Denis B Tikhonov

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (78)209.39 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Philanthotoxin-433 (PhTX-433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype by using two-electrode voltage-clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX-343, with trans-cyclopropyl-PhTX-343 being the most potent (∼28-fold) and cis-cyclopropyl-PhTX-343 least potent (∼4-fold). Both cis- and trans-cyclopropyl-PhTX-444 had intermediate potency (both ∼12-fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX-444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.
    ChemMedChem 07/2014; · 2.84 Impact Factor
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    ABSTRACT: Voltage-gated sodium and calcium channels play key roles in the physiology of excitable cells. The alpha-1 subunit of these channels folds from a polypeptide chain of four homologous repeats. In each repeat, the cytoplasmic halves of the pore-lining helices contain exceptionally conserved asparagines. Such conservation implies important roles, which are unknown. Mutations of the asparagines affect activation and inactivation gating as well as the action of pore-targeting ligands, including local anesthetics and steroidal agonists batrachotoxin and veratridine. In the absence of the open-channel structures, underlying mechanisms are unclear. Here, we modeled the pore module of Cav1.2 and Nav1.4 channels and their mutants in the open and closed states using the X-ray structures of potassium and sodium channels as templates. The energy of each model was Monte Carlo-minimized. The asparagines do not face the pore in the modeled states. In the open-channel models, the asparagine residue in a given repeat forms an inter-repeat H-bond with a polar residue, which is typically nine positions downstream from the conserved asparagine in the preceding repeat. The H-bonds, which are strengthened by surrounding hydrophobic residues, would stabilize the open channel and shape the open-pore geometry. According to our calculation, the latter is much more sensitive to mutations of the asparagines than the closed-pore geometry. Rearrangement of inter-repeat contacts may explain effects of these mutations on the voltage dependence of activation and inactivation and action of pore-targeting ligands.
    Pflügers Archiv - European Journal of Physiology 04/2014; · 4.87 Impact Factor
  • Denis B Tikhonov, Boris S Zhorov
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    ABSTRACT: The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic "blocking particle". A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.
    Biochimica et Biophysica Acta 12/2013; · 4.66 Impact Factor
  • Boris S Zhorov, Denis B Tikhonov
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    ABSTRACT: Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure-activity relationships, and effects of mutations in these ion channels.
    Trends in Pharmacological Sciences 01/2013; · 9.25 Impact Factor
  • Biophysical Journal 01/2013; 104(2):135-. · 3.67 Impact Factor
  • Denis B Tikhonov, Boris S Zhorov
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    ABSTRACT: The X-ray structure of the bacterial sodium channel NavAb provides a new template for the study of sodium and calcium channels. Unlike potassium channels, NavAb contains P2 helices in the outer-pore region. Because the sequence similarity between eukaryotic and prokaryotic sodium channels in this region is poor, the structural similarity is unclear. We analyzed it by using experimental data on tetrodotoxin block of sodium channels. Key tetrodotoxin-binding residues are outer carboxylates in repeats I, II, and IV, three positions downstream from the selectivity-filter residues. In a NavAb-based model of Nav1 channels derived from the sequence alignment without insertions/deletions, the outer carboxylates did not face the pore and therefore did not interact with tetrodotoxin. The hypothesis that the evolutionary appearance of Nav1 channels involved point deletions in an ancestral channel between the selectivity filter and the outer carboxylates allowed building of a NavAb-based model with tetrodotoxin-channel contacts similar to those proposed previously. This hypothesis also allowed us to reproduce in Nav1 the folding-stabilizing contacts between long-side chain residues in P1 and P2, which are seen in NavAb. The NavAb-based inner-pore model of Nav1 preserved major features of our previous KcsA-based models, including the access pathway for ligands through the repeat III/IV interface and their interactions with specific residues. Thus, structural properties of eukaryotic voltage-gated sodium channels that are suggested by functional data were reproduced in the NavAb-based models built by using the unaltered template structure but with adjusted sequence alignment. Sequences of eukaryotic calcium channels aligned with NavAb without insertions/deletions, which suggests that NavAb is a promising basis for the modeling of calcium channels.
