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Cyclophane and acyclic cyclophane: Novel channel blockers of N-methyl-d -aspartate receptor
Neurochemistry International 50 (2007) 443–449 www.elsevier.com/locate/neuint
Cyclophane and acyclic cyclophane: Novel channel blockers of N-methyl-D-aspartate receptor Takashi Masuko a, Hiroki Nagaoka a, Muneharu Miyake a, Koichi Metori a, Yasuo Kizawa a, Keiko Kashiwagi b, Kazuei Igarashi c, Tadashi Kusama a,* a
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan b Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Choshi, Japan c Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Received 23 May 2006; received in revised form 29 September 2006; accepted 9 October 2006 Available online 17 November 2006
Abstract The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca2+-permeable AMPA receptors (GluR1), Ca2+-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1a). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (70 mV), the IC50 values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5 0.1, 1.0 0.2, 8.0 0.8 and 4.9 0.5 mM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, 100 mV > 70 mV > 20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg2+. However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid. # 2006 Elsevier Ltd. All rights reserved. Keywords: Cyclophane; Acyclic cyclophane; Ligand-gated ion channels; N-Methyl-D-aspartate receptor; Voltage-clamp recording; Xenopus oocytes; Openchannel blockers
1. Introduction N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have structural similarities with the a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors (Dingledine et al., 1999). NMDA receptors have several characteristics that distinguish them from other glutamate receptors. These include the requirement for two different agonists (glutamate and glycine) to activate the receptor, a high permeability of the channel to Ca2+, and a voltage-dependent block of the channel by Mg2+ (Dingledine et al., 1999). NR1 is a single gene product expressed as eight
* Corresponding author. Tel.: +81 47 465 4789; fax: +81 47 465 4789. E-mail address: [email protected] (T. Kusama). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.10.003
alternatively spliced mRNAs, and NR2A, NR2B, NR2C and NR2D are distinct gene products. NMDA receptors probably consist of tetrameric and heteromeric subunit assemblies that have different physiological and pharmacological properties and are differentially distributed throughout the CNS (Danysz and Parsons, 1998). In the CNS, the NMDA receptor plays critical important role in a variety of neurophysiological phenomena, including neurodevelopment, synaptic plasticity, and excitotoxicity. It is well known that glutamate and aspartate can be neurotoxins, especially when energy supply is compromised (Greene and Greenamyre, 1996). This information has given rise to the proposal that neurodegeneration associated with a variety of acute and chronic disorders (ischemic stroke, epilepsy, Parkinson’s disease, AIDS dementia, and so on) may be caused in part by overactivation of glutamate receptors. Density
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of synaptosomal NMDA receptor exhibited by 37% increase in epileptic human neocortex, compared to non-epileptic tissue (Steffens et al., 2005). These data indicate that NMDA receptor has close relations with epileptic seizures and that NMDA receptor will be a target molecule for epileptic disease. Overactivation of NMDA receptors increase intracellular free Ca2+ levels, mediate oxidative stress and induce neuronal cell death (Nakamichi and Yoneda, 2006; Sanganahalli et al., 2006). NMDA receptor channels are blocked by a large number of structurally dissimilar blockers including Mg2+, ketamine, MK801 (dizocilpine), memantine, and various spider toxins and polyamine derivatives (Huettner and Bean, 1988; Collingridge and Lester, 1989; Chen and Huang, 1992; Igarashi et al., 1997; Dingledine et al., 1999). MK-801 and memantine inhibit NMDA receptors expressed in Xenopus oocytes and the inhibition was reduced at recombinant NMDA receptors containing NR1 N616Q in the M2 loop. The residue (N616) forms the narrowest region of channel pore. A large number of open channel blockers of NMDA receptors were shown to act in the narrowest region of channel pore (Kashiwagi et al., 2002). Recently, we reported that acyclic cyclophane ACCn (4,40 bis[2-(1,4,7,10-tetraaza-cyclododecane-1-yl)acetylaminoethoxy]diphenylmethane) inhibited the activity of NR1/ NR2A and NR1/NR2B receptors at 70 mV (Masuko et al., 2005). In this paper, we investigated the effects of cyclophanes, CPCn, CPPy and TGDMAP, and acyclic cyclophane ATGDMAP on NMDA receptor activities expressed in Xenopus oocytes. Our purpose is to develop a specific blocker for NMDA receptors, because excessive stimulus by glutamate leads to neuronal cell death in various neuronal diseases. 2. Experimental procedure 2.1. Materials Cyclophanes, CPCn (N,N0 -bis(1,4,7,10-tetraazacyclododecane-1-ylacetyl)10,26-diaza-7,13,23,29-tetraoxal[7.1.7.1]paracyclophane) and TGDMAP (1,10 {[(2R,17S,19S,34R)-8,9,11,12,25,26,28,29-octahydro-3H,10H,16H,20H,27H,3 3H-4,7:21,24-dimetheno-13,19,15:30,2,32-di[1]propen[1]yl[3]ylidynene-1H,18 H-dipyrimido[1,6-1:10 ,60 -o][1,8,15,22]tetraazacyclooctacosine-10,27-diyl]di-3, 1-propanediyl}bis4-dimethylaminopyridinium dichloride) were synthesized as described previously (Metori and Miyake, 2003; Metori et al., 2005) and the methods for synthesizing acyclic cyclophane ATGDMAP (N,N0 -(6H,12H5,11-methanodibenzo[b,f ][1,5]diazocine-2,8-diyldi-2,1-ethanediyl)-bis[N-(3(4-dimethylaminopyridinium)propyl)phenethylamine]dichloride) and another cyclophane CPPy (N,N0 -bis(3-(4-dimethylaminopyridinium)propyl)-10,26diaza-7,13,23,29-tetraoxa[7.1.7.1]paracyclophane dichloride) will be described elsewhere. MDMAP was synthesized according to the method of the previous report (Jerchel et al., 1956). The structures of these compounds shown in Fig. 1 were confirmed by mass spectrometry and 1H-NMR spectrometry. 1,2-Bis(2aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA) and collagenase were purchased from Sigma (St. Louis, MO). All other reagents used were of the highest grade commercially available.
