IHARA Makoto

Department of Applied Biological ChemistryAssociate Professor

Last Updated :2024/07/20

■Researcher comments

List of press-related appearances


■Researcher basic information


  • PhD(Kinki University)

J-Global ID

Research Keyword

  • Bioorganic chemistry   Pesticide science   構造生理学   Structural Biology   

Research Field

  • Life sciences / Bioorganic chemistry
  • Life sciences / Functional biochemistry
  • Life sciences / Structural biochemistry
  • Nanotechnology/Materials / Molecular biochemistry



  • 2018/04 - Today  Kindai UniversityFuculty of agricultureAssociate professor
  • 2013/04 - 2018/03  Kinki UniversityFaculty of AgricultureLecturer
  • 2012/12 - 2018/03  RIKENSPring-8 CenterVisiting Scientist
  • 2012/12 - 2013/03  Okayama University, Graduate school of medicine, dentistry and pharmaceutical sciencesAssistant Professor
  • 2006/10 - 2012/11  RIKEN
  • 2005/04 - 2006/09  近畿大学 研究員

■Research activity information


  • 2018/03 日本農芸化学会 農芸化学奨励賞
  • 2008 日本農学進歩賞
     環境保全型農業での選択的害虫制御に関する基礎研究 JPN
  • 2007 望月喜多司記念賞 奨励賞
     リガンド作動性イオンチャネルに対する殺虫剤の作用機構の選択性と多様性に関する電気生理学的研究 JPN
  • 2007 日本農薬学会 奨励賞
     殺虫剤のリガンド作動性イオンチャネルに対する作用機構研究 JPN


