all ASICs, which will be interesting to determine in further experiments.

3D reconstruction of astrocytic processes

Laboratory of Extrasynaptic Signalling

3D reconstruction of astrocytic processes was obtained using electron microscopy of series slices. Astrocytic processes consisted of branchlets, shafts-like structures containing organelles, and leaflets, thin organelle-free perisynaptic processes attached to the branchlets. We observed leaflets of different sizes and shapes and showed that leaflet enlargement may potentially prepare the space to accept ER cisterns and leaflet conversion to branchlets extensions. Was shown that number of glutamatergic synaptic contact to a leaflet is determined only by the leaflet size.

Publications

  1. Gavrilov N, Golyagina I, Brazhe A, Scimemi A, Turlapov V, Semyanov A (2018). Astrocytic coverage of dendritic spines, dendritic shafts, and axonal boutons in hippocampal neuropil. Front Cell Neurosci 12, 248

Kalium 2.0, a database of all known polypeptide ligands of potassium channels

Laboratory of biomolecular modeling,  Laboratory of Molecular Instruments for Neurobiology

Previously, we have created a comprehensive database of scorpion toxins acting on potassium channels, called Kalium. Now we have expanded it to include all known potassium channel ligands of peptide nature in general. Together with the Guide to PHARMACOLOGY resource, which contains information on low-molecular-mass ligands, Kalium 2.0 database provides researchers with full information on this most important group of compounds.

By tradition, our initiative has received widespread community approval, with leading international experts in the field of ion channel ligands acting as Kalium 2.0 experts. Kalium 2.0 database is available following this link.

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

Laboratory of ligand-receptor interactions,  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.

Combinatorial selective incorporation of stable13C and 15N isotopes facilitates NMR spectra analysis and allows mapping of the binding interfaces between membrane receptors and their ligands

Group of in silico analysis of membrane proteins structure,  Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Combinatorial incorporation of stable 13C and 15N isotopes into protein molecules can significantly simplify the analysis of NMR spectra. For the first time, the problem was solved and the CombLabel algorithm was developed for calculating combinatorial 13C and 15N  labeling schemes with a minimum price. The application of the program allowed to assign 50% of the NMR signals of the backbone of the second voltage-sensing domain of human sodium channel Nav1.4 (VSD-II). Leak currents through mutant variants of Nav1.4 containing Arg675Gly mutation in VSD-II lead to the development of a hereditary disease – normokalemic periodic paralysis. Hm-3 toxin from the venom of spider Heriaeus melloteei is able to block leak currents in VSD-II. By the means of NMR spectroscopy the interaction interface between VSD-II and Hm-3 toxin was determined. According to the model of the VSD-II/Hm-3 complex, based on the NMR data, the toxin binds to the extracellular S1-S2 loop, destabilizing the state of the domain, at which leak currents are observed. Using the example of the complexes of Hm-3 toxin with VSD-I and VSD-II of the Nav1.4 channel, it has been shown that arachnid toxins can interact differently with different domains within the same sodium channel.

Scorpion venom is rich in peptide blockers of voltage-gated potassium channels (KV), and we have reflected this diversity previously in Kalium, a database dedicated to such peptides. A high-affinity and selective blocker of KV1.2 channels, characteristic of the human central nervous system, was obtained from the venom of the scorpion Mesobuthus eupeus. Using molecular modeling and site-directed mutagenesis, the mechanism of selective interaction between the toxin and channels was investigated.

Molecular mechanism of action of acylpolyamines, glutamate receptor blockers

Laboratory of Molecular Instruments for Neurobiology

Spiders and wasps secrete in their venom acylpolyamines that act as high-affinity blockers of receptors for glutamate, the main excitatory neurotransmitter of the human brain. Under the leadership of Eugene Grishin in 1986, the first representative of acylpolyamines was described, namely, argiopin from the venom of the orb-weaver spider Argiope lobata. In 2018, the spatial structure of argiopin complex with a glutamate receptor was studied using cryo-electron microscopy. The obtained results will allow the creation of drugs for the treatment of neurodegenerative diseases. The study was featured on the cover of Neuron. Read more in the press release on the IBCh website.

Publications

  1. Twomey EC, Yelshanskaya MV, Vassilevski AA, Sobolevsky AI (2018). Mechanisms of Channel Block in Calcium-Permeable AMPA Receptors. Neuron 99 (5), 956–968.e4

The proton-independent activator of acid-sensing ion channels ASIC3 with unusual pharmacological properties was found in herb Laurus nobilis.