2.2. NMDA, AMPA, metabotropic glutamate receptor clones and numbering of residues The NR1 clone used in these studies is the NR1A variant (Moriyoshi et al., 1991) which lacks the 21-amino acid insert encoded by exon-5. This clone was a gift from Dr. S. Nakanishi (Osaka Bioscience Institute, Osaka, Japan). The rat and mouse NR2A and NR2B clones (Kutsuwada et al., 1992; Monyer et al.,
1992) were gifts from Dr. M. Mishina (Graduate School of Medicine, University of Tokyo, Japan) and Dr. P. H. Seeburg (Center for Molecular Biology, University of Heidelberg, Germany). The mouse NR2C and NR2D clones (Ikeda et al., 1992; Kutsuwada et al., 1992), the mouse GluR1 and GluR2 clones (Sakimura et al., 1990), and the mGluR1a clone (Masu et al., 1991) were also gifts from Dr. M. Mishina. The preparation of NR1 and NR2B mutants has been described previously (Williams et al., 1998; Kashiwagi et al., 2002, 2004). Amino acids are numbered from initiator methionine in each subunit. This differs from the numbering system used in some other laboratories, in which residues are numbered from the start of the mature peptide (Kuner et al., 1996; Beck et al., 1999).
2.3. Expression in oocytes and voltage-clamp recording Adult female Xenopus laevis (Saitama Experimental Animals Supply Co. Ltd., Saitama, Japan) were chilled on ice, and the preparation and maintenance of oocytes were carried out as described previously (Masuko et al., 1999a,b). Capped cRNAs were prepared from linearized cDNA templates using mMessage mMachine kits (Ambion, Austin, TX). Oocytes were injected with NR1A and NR2 cRNAs at a ratio of 1:5 (0.2–4 ng of NR1A plus 1–20 ng of NR2). Where indicated, oocytes were injected with cRNAs of GluR1 (50 ng), GluR1/ GluR2 (10 ng of GluR1 plus 20 ng of GluR2) or mGluR1a (20 ng) instead of NR1A and NR2 cRNAs. Macroscopic currents were recorded with a twoelectrode voltage-clamp method using Dual Electrode Voltage Clamp Amplifier CEZ-1250 (Nihon Koden, Tokyo, Japan). Electrodes were filled with 3 M KCl. Oocytes were continuously superfused (ca. 5 ml/min) with a Mg2+-free saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM BaCl2, 10 mM HEPES, pH 7.5). This solution contained BaCl2 rather than CaCl2, and, in most experiments, oocytes were injected with K+-BAPTA (100 nl of 40 mM, pH 7.5) on the day of recording to eliminate Ca2+-activated Cl currents (Leonard and Kelso, 1990; Masuko et al., 2004).
3. Results 3.1. Effects of cyclophanes, CPCn, CPPy and TGDMAP, and acyclic cyclophane ATGDMAP on glutamate receptors expressed in Xenopus oocytes The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on the activity of glutamate receptors were examined. Although 10 mM CPCn, CPPy, TGDMAP and ATGDMAP did not inhibit the activities of AMPA receptors and a metabotropic glutamate receptor, all these compounds inhibited NR1/NR2A receptor activities caused by 10 mM glutamate with 10 mM glycine (Fig. 2). These results indicate that CPCn, CPPy, TGDMAP and ATGDMAP are specific blockers for NMDA receptors. To determine the subunit-specificity of inhibition by these four compounds, the inhibitory effects at NR1/NR2 receptors containing different NR2 subunits, NR2A, NR2B, NR2C and NR2D, were investigated. The inhibition of NR1/NR2A receptors by cyclophanes and acyclic cyclophane was more pronounced than those of other NMDA receptor subtypes (Fig. 3). The inhibitions by CPCn and CPPy, both have diphenylmethane skeleton, were stronger than those by TGDMAP and ATGDMAP, both have a tro¨ger base skeleton. We studied dose-dependency of the inhibition by cyclophanes, acyclic cyclophane, cyclen and MDMAP for NR1/NR2A receptors at 70 mV. The IC50 values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5 0.1, 1.0 0.2, 8.0 0.8 and 4.9 0.5 mM, respectively (Fig. 4). The inhibitory effects of cyclen and MDMAP (up to 100 mM)
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Fig. 1. Structures of cyclophanes, acyclic cyclophane and their functional groups on side-chains.