  • Keisuke Nishikawa; Yosuke Ono; Sumito Mori; Koichi Takayama; Makoto Ihara; Kazuhiko Matsuda; Yoshiki Morimoto
    The Journal of organic chemistry 89 (6) 4128 - 4133 2024/03 [Refereed]
    Histrionicotoxin (HTX) alkaloids, which are isolated from Colombian poison dart frogs, are analgesic neurotoxins that modulate nicotinic acetylcholine receptors (nAChRs) as antagonists. Perhydrohistrionicotoxin (pHTX) is the potent synthetic analogue of HTX and possesses a 1-azaspiro[5.5]undecane skeleton common to the HTX family. Here, we show for the first time the divergent nine-step synthesis of pHTX and its three stereoisomers from the known aldehyde through a one-step construction of the 1-azaspiro[5.5]undecane framework from a linear amino ynone substrate. Surprisingly, some pHTX diastereomers exhibited antagonistic activities on the chicken α4β2-neuronal nAChRs that were more potent than pHTX.
  • Noritada Matsuo; Yukimi Sugisaka; Shiori Aoyama; Makoto Ihara; Harue Shinoyama; Munetaka Hosokawa; Yoshinobu Kamakura; Daisuke Tanaka; Yoo Tanabe; Kazuhiko Matsuda
    Journal of medicinal chemistry American Chemical Society (ACS) 66 (12) 7959 - 7968 0022-2623 2023/06 [Refereed]
    Pyrethrins from Tanacetum cinerariifolium are natural pesticides that exhibit high knockdown and killing activities against flying insects such as disease-spreading mosquitoes. Despite the increasing demand for pyrethrins, the mechanism of pyrethrin biosynthesis remains elusive. To elucidate it, we for the first time created pyrethrin mimetic phosphonates targeting the GDSL esterase/lipase (GELP or TcGLIP) underpinning pyrethrin biosynthesis. The compounds were synthesized by reacting mono-alkyl or mono-benzyl-substituted phosphonic dichloride with pyrethrolone, the alcohol moiety of pyrethrin I and II, and then p-nitrophenol. n-Pentyl (C5) and n-octyl (C8)-substituted compounds were the most potent of the (S)p,(S)c, and (R)p,(S)c diastereomers, respectively. The (S)-pyrethrolonyl group is more effective than the (R)-pyrethrolonyl group in blocking TcGLIP, consistent with the features predicted by TcGLIP models complexed with the (S)p,(S)c-C5 and (R)p,(S)c-C8 probes. The (S)p,(S)c-C5 compound suppressed pyrethrin production in T. cinerariifolium, demonstrating potential as a chemical tool for unravelling pyrethrin biosynthesis.
  • Wataru Koizumi; Shuya Otsubo; Shogo Furutani; Kunihiro Niki; Koichi Takayama; Shota Fujimura; Takanobu Maekawa; Ryosuke Koyari; Makoto Ihara; Kenji Kai; Hideo Hayashi; Mohammad Shaokat Ali; Eriko Kage-Nakadai; David B Sattelle; Kazuhiko Matsuda
    Molecular pharmacology 103 (6) 299 - 310 2023/06 [Refereed]
    The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs) but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive (L-type (UNC-38/UNC-29/UNC-63/LEV8/LEV-1)) nAChR than the nicotine-sensitive (N-type (ACR-16)) nAChR, a result consistent with in vivo studies on wild type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. Significance Statement Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.
  • Yuma Komori; Koichi Takayama; Naoki Okamoto; Masaki Kamiya; Wataru Koizumi; Makoto Ihara; Daitaro Misawa; Kotaro Kamiya; Yuto Yoshinari; Kazuki Seike; Shu Kondo; Hiromu Tanimoto; Ryusuke Niwa; David B Sattelle; Kazuhiko Matsuda
    PLoS genetics 19 (2) e1010522  2023/02 [Refereed]
    Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dβ1, and Dβ2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dβ3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dβ3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.
  • Koichi Takayama; Ryo Ito; Haruki Yamamoto; Shuya Otsubo; Rei Matsumoto; Hisanori Ojima; Yuma Komori; Kazuhiko Matsuda; Makoto Ihara
    Pesticide biochemistry and physiology Elsevier BV 187 105177 - 105177 0048-3575 2022/10 [Refereed]
    Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dβ1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dβ1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dβ1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dβ1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dβ1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dβ1 orthosteric site over the Dα1-Dα1 orthosteric site.
  • Yukimi Sugisaka; Shiori Aoyama; Konoka Kumagai; Makoto Ihara; Kazuhiko Matsuda
    Journal of agricultural and food chemistry 70 (28) 8645 - 8652 2022/07 [Refereed]
    Natural pesticides pyrethrins biosynthesized by Tanacetum cinrerariifolium are biodegradable and safer insecticides for pest insect control. TcGLIP, a GDSL lipase underpinning the ester bond formation in pyrethrins, exhibits high stereo-specificity for acyl-CoA and alcohol substrates. However, it is unknown how the enzyme recognizes the other structural features of the substrates and whether such specificity affects the product amount and composition in T. cinrerariifolium. We report here that the cysteamine moiety in (1R,3R)-chrysanthemoyl CoA and the conjugated diene moiety in (S)-pyrethrolone play key roles in the interactions with TcGLIP. CoA released from chrysanthemoyl CoA in the pyrethrin-forming reaction reduces the substrate affinity for TcGLIP by feedback inhibition. (S)-Pyrethrolone shows the highest catalytic efficiency for TcGLIP, followed by (S)-cinerolone and (S)-jasmololone, contributing, at least in part, to determine the pyrethrin compositions in T. cinerariifolium.