Laboratory of Biopharmaceuticals,  Laboratory of biomolecular NMR-spectroscopy,  Laboratory of biomolecular modeling,  Laboratory of neuroreceptors and neuroregulators

The screening of natural sources for novel ligands to ASIC ion channels resulted by a discovery of Lindoldhamine from laurel noble leaves, which can activate the ASIC3 channel at physiological pH. It has been demonstrated that acidification of extracellular media, which normally leads to the activation of the channel, is not more a necessary condition for the both human and rat ASIC3 isoforms opening. Electrophysiological experiments on heterologous expressed ASIC3 ion channels revealed differences in a modulation of human and rat isoform by lindoldhamine. Various applied protocols let to determine the binding of lindoldhamine with human ASIC3 isoform in the closed state that results in a 2-fold increase of transient current amplitude by acidic pH stimulus, however, the rat ASIC3 isoform was not affected to the ligand. Proton independent activation of the rat channel also due to a significantly lower current amplitude registered. As a result, a potent pharmacological difference among human and rat ASIC3 channels were shown during a respond to the novel alkaloid, which proves once again the ambiguity of interpretation of the animal tests data to the further drug developing for humans. The unusual pharmacological properties of lindoldhamine make possible using it as a new instrument for the ASIC channels activity studying, as well as for a study of the nervous system synaptic plasticity in total, since the decisive role of these channels in this process has been proven. The unique property of new ligand is the ability to compete with protons causing desensitization of the ASIC3 transient current. Lindoldhamine increase the amplitude of the transient current on a pH-dependent desensitization curve in contrast to known ASIC ligands, that can shift this curve towards more acidic/alkali value without amplitude change.

Toxin from the venom of the crab spider Heriaeus melloteei may serve as a hit in drug discovery for hypokalemic periodic paralysis type 2; there is no reliable medication for all cases of this disease. It is caused by mutations in the gene encoding voltage-gated sodium channels NaV1.4, characteristic of skeletal muscles. As a result of the mutations, these channels conduct aberrant currents, the muscles are unable to respond to the signals of the nervous system, and weakness develops followed by paralysis. Hm-3 toxin was found to be able to selectively inhibit such currents through voltage-sensing domain I of mutant channels. Read more in the press release on the IBCh website.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules), О.V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity

Laboratory of optical microscopy and spectroscopy of biomolecules,  Group of in silico analysis of membrane proteins structure,  Laboratory of Molecular Instruments for Neurobiology,  Group of nanobioengineering

Оksana V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling), A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

Interaction of gating modifier toxin Hm-3 with voltage-sensing domains of Nav1.4 sodium channel: structural view on the membrane-mediated binding

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Voltage-gated Na+ channels (Nav) are essential for the functioning of cardiovascular, muscular, and nervous systems. Certain mutations trigger a leak current through voltage-sensing domains (VSDs) of Nav leading to various diseases. Hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the S4 segments of VSDs in the human skeletal muscle channel NaV1.4. The gating modifier toxin Hm-3 (crab spider Heriaeus melloteei) inhibits leak currents through such mutant channels. To investigate molecular basis of Hm-3 interaction with NaV1.4 channel, we studied isolated VSD-I by NMR spectroscopy in membrane mimicking environment. Hm-3/VSD-I complex was modeled using protein-protein docking guided by NMR restrains. The toxin initially anchors onto the membrane surface and then forms the complex with the S3b-S4 loop of the VSD-I. The Hm-3 binding blocks movement of the voltage-sensor helix S4 and induces some allosteric changes that prevent development of gating-pore currents. Our report is the first NMR study of structural interactions between gating modifier toxins and Nav channels.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

Laboratory of Molecular Instruments for Neurobiology is known for systematic study of Arthropods’ venoms and derived peptides that specifically target various ion channels. Scorpions’ venom is abundant with potassium channels (Kv) blockers, and this diversity was described in previously released in Kalium database.

In cooperation with Laboratory of optical microscopy and spectroscopy of biomolecules and Group of nanobioengineering an unique screening system permitted identification in the Mesobuthus eupeus scorpion venom of Kv1.2 blocker: peptide MeKTx11-1 binging with high affinity (IC50 ≈0,2 nM) and specificity (effect on Kv1.1, 1.3 and 1.6 emerges at >100-fold higher concentrations). This peptide differs from the related MeKTx11-3 by just two residues, possessing substantially lower Kv1.2-specificity.

Finally, Group of in silico analysis of membrane proteins structure conducted a molecular modeling study of these two peptides interacting with Kv1.2 channel, immersed into an explicit lipid bilayer. This study uncovered mechanism of selective action of MeKTx11-1 peptide. The developed analysis technique will be of use for future design of selective ligands of Kv and other channels, which may be applied in fundamental studies of molecular basis of nervous system function and as drugs prototypes.