on NR1/NR2A receptor were negligible. The inhibition by CPPy was eight times stronger than that of TGDMAP, suggesting that diphenylmethane skeleton can interact with the NMDA receptor more easily than the tro¨ger base skeleton. 3.2. Noncompetitive block by cyclophanes and acyclic cyclophane for the glutamate and glycine sites on NMDA receptors To determine whether the effects of cyclophanes and acyclic cyclophane on NR1/NR2A receptors are dependent on the concentration of glutamate or glycine, the inhibitory effects of 1 mM CPCn, 1 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP on NR1/NR2A receptors were measured using 10 mM glycine with 3, 30 and 300 mM glutamate (Fig. 5A) or 10 mM glutamate with 3, 30 and 300 mM glycine (Fig. 5B). The inhibition by cyclophanes and acyclic cyclophane was
independent on the concentration (3–300 mM) of glycine or glutamate, indicating noncompetitive block for the glutamate or glycine site on NMDA receptors. 3.3. Voltage-dependent inhibition by cyclophanes and acyclic cyclophane To clarify the mechanism of inhibition by cyclophanes and acyclic cyclophane, we tried to determine whether CPCn, CPPy, TGDMAP and ATGDMAP produced voltage-dependent inhibition of NMDA receptors or not. We studied the effects of CPCn, CPPy, TGDMAP and ATGDMAP on NR1/NR2A and NR1/ NR2B expressed in Xenopus oocytes voltage-clamped at 20, 70 and 100 mV (Fig. 6). The inhibitions by CPCn, CPPy, TGDMAP and ATGDMAP became prominent at 100 mV compared to those at 20 mV. The results suggest that inhibition by cyclophanes and acyclic cyclophane is voltage-dependent.
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Fig. 4. Inhibitory curves of cyclophanes, acyclic cyclophane and their sidechains at NR1/NR2A receptor currents at 70 mV. Concentration–inhibition curves for CPCn, CPPy, TGDMAP, ATGDMAP, cyclen and MDMAP were determined at NR1/NR2A receptors, voltage-clamped at 70 mV. Responses to 10 mM glutamate with 10 mM glycine measured in the presence of these compounds were expressed as a percentage of the control response at each receptor type. The values are presented as mean S.E.M. from four to six oocytes.
Fig. 2. Effects of cyclophanes and acyclic cyclophane on glutamate receptors at 70 mV. The effects of 10 mM CPCn, CPPy, TGDMAP and ATGDMAP were determined in oocytes expressing NMDA (NR1/NR2A), Ca2+-permeable AMPA (GluR1), Ca2+-nonpermeable AMPA (GluR1/GluR2) and metabotropic glutamate (mGluR1a) receptors, voltage-clamped at 70 mV. NR1/NR2A and mGluR1a receptor currents were evoked by 10 mM glutamate with 10 mM glycine and 100 mM glutamate, respectively. GluR1 and GluR1/GluR2 receptor currents were evoked by 100 mM kainate. (A) Representative traces showing the effects of 10 mM CPCn on NR1/NR2A, GluR1, GluR1/GluR2 and mGluR1a receptors, respectively. (B) Macroscopic currents in the presence of cyclophanes and acyclic cyclophane were expressed as a percentage of the control response at each glutamate receptor. The values are presented as mean S.E.M. from four to six oocytes.
pore, because inhibition by CPCn was voltage-dependent. The narrowest region composed of M2 loops of NR1 and NR2 subunits is a critical determinant of divalent cation permeability and channel block by Mg2+ or other organic channel blockers. The potency of many open channel blockers, including MK801, polyamine derivatives such as TB-34, and memantine, was greatly reduced in the mutants NR1 (N616Q), NR2B (N615Q) and NR2B (N616Q) (Kashiwagi et al., 1997, 2002). However, inhibition by CPCn was not affected in NR1/NR2B receptor mutants containing NR1 (N616Q), NR2B (N615Q) and NR2B (N616Q) (Fig. 7). Furthermore, the inhibitory potency of CPCn did not change in NMDA receptor mutants containing NR1
3.4. Identification of amino acid residues involved in the block by CPCn To identify amino acid residues that are involved in the channel block of NMDA receptors by CPCn, we examined the effects of mutations on the residues composing the channel
Fig. 3. Effects of cyclophanes, acyclic cyclophane and their side-chains on NMDA receptors at 70 mV. The effects of 10 mM CPCn, CPPy, TGDMAP, ATGDMAP, cyclen and MDMAP were determined in oocytes expressing NR1/ NR2 receptors containing NR2A, NR2B, NR2C and NR2D, voltage-clamped at 70 mV. Responses to 10 mM glutamate with 10 mM glycine measured in the presence of these compounds were expressed as a percentage of the control response at each receptor type. The values are presented as mean S.E.M. from four to six oocytes for each subunit combination.
Fig. 5. Effects of cyclophanes and acyclic cyclophane in the presence of various concentrations of agonists on NR1/NR2A receptor currents at 70 mV. The inhibitions by 1 mM CPCn, 1 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP were determined in oocytes expressing NR1/NR2A receptors, voltage-clamped at 70 mV: (A) 3, 30, or 300 mM glutamate with 10 mM glycine; (B) 3, 30, or 300 mM glycine with 10 mM glutamate. The values are presented as mean S.E.M. from four to six oocytes for each subunit combination.
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Fig. 6. Voltage-dependent inhibition by cyclophanes and acyclic cyclophane of NMDA receptor currents. The effects of 1 mM CPCn, 1 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP on NR1/NR2A receptors, and 3 mM CPCn, 3 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP on NR1/NR2B receptors, were measured at 20, 70 and 100 mV. The values are presented as mean S.E.M. from four to six oocytes for each subunit combination.