  • Makoto Ihara; Keiji Tanaka; Kenji Kai; Hideo Hayashi; Kazuhiko Matsuda
    Pesticide biochemistry and physiology Elsevier BV 183 105074 - 105074 0048-3575 2022/05 [Refereed]
    Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.
  • Qiang Wang; Peng Xu; Felipe Andreazza; Yahui Liu; Yoshiko Nomura; Phil Duran; Lan Jiang; Mengli Chen; Genki Takamatsu; Makoto Ihara; Kazuhiko Matsuda; Rufus Isaacs; Eugenio E Oliveira; Yuzhe Du; Ke Dong
    PLoS genetics 17 (7) e1009677  2021/07 [Refereed]
    Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.
  • Feng Liu; Qiang Wang; Peng Xu; Felipe Andreazza; Wilson R Valbon; Elizabeth Bandason; Mengli Chen; Ru Yan; Bo Feng; Leticia B Smith; Jeffrey G Scott; Genki Takamatsu; Makoto Ihara; Kazuhiko Matsuda; James Klimavicz; Joel Coats; Eugenio E Oliveira; Yuzhe Du; Ke Dong
    Nature communications 12 (1) 2553 - 2553 2021/05 
    Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-β-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.
  • Makoto Ihara; Shogo Furutani; Sho Shigetou; Shota Shimada; Kunihiro Niki; Yuma Komori; Masaki Kamiya; Wataru Koizumi; Leo Magara; Mai Hikida; Akira Noguchi; Daiki Okuhara; Yuto Yoshinari; Shu Kondo; Hiromu Tanimoto; Ryusuke Niwa; David B Sattelle; Kazuhiko Matsuda
    Proceedings of the National Academy of Sciences of the United States of America 117 (28) 16283 - 16291 2020/07 [Refereed]
    The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.
  • Sho Shigetou; Shota Shimada; Ihara Makoto; Kazuhiko Matsuda
    Pesticide biochemistry and physiology 166 104545 - 104545 2020/06 [Refereed]
    Neonicotinoids targeting insect nicotinic acetylcholine (ACh) receptors (insect nAChRs) are used for crop protection, but there is a concern about adverse effects on pollinators such as honeybees (Apis mellifera). Thus, we investigated the agonist actions of neonicotinoids (imidacloprid, thiacloprid and clothianidin) on A. mellifera α1 (Amα1)/chicken β2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(β2)2 and (Amα1)2(β2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(β2)2 and (Amα1)2(β2)3 nAChRs with similar current amplitude. We investigated the agonist activity of imidacloprid, thiacloprid and clothianidin for the two hybrid nAChRs and found that: 1) imidacloprid showed higher affinity than clothianidin, whereas clothianidin showed higher efficacy than imidacloprid for the nAChRs; 2) Thiacloprid showed the highest agonist affinity and the lowest efficacy for the nAChRs. The Amα1/β2 subunit ratio influenced the efficacy of imidacloprid and thiacloprid, but hardly affected that of clothianidin. Hydrogen bond formation by the NH group in clothianidin with the main chain carbonyl of the loop B may account, at least in part, for the unique agonist actions of clothianidin on the hybrid nAChRs tested.
  • Shota Shimada; Masaki Kamiya; Sho Shigetou; Kakeru Tomiyama; Yuma Komori; Leo Magara; Makoto Ihara; Kazuhiko Matsuda
    Scientific reports 10 (1) 7529 - 7529 2020/05 
    Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit.
  • Teruyuki Tahara; Ami Watanabe; Maho Yutani; Yuko Yamano; Mami Sagara; Shizu Nagai; Keita Saito; Mitsuaki Yamashita; Makoto Ihara; Akira Iida
    Bioorganic & medicinal chemistry 28 (6) 115347 - 115347 0968-0896 2020/03 [Refereed]
    © 2020 Elsevier Ltd The extract of Tabebuia avellanedae has been used as a folk medicine, and the various biological activities of T. avellanedae have been extensively studied. However, few studies have reported which natural products play a role in their biological effects. In this study, we evaluated representative naphthoquinones isolated from T. avellanedae and found that furanonaphthoquinones were the key structures required to exhibit STAT3 phosphorylation inhibitory activities. Our SAR analysis indicated that removal of a hydroxyl group enhanced the STAT3 phosphorylation inhibitory activity. In addition, the combined results of a mobility shift assay, SH2 domain binding assay, and docking simulation by Autodock 4.2.6 suggested that (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (1) could directly bind to the hinge region of STAT3.
  • Nozomu Sakurai; Hossein Mardani-Korrani; Masaru Nakayasu; Kazuhiko Matsuda; Kumiko Ochiai; Masaru Kobayashi; Yusuke Tahara; Takeshi Onodera; Yuichi Aoki; Takashi Motobayashi; Masakazu Komatsuzaki; Makoto Ihara; Daisuke Shibata; Yoshiharu Fujii; Akifumi Sugiyama
    Frontiers in genetics 11 114 - 114 2020/02 