    Molecular pharmacology 04/2012; 82(1):97-104. · 4.53 Impact Factor
  • Maxim V Nikolaev, Lev G Magazanik, Denis B Tikhonov
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    ABSTRACT: The NMDA type of ionotropic glutamate receptors plays a unique role in synaptic functions because of high permeability for calcium and because of a voltage-dependent block by endogenous Mg(2+). Activity and voltage dependence of the NMDA receptor channel block by organic cations are strongly affected by competition with magnesium ions for the binding site in the channel pore. It complicates prediction of action of NMDA receptor channel blockers in vivo. In the present work we studied the NMDA receptor channel block in the presence of Mg(2+) by several organic blockers with different characteristics of voltage dependence and mechanism of action. The action of NMDA receptor channel antagonists was studied in native NMDA receptors of hippocampus CA1 pyramidal neurons isolated from rat brain slices. It was demonstrated that the IC(50) values of NMDA receptor channel blockers at -30 mV are increased 1.5-5 times compared with magnesium-free conditions. The voltage dependence of the channel block is decreased, abolished or even inversed in the presence of magnesium. Although simple competition between magnesium ion and organic channel blockers provides a general explanation of the observed effects, certain disagreements were revealed. Diversity in Mg(2+) effects on the NMDAR channel block by different organic cations reported herein likely reflects interaction of NMDAR channel blockers with additional binding site(s) and suggests that individual analysis in the presence of Mg(2+) is required for newly developed NMDAR channel blocking drugs.
    Neuropharmacology 04/2012; 62(5-6):2078-85. · 4.11 Impact Factor
  • Denis B. Tikhonov, Boris S. Zhorov
    Biophysical Journal 01/2012; 102(3):327-. · 3.67 Impact Factor
  • Denis B. Tikhonov, Boris S. Zhorov
    Biophysical Journal 01/2012; 102(3):679-. · 3.67 Impact Factor
  • Oleg I Barygin, Eugene V Grishin, Denis B Tikhonov
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    ABSTRACT: Binding of argiotoxin in the closed state of Ca(2+)-permeable AMPA receptor channels was studied using electrophysiological and molecular modeling approaches. Experimental study unambiguously revealed that argiotoxin is trapped in the closed AMPA receptor channels after agonist dissociation. Docking of the argiotoxin to the channel model based on recently published X-ray structure demonstrated that the drug can be effectively accommodated in the cavity of the closed channel only if the terminal moiety of the molecule penetrates in the narrow portion of the pore below the selectivity filter. Combining these results, we conclude that the selectivity filter of the AMPA receptor channels is not sterically occluded in the closed state.
    Biochemistry 08/2011; 50(38):8213-20. · 3.38 Impact Factor
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    ABSTRACT: This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favour the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR-mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes – such as high-frequency synaptic activity or increase of the steady-state glutamate concentration – to be examined.
    The Journal of Physiology 04/2011; 589(Pt 7):1587-601. · 4.38 Impact Factor
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    ABSTRACT: This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N(1)-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favor the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes - such as high-frequency synaptic activity or increase of the steady-state glutamate concentration - to be examined.
    The Journal of Physiology 01/2011; · 4.38 Impact Factor
  • T. B. Tikhonova, L. G. Magazanik, D. B. Tikhonov
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    ABSTRACT: The ionic composition of the medium can influence the actions of many pharmacological agents. We report here our studies of the effects of the extracellular sodium concentration on blockade of calcium-permeable ion channels of AMPA receptors by the cationic phenylcyclohexyl derivative IEM-1925. Experiments were performed using native receptors from striatal giant interneurons isolated from rat brain slices. Recordings were made by whole-cell patch voltage clamping. Replacement of some of the extracellular sodium with sucrose was found to increase the blocking action of IEM-1925. This effect depended on the membrane potential, increasing on hyperpolarization. Analysis of the kinetics of the interaction of IEM-1925 with channels showed that decreases in the extracellular sodium concentration had no effect on the stability of blocker–channel complexes, increases in blockade occurring because of increases in the association rate constant. These results lead to the conclusion that current-carrying sodium ions complete with the blocking IEM-1925 cation for the binding site in the channel. KEY WORDSsynaptic transmission–glutamate–AMPA–channel blockade–ionic composition of the extracellular medium
    Neuroscience and Behavioral Physiology 01/2011; 41(6):647-653.