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(E598Q), NR1 (T602G) and NR1 (E621Q), located in the intracellular regions of NMDA receptor. On the other hand, the degree of inhibition of CPCn was reduced in mutants containing NR1 (N650A), located on the top of M1, and NR1 (D669N), located in the extracellular regions, although the inhibitory effects were not changed at the mutant receptors containing NR1 (N650Q) and NR1 (D669E) receptors. The acidic group at NR1 D669 and amide group at NR1 N650 may be involved in the interaction with CPCn. The results suggest that CPCn directly interacts with surface amino acid residues around the vestibule of the ion channel and extracellular region, like a lid, and does not reach the narrowest region of channel of NMDA receptors. 4. Discussion NMDA receptors play some crucial physiological roles in various forms of synaptic plasticity such as those involved in learning and memory (Collingridge and Singer, 1990; Danysz
Fig. 7. Effects of CPCn on wild type NR1/NR2B and mutant receptors in the channel pore region. (A) Schematic illustration of the NR1 and NR2 subunits, which contain three transmembrane segments (M1, M3, and M4), a re-entrant loop (M2), an extracellular N-terminal domain, extracellular loop between M3 and M4, an intracellular loop between M1 and M2, and intracellular C-terminal domain. The inhibitory effects of CPCn were examined on the mutants at the indicated amino acid residues in the figure. (B) Representative traces showing block by CPCn at wild type NR1/NR2B receptors, mutant NR1 (N650A)/NR2B and NR1 (D669N)/NR2B receptors, respectively. (C) The effects of 10 mM CPCn were determined in oocytes expressing wild type and mutant NR1/NR2B receptors, voltage-clamped at 70 mV. The values are presented as mean S.E.M. from four to six oocytes for each mutant.
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et al., 1995). NMDA receptor antagonists is expected for a wide range of therapeutic applications ranging from acute neurodegeneration (e.g. stroke and trauma), chronic neurodegeneration (e.g. Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, ALS) to symptomatic treatment (e.g. epilepsy, drug dependence, depression, anxiety, chronic pain) (Parsons et al., 1999). In fact, memantine has been used in patients of Alzheimer’s disease. Functional modulation of NMDA receptors can be achieved through actions at different recognition sites such as: the agonist binding site, in which 1aminocyclophane carboxylic acid binds (Nahum-Levy et al., 1999), the channel-forming site, in which memantine binds, and the regulatory domain (N-terminal domain), in which polyamines and Zn2+ bind (Masuko et al., 1999b; Paoletti et al., 2000). In this paper, we demonstrate that cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) directly inhibit macroscopic currents at heteromeric NMDA receptors. Furthermore, we find that these compounds act at the vestibule of channel pore and extracellular region, but not the narrowest region, composed of NR1 N616, NR2B N615 and NR2B N616 residues, of NMDA receptor channels. NMDA receptor antagonist, MK-801 acts in the narrowest region of the channel pore, and get trapped when the ion channels close. Therefore, dissociation of MK-801 from NMDA receptor is very slowly and NMDA receptor activities do not recover completely. All the compounds synthesized initially in this category showed toxicity mainly because of their high affinity for NMDA receptors. The cyclophane acts at extracellular month of the channel and therefore easily dissociate by washing (Fig. 2). We expect that easily dissociable antagonists, like memantine, will become available for the therapeutic use. The inhibitory potency of CPCn on NR1/NR2A receptor was greater than that of the Alzheimer’s therapeutic drug, memantine (1.0 mM) under our experimental conditions. In conclusion, the newly synthesized cyclophanes are promising compounds as a novel class of NMDA channel blocker which binds to the sites within the vestibule and the extracellular regions of the channel. The binding site was different from those for other NMDA receptor channel blockers including memantine, MK-801, polyamine derivatives and Mg2+ (Kashiwagi et al., 2004). Experiments are in progress to synthesize various smaller cyclophanes having inhibitory effects on NMDA receptors, because CPCn, CPPy, TGDMAP and ATGDMAP still have higher molecular weight to pass the blood brain barrier. Acknowledgments We thank Dr. A.J. Michael for help in preparing the manuscript. Thanks are also due to Drs. S. Nakanishi, P.H. Seeburg and M. Mishina for the kind supply of glutamate receptor clones. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (T.M.) and was partly supported by a grant from ‘‘Academic Frontier’’ Project for Private Universities: matching fund subsidy from MEXT 2002–2006.