    Inter-organismal communications below ground, such as plant–microbe interactions in the rhizosphere, affect plant growth. Metabolites are shown to play important roles in biological communication, but there still remain a large number of metabolites in soil to be uncovered. Metabolomics, a technique for the comprehensive analysis of metabolites in samples, may uncover the molecules that intermediate these interactions. We conducted a multivariate analysis using liquid chromatography (LC)—mass spectrometry (MS)-based untargeted metabolomics in several soil samples and also targeted metabolome analysis for the identification of the candidate compounds in soil. We identified okaramine A, B, and C in the rhizosphere soil of hairy vetch. Okaramines are indole alkaloids first identified in soybean pulp (okara) inoculated with Penicillium simplicissimum AK-40 and are insecticidal. Okaramine B was detected in the rhizosphere from an open field growing hairy vetch. Okaramine B was also detected in both bulk and rhizosphere soils of soybean grown following hairy vetch, but not detected in soils of soybean without hairy vetch growth. These results suggested that okaramines might be involved in indirect defense of plants against insects. To our knowledge, this is the first report of okaramines in the natural environment. Untargeted and targeted metabolomics would be useful to uncover the chemistry of the rhizosphere.
  • Mengli Chen; Yuzhe Du; Guonian Zhu; Genki Takamatsu; Makoto Ihara; Kazuhiko Matsuda; Boris S Zhorov; Ke Dong
    Pesticide biochemistry and physiology 151 82 - 89 0048-3575 2018/10 [Refereed]
    © 2018 Elsevier Inc. Pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II are six closely related insecticidal active esters, known as pyrethrins, found in the pyrethrum extract from the dry flowers of Tanacetum cinerariifolium. The chemical structures of the six compounds differ only in the terminal moieties at the acid and alcohol ends, but the compounds’ in vivo toxicities are substantially different. Pyrethrins are lead compounds for pyrethroids, a large family of synthetic insecticides that alter nerve functions by prolonging the opening of voltage-gated sodium channels. However, data on the mechanism of action of natural pyrethrins are very limited. In this study, we examined the actions of all six pyrethrins on cockroach sodium channels expressed in Xenopus oocytes. Although the six compounds showed comparable potencies in inhibiting the inactivation of sodium channels, they had greatly variable potencies in inhibiting channel deactivation. Furthermore, unlike pyrethroids, the action of pyrethrins neither depend on nor were enhanced by repeated channel activation. We created a NavMs-based model of the cockroach sodium channel, in which pyrethrin II was docked at the pyrethroid receptor site 1 (PyR1), and proposed a rationale for the observed structure-activity relationship of the six pyrethrins. Our study sheds light on the molecular mechanism of pyrethrum action on sodium channels and reveled differences in the modes of action of the six bioactive constitutes of pyrethrum.
  • Mai Hikida; Shota Shimada; Ryo Kurata; Sho Shigetou; Makoto Ihara; David B Sattelle; Kazuhiko Matsuda
    Pesticide biochemistry and physiology Elsevier {BV} 151 47 - 52 0048-3575 2018/10 [Refereed]
    © 2018 Elsevier Inc. Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken β2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.
  • Aiichiro Muraoka; Yoshinori Matsuura; Hisashi Naitow; Makoto Ihara; Naoki Kunishima
    Analytical biochemistry Elsevier {BV} 557 46 - 58 0003-2697 2018/09 [Refereed]
    © 2018 Elsevier Inc. It is known that the crystallizability of protein molecules may be improved by replacing their surface lysine residues with other residue types. Here an experimental method to identify surface lysine residues by NHS-biotin chemical modification combined with MALDI-TOF MS was proposed and was evaluated using PH1033 protein from Pyrococcus horikoshii. Interestingly, the biotinylation experiment with a protein-reagent molar ratio of 1:1 revealed that only seven of twenty-two lysine residues in the protein comprising 144 residues were labeled. To investigate the result, we analyzed structures from a molecular-dynamics simulation mimicking the experiment. A logistic regression analysis revealed that the biotinylation was significantly correlated with four factors relevant to the local environment of lysine residues: the solvent accessibility, the electrostatic energy, the number of hydrogen bonds, and the estimated pKa value. This result is overall in agreement with that from the same analysis on the crystal structure. However, reflecting the flexibility of the protein molecule in solution state, the factors except for the electrostatic energy were highly variable in the MD structures depending upon the protonation state of Tyr87. The present procedure of biotin-labeling can avoid lysine residues with extensive intramolecular interactions that are incompatible with the rational design of protein crystals.
  • Shogo Furutani; Makoto Ihara; Kristin Lees; Steven D Buckingham; Frederick A Partridge; Jonathan A David; Rohit Patel; Scott Warchal; Ian R Mellor; Kazuhiko Matsuda; David B Sattelle
    International journal for parasitology. Drugs and drug resistance Elsevier {BV} 8 (2) 350 - 360 2018/08 [Refereed]