  • Denis B Tikhonov, Boris S Zhorov
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    ABSTRACT: In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184-197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra- and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose π-electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outer-pore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.
    Journal of Biological Chemistry 11/2010; 286(4):2998-3006. · 4.65 Impact Factor
  • Oleg I Barygin, Natalia V Luchkina, Denis B Tikhonov
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    ABSTRACT: Polyamine-containing toxins and synthetic dicationic derivatives of adamantane and phenylcyclohexyl selectively antagonize Ca(2+)-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor channels. These compounds demonstrate voltage-dependent open-channel block and are trapped by closed channels. In this study, we describe an alternative mechanism of non-competitive AMPA receptor inhibition caused by 9-aminoacridine and some of its derivatives. These compounds exhibit similar potency against Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors. The inhibition is largely voltage-independent, binding and unbinding do not require presence of agonist. We conclude that 9-aminoacridine binds to a shallow site in the AMPA receptor, which is located above the activation gate. A comparison of three-dimensional structures of the antagonists suggests that the 'V-like' shape of the hydrophobic headgroup favors voltage-dependent binding to the deep site in the channel pore, whereas the compounds possessing flat aromatic headgroups preferably bind to the shallow site. The characterization of the novel mechanism of AMPA receptor channel antagonism opens a way to develop a new family of pharmacological agents, which can be of scientific and practical importance.
    Journal of Neurochemistry 10/2010; 115(6):1621-32. · 3.97 Impact Factor
  • D B Tikhonov, L G Magazanik
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    ABSTRACT: Dysfunctions of glutamatergic synaptic neurotransmission often accompany various CNS disorders. Action of excessive glutamate, which causes excitotoxic effects by neuron depolarization and massive calcium influx can lead to cell death. Despite obvious importance of development of anti-glutamic neuroprotectors, among great number of known antagonists of ionotropic glutamate receptors only memantine is used in medicinal practice. One of the sources of numerous side effects caused by glutamate receptor antagonists is that the drugs usually inhibit receptors, which mediate both normal and pathological CNS processes. A possible approach to overcoming the problem is to develop the drugs whose action is enhanced in potentially pathological conditions such as high-frequency activation, high glutamate concentration, depolarized membrane, etc. Action of many classes of antagonists depends on pattern of receptor activation and on membrane voltage. In the present work, we discuss several peculiarities of channel blocking mechanisms from the viewpoint of neuroprotector development. In particular, we compare channel blockers which demonstrate different types of interaction with the channel gating machinery, we consider different types of voltage dependence and consider action of channel blockers, which can permeate through the channel. We conclude that meticulous analysis of the mechanism of action of the glutamate receptor channel antagonists could help to approach predicting of in vivo action using in vitro data.
    Rossiĭskii fiziologicheskiĭ zhurnal imeni I.M. Sechenova / Rossiĭskaia akademiia nauk 07/2010; 96(7):726-39.