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Nakamichi, N., Yoneda, Y., 2006. Maturation-dependent reduced responsiveness of intracellular free Ca2+ ions to repeated stimulation by Nmethyl-D-aspartate in cultured rat cortical neurons. Neurochem. Int. 49, 230–237. Paoletti, P., Perin-Dureau, F., Fayyazuddin, A., Le Goff, A., Callebaut, I., Neyton, J., 2000. Molecular organization of a zinc binding N-terminal modulatory domain in a NMDA receptor subunit. Neuron 28, 911–925. Parsons, C.G., Danysz, W., Quack, G., 1999. Memantine is a clinically well tolerated N-methyl-D-aspartate (NMDA) receptor antagonist—a review of preclinical data. Neuropharmacology 38, 735–767. Sakimura, K., Bujo, H., Kushiya, E., Araki, K., Yamazaki, M., Yamazaki, M., Meguro, H., Warashina, A., Numa, S., Mishina, M., 1990. Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate. FEBS Lett. 272, 73–80. Sanganahalli, G.B., Joshi, G.P., Joshi, B.N., 2006. NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices. Neurochem. Int. 49, 475–480. Steffens, M., Huppertz, J.-H., Zentner, J., Chauzit, E., Feuerstein, J.T., 2005. Unchanged glutamine synthetase activity and increased NMDA receptor density in epileptic human neocortex: implications for the pathophysiology of epilepsy. Neurochem. Int. 47, 379–384. Williams, K., Pahk, A.J., Kashiwagi, K., Masuko, T., Nguyen, N.D., Igarashi, K., 1998. The selectivity filter of the N-methyl-D-aspartate receptor: a tryptophan residue controls block and permeation of Mg2+. Mol. Pharmacol. 53, 933–941.
Cyclophane and acyclic cyclophane: Novel channel blockers of N-methyl-D-aspartate receptor Takashi Masuko a, Hiroki Nagaoka a, Muneharu Miyake a, Koichi Metori a, Yasuo Kizawa a, Keiko Kashiwagi b, Kazuei Igarashi c, Tadashi Kusama a,* a
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan b Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Choshi, Japan c Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Received 23 May 2006; received in revised form 29 September 2006; accepted 9 October 2006 Available online 17 November 2006
Abstract The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca2+-permeable AMPA receptors (GluR1), Ca2+-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1a). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (70 mV), the IC50 values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5 0.1, 1.0 0.2, 8.0 0.8 and 4.9 0.5 mM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, 100 mV > 70 mV > 20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg2+. However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid. # 2006 Elsevier Ltd. All rights reserved. Keywords: Cyclophane; Acyclic cyclophane; Ligand-gated ion channels; N-Methyl-D-aspartate receptor; Voltage-clamp recording; Xenopus oocytes; Openchannel blockers
1. Introduction N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have structural similarities with the a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors (Dingledine et al., 1999). NMDA receptors have several characteristics that distinguish them from other glutamate receptors. These include the requirement for two different agonists (glutamate and glycine) to activate the receptor, a high permeability of the channel to Ca2+, and a voltage-dependent block of the channel by Mg2+ (Dingledine et al., 1999). NR1 is a single gene product expressed as eight
* Corresponding author. Tel.: +81 47 465 4789; fax: +81 47 465 4789. E-mail address: [email protected] (T. Kusama). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.10.003
alternatively spliced mRNAs, and NR2A, NR2B, NR2C and NR2D are distinct gene products. NMDA receptors probably consist of tetrameric and heteromeric subunit assemblies that have different physiological and pharmacological properties and are differentially distributed throughout the CNS (Danysz and Parsons, 1998). In the CNS, the NMDA receptor plays critical important role in a variety of neurophysiological phenomena, including neurodevelopment, synaptic plasticity, and excitotoxicity. It is well known that glutamate and aspartate can be neurotoxins, especially when energy supply is compromised (Greene and Greenamyre, 1996). This information has given rise to the proposal that neurodegeneration associated with a variety of acute and chronic disorders (ischemic stroke, epilepsy, Parkinson’s disease, AIDS dementia, and so on) may be caused in part by overactivation of glutamate receptors. Density
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of synaptosomal NMDA receptor exhibited by 37% increase in epileptic human neocortex, compared to non-epileptic tissue (Steffens et al., 2005). These data indicate that NMDA receptor has close relations with epileptic seizures and that NMDA receptor will be a target molecule for epileptic disease. Overactivation of NMDA receptors increase intracellular free Ca2+ levels, mediate oxidative stress and induce neuronal cell death (Nakamichi and Yoneda, 2006; Sanganahalli et al., 2006). NMDA receptor channels are blocked by a large number of structurally dissimilar blockers including Mg2+, ketamine, MK801 (dizocilpine), memantine, and various spider toxins and polyamine derivatives (Huettner and Bean, 1988; Collingridge and Lester, 1989; Chen and Huang, 1992; Igarashi et al., 1997; Dingledine et al., 1999). MK-801 and memantine inhibit NMDA receptors expressed in Xenopus oocytes and the inhibition was reduced at recombinant NMDA receptors containing NR1 N616Q in the M2 loop. The residue (N616) forms the narrowest region of channel pore. A large number of open channel blockers of NMDA receptors were shown to act in the narrowest region of channel pore (Kashiwagi et al., 2002). Recently, we reported that acyclic cyclophane ACCn (4,40 bis[2-(1,4,7,10-tetraaza-cyclododecane-1-yl)acetylaminoethoxy]diphenylmethane) inhibited the activity of NR1/ NR2A and NR1/NR2B receptors at 70 mV (Masuko et al., 2005). In this paper, we investigated the effects of cyclophanes, CPCn, CPPy and TGDMAP, and acyclic cyclophane ATGDMAP on NMDA receptor activities expressed in Xenopus oocytes. Our purpose is to develop a specific blocker for NMDA receptors, because excessive stimulus by glutamate leads to neuronal cell death in various neuronal diseases. 2. Experimental procedure 2.1. Materials Cyclophanes, CPCn (N,N0 -bis(1,4,7,10-tetraazacyclododecane-1-ylacetyl)10,26-diaza-7,13,23,29-tetraoxal[7.1.7.1]paracyclophane) and TGDMAP (1,10 {[(2R,17S,19S,34R)-8,9,11,12,25,26,28,29-octahydro-3H,10H,16H,20H,27H,3 3H-4,7:21,24-dimetheno-13,19,15:30,2,32-di[1]propen[1]yl[3]ylidynene-1H,18 H-dipyrimido[1,6-1:10 ,60 -o][1,8,15,22]tetraazacyclooctacosine-10,27-diyl]di-3, 1-propanediyl}bis4-dimethylaminopyridinium dichloride) were synthesized as described previously (Metori and Miyake, 2003; Metori et al., 2005) and the methods for synthesizing acyclic cyclophane ATGDMAP (N,N0 -(6H,12H5,11-methanodibenzo[b,f ][1,5]diazocine-2,8-diyldi-2,1-ethanediyl)-bis[N-(3(4-dimethylaminopyridinium)propyl)phenethylamine]dichloride) and another cyclophane CPPy (N,N0 -bis(3-(4-dimethylaminopyridinium)propyl)-10,26diaza-7,13,23,29-tetraoxa[7.1.7.1]paracyclophane dichloride) will be described elsewhere. MDMAP was synthesized according to the method of the previous report (Jerchel et al., 1956). The structures of these compounds shown in Fig. 1 were confirmed by mass spectrometry and 1H-NMR spectrometry. 1,2-Bis(2aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA) and collagenase were purchased from Sigma (St. Louis, MO). All other reagents used were of the highest grade commercially available.