    © 2018 A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50–400 nA currents in response to 100 μM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ± 0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 μM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 μM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ± 0.04) and the phenylpyrazole fipronil (pIC50 6.90 ± 0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ± 0.43) and may serve as a candidate lead compound for the development of new acaricides.
  • Makoto Ihara; Mai Hikida; Hiroyuki Matsushita; Kyosuke Yamanaka; Yuya Kishimoto; Kazuki Kubo; Shun Watanabe; Mifumi Sakamoto; Koutaro Matsui; Akihiro Yamaguchi; Daiki Okuhara; Shogo Furutani; David B Sattelle; Kazuhiko Matsuda
    British journal of pharmacology Wiley-Blackwell 175 (11) 1999 - 2012 0007-1188 2018/06 [Refereed]
    © 2017 The British Pharmacological Society Background and Purpose: Neonicotinoid insecticides interact with the orthosteric site formed at subunit interfaces of insect nicotinic ACh (nACh) receptors. However, their interactions with the orthosteric sites at α–non α and α–α subunit interfaces remain poorly understood. The aim of this study was to elucidate the mechanism of neonicotinoid actions using the Drosophila Dα1-chicken β2 hybrid nACh receptor. Experimental Approach: Computer models of the (Dα1) 3 (β2) 2 nACh receptor in complex with imidacloprid and thiacloprid were generated. Amino acids in the Dα1 subunit were mutated to corresponding amino acids in the human α4 subunit to examine their effects on the agonist actions of neonicotinoids on (Dα1) 3 (β2) 2 and (Dα1) 2 (β2) 3 nACh receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. Key Results: The (Dα1) 3 (β2) 2 nACh receptor models indicated that amino acids in loops D, E and G probably determine the effects of neonicotinoids. The amino acid mutations tested had minimal effects on the EC 50 for ACh. However, the R57S mutation in loop G, although having minimal effect on imidacloprid's actions, reduced the affinity of thiacloprid for the (Dα1) 3 (β2) 2 nACh receptor, while scarcely affecting thiacloprid's action on the (Dα1) 2 (β2) 3 nACh receptor. Both the K140T and the combined R57S;K140T mutations reduced neonicotinoid efficacy but only for the (Dα1) 3 (β2) 2 nACh receptor. Combining the E78K mutation with the R57S;K140T mutations resulted in a selective reduction of thiacloprid's affinity for the (Dα1) 3 (β2) 2 nACh receptor. Conclusions and Implications: These findings suggest that a triangle of residues from loops D, E and G contribute to the selective actions of neonicotinoids on insect-vertebrate hybrid nACh receptors. Linked Articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
  • Naoki Kato; Shogo Furutani; Junnosuke Otaka; Akira Noguchi; Kiyomi Kinugasa; Kenji Kai; Hideo Hayashi; Makoto Ihara; Shunji Takahashi; Kazuhiko Matsuda; Hiroyuki Osada
    ACS chemical biology 13 (3) 561 - 566 1554-8929 2018/03 [Refereed]
    © 2018 American Chemical Society. Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. To explore the biosynthetic potential of okaramines, gene knockout experiments of an okaramine-producer fungus were performed. The deletion mutants of the oxygenase genes okaB, okaD, okaE, and okaG provided analogues that were unlikely to be accumulated in the normal biosynthetic process of the wild-type strain. Analysis of the structure-activity relationships of okaramines collected from the fungal cultures revealed that 1,4-dihydroazocine and N-aliphatic group attached to the indole were crucial for GluCl-activating activity. This provided insights into further derivatization of the complex structure of okaramines in order to facilitate the development of new insecticides.
  • Daiki Okuhara; Shogo Furutani; Katsuhiko Ito; Makoto Ihara; Kazuhiko Matsuda
    Molecular pharmacology 92 (4) 491 - 499 0026-895X 2017/10 [Refereed]
    Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics. The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl-1 gene from the brain, third thoracic ganglion (T3G), and midgut of larvae of silkworm Bombyx mori. It was found that .50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half-maximal activation at a pH of 7.87, and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants.
  • Shogo Furutani; Daiki Okuhara; Anju Hashimoto; Makoto Ihara; Kenji Kai; Hideo Hayashi; David B Sattelle; Kazuhiko Matsuda
    Bioscience, biotechnology, and biochemistry Informa {UK} Limited 81 (10) 1861 - 1867 0916-8451 2017/10 [Refereed]
    © 2017 Japan Society for Bioscience, Biotechnology, and Agrochemistry. Okaramines produced by Penicillium simplicissimum AK-40 activate L-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to L-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.
  • Yasumichi Onozaki; Ryo Horikoshi; Ikuya Ohno; Shigeki Kitsuda; Kathleen A Durkin; Tomonori Suzuki; Chiaki Asahara; Natsuko Hiroki; Rena Komabashiri; Rikako Shimizu; Shogo Furutani; Makoto Ihara; Kazuhiko Matsuda; Masaaki Mitomi; Shinzo Kagabu; Katsuhito Uomoto; Motohiro Tomizawa
    Journal of agricultural and food chemistry 65 (36) 7865 - 7873 0021-8561 2017/09 [Refereed]