  • Ricky C K Cheng, Denis B Tikhonov, Boris S Zhorov
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    ABSTRACT: Calcium channels play crucial physiological roles. In the absence of high-resolution structures of the channels, the mechanism of ion permeation is unknown. Here we used a method proposed in an accompanying paper (Cheng and Zhorov in Eur Biophys J, 2009) to predict possible chelation patterns of calcium ions in a structural model of the L-type calcium channel. We compared three models in which two or three calcium ions interact with the four selectivity filter glutamates and a conserved aspartate adjacent to the glutamate in repeat II. Monte Carlo energy minimizations yielded many complexes with calcium ions bound to at least two selectivity filter carboxylates. In these complexes calcium-carboxylate attractions are counterbalanced by calcium-calcium and carboxylate-carboxylate repulsions. Superposition of the complexes suggests a high degree of mobility of calcium ions and carboxylate groups of the glutamates. We used the predicted complexes to propose a permeation mechanism that involves single-file movement of calcium ions. The key feature of this mechanism is the presence of bridging glutamates that coordinate two calcium ions and enable their transitions between different chelating patterns involving four to six oxygen atoms from the channel protein. The conserved aspartate is proposed to coordinate a calcium ion incoming to the selectivity filter from the extracellular side. Glutamates in repeats III and IV, which are most distant from the repeat II aspartate, are proposed to coordinate the calcium ion that leaves the selectivity filter to the inner pore. Published experimental data and earlier proposed permeation models are discussed in view of our model.
    Biophysics of Structure and Mechanism 04/2010; 39(5):839-53. · 2.44 Impact Factor
  • Serge N Moralev, Denis B Tikhonov
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    ABSTRACT: It is known than the most potent homologues in various series of O,O-dialkylphosphates are the dibutyl or diamyl derivatives toward mammalian cholinesterases (ChEs) (both Acetyl- and Butyryl-ChEs), and the dimethyl or diethyl ones toward insect AChEs. To investigate the ChE interaction with organophosphorus inhibitors (OPIs) in more detail, we have performed in silico docking of the series of O,O-dialkylfluorophosphates into active center of different ChEs - both from mammals (human and mouse AChEs and horse BChE), and from insects (spring grain aphid AChE belonging to AChE-1 type, and housefly AChE belonging to AChE-2 type). According to the modeling results, one radical is directed to the anionic site W84, another to the acyl pocket. In addition to well-known residues 288 and 290 (Torpedo AChE sequence numbering), we showed an essential influence of residue 400 - a short alkyl residue in mammalian ChEs and phenylalanine in insect ChEs. Phenylalanine in this position creates sterical hindrance for proper orientation of the OPI molecule, which increases the distance between the catalytic serine gamma-oxygen and phosphorus, and decreases the angle of nucleophylic attack. This suggestion was supported by docking of dibutylfluorophosphate into the active center of AChEs with in silico mutations. Thus, we suggest both the angle of nucleophylic attack and the distance between the catalytic serine and phosphorus atom as measures of productivity of OPI binding.
    Chemico-biological interactions 03/2010; 187(1-3):153-6. · 2.46 Impact Factor
  • Denis B. Tikhonov, Boris S. Zhorov
    Biophysical Journal 01/2010; 98. · 3.67 Impact Factor
  • D B Tikhonov, L G Magazanik
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    ABSTRACT: This article provides a review of approaches to identification of the pathways of the molecular evolution of glutamate receptors. Extensive evidence has now accumulated on the homology of glutamate-binding proteins with the ability to function as ligand-activated channels. However, knowledge of the amino acid sequences of the polypeptides forming these channels is a necessary but insufficient condition for identifying their origin and changes during evolution. Natural selection of protein molecules appears to have identified and fixed their functional nature. Molecular and functional approaches should therefore complement each other in studies of protein evolution. Studies of glutamate receptor channels in vertebrates and invertebrates provide an example showing how knowledge of the spatial organization and the details of the mechanisms of operation allows relationships to be identified and possible pathways of the molecular evolution of receptors to be established.
    Neuroscience and Behavioral Physiology 10/2009; 39(8):763-73.

Publication Stats

776 Citations
209.39 Total Impact Points

Institutions

  • 1998–2014
    • Russian Academy of Sciences
      • Institute of Evolutionary Physiology and Biochemistry
      Moskva, Moscow, Russia
  • 2004–2013
    • McMaster University
      • Department of Biochemistry and Biomedical Sciences
      Hamilton, Ontario, Canada
  • 2006–2007
    • University at Albany, The State University of New York
      New York City, New York, United States