2.2. NMDA, AMPA, metabotropic glutamate receptor clones and numbering of residues The NR1 clone used in these studies is the NR1A variant (Moriyoshi et al., 1991) which lacks the 21-amino acid insert encoded by exon-5. This clone was a gift from Dr. S. Nakanishi (Osaka Bioscience Institute, Osaka, Japan). The rat and mouse NR2A and NR2B clones (Kutsuwada et al., 1992; Monyer et al.,
1992) were gifts from Dr. M. Mishina (Graduate School of Medicine, University of Tokyo, Japan) and Dr. P. H. Seeburg (Center for Molecular Biology, University of Heidelberg, Germany). The mouse NR2C and NR2D clones (Ikeda et al., 1992; Kutsuwada et al., 1992), the mouse GluR1 and GluR2 clones (Sakimura et al., 1990), and the mGluR1a clone (Masu et al., 1991) were also gifts from Dr. M. Mishina. The preparation of NR1 and NR2B mutants has been described previously (Williams et al., 1998; Kashiwagi et al., 2002, 2004). Amino acids are numbered from initiator methionine in each subunit. This differs from the numbering system used in some other laboratories, in which residues are numbered from the start of the mature peptide (Kuner et al., 1996; Beck et al., 1999).
2.3. Expression in oocytes and voltage-clamp recording Adult female Xenopus laevis (Saitama Experimental Animals Supply Co. Ltd., Saitama, Japan) were chilled on ice, and the preparation and maintenance of oocytes were carried out as described previously (Masuko et al., 1999a,b). Capped cRNAs were prepared from linearized cDNA templates using mMessage mMachine kits (Ambion, Austin, TX). Oocytes were injected with NR1A and NR2 cRNAs at a ratio of 1:5 (0.2–4 ng of NR1A plus 1–20 ng of NR2). Where indicated, oocytes were injected with cRNAs of GluR1 (50 ng), GluR1/ GluR2 (10 ng of GluR1 plus 20 ng of GluR2) or mGluR1a (20 ng) instead of NR1A and NR2 cRNAs. Macroscopic currents were recorded with a twoelectrode voltage-clamp method using Dual Electrode Voltage Clamp Amplifier CEZ-1250 (Nihon Koden, Tokyo, Japan). Electrodes were filled with 3 M KCl. Oocytes were continuously superfused (ca. 5 ml/min) with a Mg2+-free saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM BaCl2, 10 mM HEPES, pH 7.5). This solution contained BaCl2 rather than CaCl2, and, in most experiments, oocytes were injected with K+-BAPTA (100 nl of 40 mM, pH 7.5) on the day of recording to eliminate Ca2+-activated Cl currents (Leonard and Kelso, 1990; Masuko et al., 2004).
3. Results 3.1. Effects of cyclophanes, CPCn, CPPy and TGDMAP, and acyclic cyclophane ATGDMAP on glutamate receptors expressed in Xenopus oocytes The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on the activity of glutamate receptors were examined. Although 10 mM CPCn, CPPy, TGDMAP and ATGDMAP did not inhibit the activities of AMPA receptors and a metabotropic glutamate receptor, all these compounds inhibited NR1/NR2A receptor activities caused by 10 mM glutamate with 10 mM glycine (Fig. 2). These results indicate that CPCn, CPPy, TGDMAP and ATGDMAP are specific blockers for NMDA receptors. To determine the subunit-specificity of inhibition by these four compounds, the inhibitory effects at NR1/NR2 receptors containing different NR2 subunits, NR2A, NR2B, NR2C and NR2D, were investigated. The inhibition of NR1/NR2A receptors by cyclophanes and acyclic cyclophane was more pronounced than those of other NMDA receptor subtypes (Fig. 3). The inhibitions by CPCn and CPPy, both have diphenylmethane skeleton, were stronger than those by TGDMAP and ATGDMAP, both have a tro¨ger base skeleton. We studied dose-dependency of the inhibition by cyclophanes, acyclic cyclophane, cyclen and MDMAP for NR1/NR2A receptors at 70 mV. The IC50 values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5 0.1, 1.0 0.2, 8.0 0.8 and 4.9 0.5 mM, respectively (Fig. 4). The inhibitory effects of cyclen and MDMAP (up to 100 mM)
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Fig. 1. Structures of cyclophanes, acyclic cyclophane and their functional groups on side-chains.