    © 2017 American Chemical Society. A novel chemotype insecticide flupyrimin (FLP) [N-[ (E)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloprid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [3H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [3H]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat α4β2 nAChR. Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.
  • Shogo Furutani; Makoto Ihara; Kenji Kai; Keiji Tanaka; David B. Sattelle; Hideo Hayashi; Kazuhiko Matsuda
    NEUROTOXICOLOGY ELSEVIER SCIENCE BV 60 240 - 244 0161-813X 2017/05 [Refereed]
    The okaramine indole alkaloids were recently shown to be more selective than ivermectin in activating the glutamate-gated chloride channels of the silkworm larvae of Bombyx mori (BmGluCls). Those studies were carried out using the exon 3b variant as a representative of BmGluCls. However, it remains unknown whether okaramines are similarly effective on other silkworm GluCl variants and whether they share the same binding site as ivermectin on GluCls. To begin to address these questions, we examined the potency of four okaramines on the exon 3c variant of BmGluCls by two-electrode voltage clamp voltage recordings of glutamate-induced chloride currents. The potency of okaramines in activating the exon 3c BmGluCI agreed well with findings on the exon 3b BmGluCl and insecticidal potency. Okaramine B (10 mu M) reduced the maximum binding (B-max) but not the dissociation constant (K-D) of [H-3]ivermectin in studies on plasma membrane fractions of HEK293 cells expressing the exon 3c variant. These findings indicate that activation of GluCls is important in the insecticidal actions of okaramines. (C) 2016 Elsevier B.V. All rights reserved.
  • Koji Kobashi; Takaaki Harada; Yoshihiro Adachi; Miho Mori; Makoto Ihara; Daisuke Hayasaka
    Ecotoxicology and environmental safety 138 122 - 129 0147-6513 2017/04 [Refereed]
    © 2016 Elsevier Inc. There are growing concerns about the impacts of neonicotinoid insecticides on ecosystems worldwide, and yet ecotoxicity of many of these chemicals at community or ecosystem levels have not been evaluated under realistic conditions. In this study, effects of two neonicotinoid insecticides, imidacloprid and dinotefuran, on aquatic insect assemblages were evaluated in experimental rice mesocosms. During the 5-month period of the rice-growing season, residual concentrations of imidacloprid were 5–10 times higher than those of dinotefuran in both soil and water. Imidacloprid treatment (10 kg/ha) reduced significantly the populations of, Crocothemis servilia mariannae and Lyriothemis pachygastra nymphs, whereas those of Orthetrum albistylum speciosum increased slightly throughout the experimental period. However, Notonecta triguttata, which numbers were high from the start, later declined, indicating possible delayed chronic toxicity, while Guignotus japonicus disappeared. In contrast, dinotefuran (10 kg/ha) did not decrease the populations of any species, but rather increased the abundance of some insects, particularly Chironominae spp. larvae and C. servilia mariannae nymphs, with the latter being 1.7x higher than those of controls. This was an indirect effect resulting from increased prey (e.g., chironomid larvae) and lack of competition with other dragonfly species. The susceptibilities of dragonfly nymphs to neonicotinoids, particularly imidacloprid, were consistent with those reported elsewhere. In general, imidacloprid had higher impacts on aquatic insects compared to dinotefuran.
  • S. Furutani; M. Ihara; K. Kai; H. Hayashi; K. Matsuda
    ACS Symposium Series 1264 125 - 131 0097-6156 2017 
    Although some fungi produce mycotoxins that render food on which they grow inedible, other fungi produce metabolites that, in addition, to human and animal healthcare, are useful in pest control. Penicillium simplicissimum AK-40 produces okaramines as an insecticide when grown on okara, a by-product of soybean curd production. Okaramines selectively activate glutamate-gated chloride channels expressed only in the nervous system of invertebrates. Asperparalines from Aspergillus japonicus JV-23 and chrodrimanins from Talaromyces sp. YO-2, both of which were isolated similarly to okaramines, selectively block insect nicotinic acetylcholine and γ-aminobutyric acid receptors, respectively. These results suggest that fungi induced by certain plant factors have potential for producing next-generation pesticide leads.
  • Yuri Nakatani; Shogo Furutani; Makoto Ihara; Kazuhiko Matsuda
    Pesticide biochemistry and physiology Elsevier {BV} 126 1 - 5 0048-3575 2016/01 [Refereed]
    © 2015 Elsevier Inc.. The pH-sensitive chloride channels (pHCls) are expressed widely in the insect nervous system, but their physiological roles and pesticide sensitivity in Lepidoptera are poorly understood. Here, cDNAs of pHCl variants "A" and "B" were isolated from the head of silkworm (Bombyx mori) larvae and expressed in Xenopus laevis oocytes to characterize their functions and examine their pesticide sensitivity. Variant "A" possesses four entire transmembrane domains (TMs), while variant "B" lacks a part of the TMand the TM3-TM4 linker. Only the A variant formed a chloride channel in oocytes which was activated in response to an increase of pH in the extracellular solution.Neither fipronil nor ?-benzenehexachloride had a significant blocking effect on the A variantwhen tested at 10 μM. By contrast,macrolide ivermectin activated it at acidic pH but blocked it at pH 7.6 at concentrations higher than 300 nM, indicating a likely contribution to in vivo toxicity.