The structure of the two components of the lipopeptide antibiotic crystallomycin from a sample obtained 60 years ago has been established. The identity of the components of two crystallomycin components to these of aspartocin (the structure of which has been elucidated recently) has been found. The antibiotic exhibits Ca2+ -dependent activity against gram-positive bacteria. The conformations of crystallomycin 2 in solution were investigated using NMR.

The amino acid 4-chloro-L-kinurenin, previously found in natural products only once, was found in the peptide antibiotic INA-5812. We first described the fluorescent properties of 4-chloro-L-kinurenin and its use as an energy donor for the excitation of other fluorophores.

The structure of two new macrolide antibiotics, astolides A and B, has been established using various 2D NMR techniques. Astolide molecules contain simultaneously a membrane-active polyol macrolide and a redox-active naphthoquinone residue as aglycones. The presence of a hydroxyl group at position 18 dramatically changes the spectrum of biological activity in comparison with the known analogues – antifungal activity increases and cytotoxicity reduces.

Publications

  1. Alferova VA, Shuvalov MV, Suchkova TA, Proskurin GV, Aparin IO, Rogozhin EA, Novikov RA, Solyev PN, Chistov AA, Ustinov AV, Tyurin AP, Korshun VA (2018). 4-Chloro-l-kynurenine as fluorescent amino acid in natural peptides. Amino Acids 50 (12), 1697–1705
  2. Alferova VA, Novikov RA, Bychkova OP, Rogozhin EA, Shuvalov MV, Prokhorenko IA, Sadykova VS, Kulko AB, Dezhenkova LG, Stepashkina EA, Efremov MA, Sineva ON, Kudryakova GK, Peregudov AS, Solyev PN, Tkachev YV, Fedorova GB, Terekhova LP, Tyurin AP, Trenin AS, Korshun VA (2018). Astolides A and B, antifungal and cytotoxic naphthoquinone-derived polyol macrolactones from Streptomyces hygroscopicus. Tetrahedron 74 (52), 7442–7449
  3. Jiang ZK, Tuo L, Huang DL, Osterman IA, Tyurin AP, Liu SW, Lukyanov DA, Sergiev PV, Dontsova OA, Korshun VA, Li FN, Sun CH (2018). Diversity, novelty, and antimicrobial activity of endophytic actinobacteria from mangrove plants in Beilun Estuary National Nature Reserve of Guangxi, China. Front Microbiol 9 (MAY), 868
  4. Tyurin AP, Alferova VA, Paramonov AS, Shuvalov MV, Malanicheva IA, Grammatikova NE, Solyev PN, Liu S, Sun C, Prokhorenko IA, Efimenko TA, Terekhova LP, Efremenkova OV, Shenkarev ZO, Korshun VA (2018). Crystallomycin revisited after 60 years: Aspartocins B and C. Medchemcomm 9 (4), 667–675

The multidirectional mechanism of action of peptide modulators of TRPV1 receptor in different conditions of receptor activation has been established.

Laboratory of neuroreceptors and neuroregulators

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

Synthetic fragment of receptor for advanced glycation end products prevents memory loss and protects brain neurons in animals with experimentally induced Alzheimer’s disease.

Laboratory of synthetic vaccines

It was established the relationship between ability of synthetic fragments of receptor for advanced glycation end products administrated intranasally as well as immunized to prevent memory loss in animals with experimentally induced Alzheimer’s disease and to prevent neuronal death and to decreased the amyloid-beta level in the brain. Using fluorescent labeled peptide, we revealed its penetration in the brain of mice and colocalization of peptide with amiloyd-beta  plaques. It was shown that amyloid-beta can be one of the molecular target of active peptide. These findings provide a new approach for design of anti-Alzheimer’s disease therapy.

Venoms of many spiders contain two-domain toxins that unite in their structure modules, which are similar to "simple" single-domain toxins. We conducted a detailed structural study of those toxins that consist of disulfide-rich (similar to ordinary neurotoxins) and linear (similar to conventional cytotoxins) modules. Linear modules can serve for the association of two-domain toxins with membranes due to the formation of amphiphilic helices, characteristic of membrane-active peptides. We propose a "membrane access" mode of action for two-domain toxins: linear modules interact with lipid bilayers, whereas disulfide-rich modules bind to protein receptors.

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

Laboratory of biomolecular NMR-spectroscopy,  Laboratory of ligand-receptor interactions,  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.

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.

Laboratory of neuroreceptors and neuroregulators

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.

ω-Tbo-IT1 — selective inhibitor of insect channels isolated from Tibellus oblongus spider venom

Laboratory of neuroreceptors and neuroregulators

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, 17232

A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers

Laboratory of neuroreceptors and neuroregulators

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.