on NR1/NR2A receptor were negligible. The inhibition by CPPy was eight times stronger than that of TGDMAP, suggesting that diphenylmethane skeleton can interact with the NMDA receptor more easily than the tro¨ger base skeleton. 3.2. Noncompetitive block by cyclophanes and acyclic cyclophane for the glutamate and glycine sites on NMDA receptors To determine whether the effects of cyclophanes and acyclic cyclophane on NR1/NR2A receptors are dependent on the concentration of glutamate or glycine, the inhibitory effects of 1 mM CPCn, 1 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP on NR1/NR2A receptors were measured using 10 mM glycine with 3, 30 and 300 mM glutamate (Fig. 5A) or 10 mM glutamate with 3, 30 and 300 mM glycine (Fig. 5B). The inhibition by cyclophanes and acyclic cyclophane was
independent on the concentration (3–300 mM) of glycine or glutamate, indicating noncompetitive block for the glutamate or glycine site on NMDA receptors. 3.3. Voltage-dependent inhibition by cyclophanes and acyclic cyclophane To clarify the mechanism of inhibition by cyclophanes and acyclic cyclophane, we tried to determine whether CPCn, CPPy, TGDMAP and ATGDMAP produced voltage-dependent inhibition of NMDA receptors or not. We studied the effects of CPCn, CPPy, TGDMAP and ATGDMAP on NR1/NR2A and NR1/ NR2B expressed in Xenopus oocytes voltage-clamped at 20, 70 and 100 mV (Fig. 6). The inhibitions by CPCn, CPPy, TGDMAP and ATGDMAP became prominent at 100 mV compared to those at 20 mV. The results suggest that inhibition by cyclophanes and acyclic cyclophane is voltage-dependent.
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Fig. 4. Inhibitory curves of cyclophanes, acyclic cyclophane and their sidechains at NR1/NR2A receptor currents at 70 mV. Concentration–inhibition curves for CPCn, CPPy, TGDMAP, ATGDMAP, cyclen and MDMAP were determined at NR1/NR2A receptors, voltage-clamped at 70 mV. Responses to 10 mM glutamate with 10 mM glycine measured in the presence of these compounds were expressed as a percentage of the control response at each receptor type. The values are presented as mean S.E.M. from four to six oocytes.
Fig. 2. Effects of cyclophanes and acyclic cyclophane on glutamate receptors at 70 mV. The effects of 10 mM CPCn, CPPy, TGDMAP and ATGDMAP were determined in oocytes expressing NMDA (NR1/NR2A), Ca2+-permeable AMPA (GluR1), Ca2+-nonpermeable AMPA (GluR1/GluR2) and metabotropic glutamate (mGluR1a) receptors, voltage-clamped at 70 mV. NR1/NR2A and mGluR1a receptor currents were evoked by 10 mM glutamate with 10 mM glycine and 100 mM glutamate, respectively. GluR1 and GluR1/GluR2 receptor currents were evoked by 100 mM kainate. (A) Representative traces showing the effects of 10 mM CPCn on NR1/NR2A, GluR1, GluR1/GluR2 and mGluR1a receptors, respectively. (B) Macroscopic currents in the presence of cyclophanes and acyclic cyclophane were expressed as a percentage of the control response at each glutamate receptor. The values are presented as mean S.E.M. from four to six oocytes.
pore, because inhibition by CPCn was voltage-dependent. The narrowest region composed of M2 loops of NR1 and NR2 subunits is a critical determinant of divalent cation permeability and channel block by Mg2+ or other organic channel blockers. The potency of many open channel blockers, including MK801, polyamine derivatives such as TB-34, and memantine, was greatly reduced in the mutants NR1 (N616Q), NR2B (N615Q) and NR2B (N616Q) (Kashiwagi et al., 1997, 2002). However, inhibition by CPCn was not affected in NR1/NR2B receptor mutants containing NR1 (N616Q), NR2B (N615Q) and NR2B (N616Q) (Fig. 7). Furthermore, the inhibitory potency of CPCn did not change in NMDA receptor mutants containing NR1
3.4. Identification of amino acid residues involved in the block by CPCn To identify amino acid residues that are involved in the channel block of NMDA receptors by CPCn, we examined the effects of mutations on the residues composing the channel
Fig. 3. Effects of cyclophanes, acyclic cyclophane and their side-chains on NMDA receptors at 70 mV. The effects of 10 mM CPCn, CPPy, TGDMAP, ATGDMAP, cyclen and MDMAP were determined in oocytes expressing NR1/ NR2 receptors containing NR2A, NR2B, NR2C and NR2D, voltage-clamped at 70 mV. Responses to 10 mM glutamate with 10 mM glycine measured in the presence of these compounds were expressed as a percentage of the control response at each receptor type. The values are presented as mean S.E.M. from four to six oocytes for each subunit combination.