  • Koji Sakamori; Naoaki Ono; Makoto Ihara; Hideyuki Suzuki; Hideyuki Matsuura; Ken Tanaka; Daisaku Ohta; Shigehiko Kanaya; Kazuhiko Matsuda
    Plant signaling & behavior 11 (4) e1149675  1559-2316 2016 [Refereed]
    © 2016 Taylor & Francis Group, LLC. Natural pyrethrins are used to control household and agricultural pests, and it is of value to understand biosynthesis in Tanacetum cinerariifolium for enhanced production. We previously found that a blend of four green leaf volatiles (GLVs) and (E)-b-farnesene emitted by T. cinerariifolium seedlings enhanced gene expressions of certain biosynthetic enzymes in unwounded seedlings; however, the extent to which such a regulation facilitates pyrethrin biosynthesis remains unknown. Here we have investigated the effects of the blend of the volatile organic compounds (VOCs) on gene expressions of seven biosynthetic enzymes. VOC treatment resulted in enhanced chrysanthemyl diphosphate synthase (CDS), chrysanthemic acid synthase (CAS), Tanacetum cinerariifolium GDSL lipase (TcGLIP) and acyl-Coenzyme A oxidase 1 (ACX1) gene expressions that reached a peak at a 12 h VOC treatment, whereas the treatment minimally influenced the expressions of other biosynthetic genes. In undifferentiated Tanacetum tissues, such VOCinduced amplification of CDS, CAS, TcGLIP and ACX1 gene expressions were markedly reduced, suggesting that a high-resolution, VOC-mediated communication is an event selective to differentiated plants.
  • Makoto Ihara; David B Sattelle; Kazuhiko Matsuda
    Pesticide biochemistry and physiology Elsevier {BV} 121 47 - 52 0048-3575 2015/06 
    © 2015 Elsevier Inc. Neonicotinoid insecticides interact with the orthosteric site on the extracellular ligand binding domain (LBD) of nicotinic acetylcholine receptors (nAChRs), typically activating the cation permeable ion channels. In nAChRs consisting of two α and three non-α subunits, LBDs contain six loops (loops A, B and C on the α subunit and loops D, E and F on the non-α subunit) which make up the orthosteric binding site at the α/non-α subunit interfaces. Recently, an additional site (loop G) on the β1 strand has been identified. Also, when the α/non-α subunit ratio is 3/2, another binding site is generated at the interface of two adjacent α subunits. Roles for loop G and the α-α interface in the interactions with neonicotinoids are discussed with reference to recent structural and physiological data.
  • Yan Xu; Shogo Furutani; Makoto Ihara; Yun Ling; Xinling Yang; Kenji Kai; Hideo Hayashi; Kazuhiko Matsuda
    PloS one Public Library of Science ({PLoS}) 10 (4) e0122629  2015/04 [Refereed]
    © 2015 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.
  • Shogo Furutani; Makoto Ihara; Yuri Nishino; Miki Akamatsu; Andrew K Jones; David B Sattelle; Kazuhiko Matsuda
    Molecular pharmacology AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS 86 (6) 686 - 95 0026-895X 2014/12 [Refereed]
    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for L-glutamate and ivermectin (IVM); however, I-max (the maximum L-glutamate- and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest L-glutamate- and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on L-glutamate and IVM actions. Binding assays using [H-3] IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced B-max but not K-d values of [H-3] IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the L-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2-TM3 linker, influencing the receptor response.
  • Makoto Ihara; Toshihide Okajima; Atsuko Yamashita; Takuma Oda; Takuya Asano; Mikana Matsui; David B Sattelle; Kazuhiko Matsuda
    Molecular pharmacology AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS 86 (6) 736 - 46 0026-895X 2014/12 [Refereed]
    Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the beta 1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the alpha 7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.
  • Shogo Furutani; Yuri Nakatani; Yuka Miura; Makoto Ihara; Kenji Kai; Hideo Hayashi; Kazuhiko Matsuda
    Scientific reports NATURE PUBLISHING GROUP 4 6190 - 6190 2045-2322 2014/08 [Refereed]
    In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) gamma-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 mu M on human alpha 1 beta 2 gamma 2 GABACl and alpha 1 beta glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.
  • Makoto Ihara; Yoshitaka Takano; Atsuko Yamashita
    Protein science : a publication of the Protein Society WILEY-BLACKWELL 23 (7) 923 - 31 0961-8368 2014/07 [Refereed]