Fig. 5. Effects of cyclophanes and acyclic cyclophane in the presence of various concentrations of agonists on NR1/NR2A receptor currents at 70 mV. The inhibitions by 1 mM CPCn, 1 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP were determined in oocytes expressing NR1/NR2A receptors, voltage-clamped at 70 mV: (A) 3, 30, or 300 mM glutamate with 10 mM glycine; (B) 3, 30, or 300 mM glycine with 10 mM glutamate. The values are presented as mean S.E.M. from four to six oocytes for each subunit combination.
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Fig. 6. Voltage-dependent inhibition by cyclophanes and acyclic cyclophane of NMDA receptor currents. The effects of 1 mM CPCn, 1 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP on NR1/NR2A receptors, and 3 mM CPCn, 3 mM CPPy, 10 mM TGDMAP and 10 mM ATGDMAP on NR1/NR2B receptors, were measured at 20, 70 and 100 mV. The values are presented as mean S.E.M. from four to six oocytes for each subunit combination.
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(E598Q), NR1 (T602G) and NR1 (E621Q), located in the intracellular regions of NMDA receptor. On the other hand, the degree of inhibition of CPCn was reduced in mutants containing NR1 (N650A), located on the top of M1, and NR1 (D669N), located in the extracellular regions, although the inhibitory effects were not changed at the mutant receptors containing NR1 (N650Q) and NR1 (D669E) receptors. The acidic group at NR1 D669 and amide group at NR1 N650 may be involved in the interaction with CPCn. The results suggest that CPCn directly interacts with surface amino acid residues around the vestibule of the ion channel and extracellular region, like a lid, and does not reach the narrowest region of channel of NMDA receptors. 4. Discussion NMDA receptors play some crucial physiological roles in various forms of synaptic plasticity such as those involved in learning and memory (Collingridge and Singer, 1990; Danysz
Fig. 7. Effects of CPCn on wild type NR1/NR2B and mutant receptors in the channel pore region. (A) Schematic illustration of the NR1 and NR2 subunits, which contain three transmembrane segments (M1, M3, and M4), a re-entrant loop (M2), an extracellular N-terminal domain, extracellular loop between M3 and M4, an intracellular loop between M1 and M2, and intracellular C-terminal domain. The inhibitory effects of CPCn were examined on the mutants at the indicated amino acid residues in the figure. (B) Representative traces showing block by CPCn at wild type NR1/NR2B receptors, mutant NR1 (N650A)/NR2B and NR1 (D669N)/NR2B receptors, respectively. (C) The effects of 10 mM CPCn were determined in oocytes expressing wild type and mutant NR1/NR2B receptors, voltage-clamped at 70 mV. The values are presented as mean S.E.M. from four to six oocytes for each mutant.
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et al., 1995). NMDA receptor antagonists is expected for a wide range of therapeutic applications ranging from acute neurodegeneration (e.g. stroke and trauma), chronic neurodegeneration (e.g. Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, ALS) to symptomatic treatment (e.g. epilepsy, drug dependence, depression, anxiety, chronic pain) (Parsons et al., 1999). In fact, memantine has been used in patients of Alzheimer’s disease. Functional modulation of NMDA receptors can be achieved through actions at different recognition sites such as: the agonist binding site, in which 1aminocyclophane carboxylic acid binds (Nahum-Levy et al., 1999), the channel-forming site, in which memantine binds, and the regulatory domain (N-terminal domain), in which polyamines and Zn2+ bind (Masuko et al., 1999b; Paoletti et al., 2000). In this paper, we demonstrate that cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) directly inhibit macroscopic currents at heteromeric NMDA receptors. Furthermore, we find that these compounds act at the vestibule of channel pore and extracellular region, but not the narrowest region, composed of NR1 N616, NR2B N615 and NR2B N616 residues, of NMDA receptor channels. NMDA receptor antagonist, MK-801 acts in the narrowest region of the channel pore, and get trapped when the ion channels close. Therefore, dissociation of MK-801 from NMDA receptor is very slowly and NMDA receptor activities do not recover completely. All the compounds synthesized initially in this category showed toxicity mainly because of their high affinity for NMDA receptors. The cyclophane acts at extracellular month of the channel and therefore easily dissociate by washing (Fig. 2). We expect that easily dissociable antagonists, like memantine, will become available for the therapeutic use. The inhibitory potency of CPCn on NR1/NR2A receptor was greater than that of the Alzheimer’s therapeutic drug, memantine (1.0 mM) under our experimental conditions. In conclusion, the newly synthesized cyclophanes are promising compounds as a novel class of NMDA channel blocker which binds to the sites within the vestibule and the extracellular regions of the channel. The binding site was different from those for other NMDA receptor channel blockers including memantine, MK-801, polyamine derivatives and Mg2+ (Kashiwagi et al., 2004). Experiments are in progress to synthesize various smaller cyclophanes having inhibitory effects on NMDA receptors, because CPCn, CPPy, TGDMAP and ATGDMAP still have higher molecular weight to pass the blood brain barrier. Acknowledgments We thank Dr. A.J. Michael for help in preparing the manuscript. Thanks are also due to Drs. S. Nakanishi, P.H. Seeburg and M. Mishina for the kind supply of glutamate receptor clones. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (T.M.) and was partly supported by a grant from ‘‘Academic Frontier’’ Project for Private Universities: matching fund subsidy from MEXT 2002–2006.
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