    Transient receptor potential (TRP) channels are members of the voltage gated ion channel superfamily and display the unique characteristic of activation by diverse stimuli. We performed an expression analysis of fungal TRP channels, which possess relatively simple structures yet share the common functional characteristics with the other members, using a green fluorescent protein-based screening methodology. The analysis revealed that all the tested fungal TRP channels were severely digested in their cytosolic regions during expression, implying the common flexibility of this region, as observed in the recent structural analyses of the fungal member, TRPGz. These characteristics are likely to be important for their diverse functions.
  • Makoto Ihara; Naoya Shimazu; Mai Utsunomiya; Miki Akamatsu; David B Sattelle; Kazuhiko Matsuda
    Bioscience, biotechnology, and biochemistry TAYLOR & FRANCIS LTD 78 (4) 543 - 9 0916-8451 2014/04 [Refereed]
    Polymorphisms are sometimes observed in native insect nicotinic acetylcholine receptor (nAChR) subunits, which are important insecticide targets, yet little is known of their impact on insecticide actions. Here we investigated the effects of a polymorphism involving the substitution of histidine108 by leucine in the Drosophila melanogaster D alpha 1 subunit on the agonist actions of the neurotransmitter acetylcholine (ACh) and two commercial neonicotinoid insecticides (imidacloprid and clothianidin). There was no significant impact of the H108L substitution on either the ACh EC50, the concentration leading to a half maximal ACh response, or the maximum current amplitude in response at 10 mu M ACh, of the D alpha 1-chicken beta 2 nAChR expressed in Xenopus laevis oocytes. However, the response amplitudes to imidacloprid and clothianidin were significantly enhanced, indicating a role of His108 in the selective interactions of D alpha 1 with these neonicotinoids.
  • Makoto Ihara; Shin Hamamoto; Yohei Miyanoiri; Mitsuhiro Takeda; Masatsune Kainosho; Isamu Yabe; Nobuyuki Uozumi; Atsuko Yamashita
    The Journal of biological chemistry American Society for Biochemistry and Molecular Biology 288 (21) 15303 - 17 0021-9258 2013/05 [Refereed]
    Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Yuji Ashikawa; Makoto Ihara; Noriko Matsuura; Yuko Fukunaga; Yuko Kusakabe; Atsuko Yamashita
    Protein science : a publication of the Protein Society WILEY-BLACKWELL 20 (10) 1720 - 34 0961-8368 2011/10 [Refereed]
    Applications of the GFP-fusion technique have greatly facilitated evaluations of the amounts and qualities of sample proteins used for structural analyses. In this study, we applied the GFP-based sample evaluation to secreted protein expression by insect cells. We verified that a GFP variant, GFPuv, retains proper folding and monodispersity within all expression spaces in Sf9 cells, such as the cytosol, organelles, and even the extracellular space after secretion, and thus can serve as a proper folding reporter for recombinant proteins. We then applied the GFPuv-based system to the extracellular domains of class C G-protein coupled receptors (GPCRs) and examined their localization, folding, and oligomerization upon insect cell expression. The extracellular domain of metabotropic glutamate receptor 1 (mGluR1) exhibited good secreted expression by Sf9 cells, and the secreted proteins formed dimer with a monodisperse hydrodynamic state favorable for crystallization, consistent with the results from previous successful structural analyses. In contrast, the extracellular domains of sweet/umami taste receptors (T1R) almost completely remained in the cell. Notably, the T1R and mGluR1 subfractions that remained in the cellular space showed polydisperse hydrodynamic states with large aggregated fractions, without forming dimers. These results indicated that the proper folding and oligomerization of the extracellular domains of the class C GPCR are achieved through the secretory pathway.
  • Makoto Ihara; Noriko Matsuura; Atsuko Yamashita
    Analytical biochemistry ACADEMIC PRESS INC ELSEVIER SCIENCE 412 (2) 217 - 23 0003-2697 2011/05 [Refereed]
    An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins. (C) 2011 Elsevier Inc. All rights reserved.
  • Makoto Ihara; Toshihide Okajima; Atsuko Yamashita; Takuma Oda; Koichi Hirata; Hisashi Nishiwaki; Takako Morimoto; Miki Akamatsu; Yuji Ashikawa; Shun'ichi Kuroda; Ryosuke Mega; Seiki Kuramitsu; David B Sattelle; Kazuhiko Matsuda
    Invertebrate neuroscience : IN 2 8 (2) 71 - 81 1354-2516 2008/06 [Refereed]
    Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-π interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs. © 2008 The Author(s).
  • Kenzo Fujimoto; Yoshinaga Yoshimura; Makoto Ihara; Kazuhiko Matsuda; Yuko Takeuchi; Takaaki Aoki; Toru Ide
    Bioorganic & medicinal chemistry letters PERGAMON-ELSEVIER SCIENCE LTD 18 (3) 1106 - 9 0960-894X 2008/02 [Refereed]
    We synthesized a novel fluorescent analogue of acetylcholine, Cy3-3-acyleholine. The molecular weight of the products agreed with structural predictions. Discrete intensity changes of fluorescent spots due to a single ligand binding/unbinding to nAChR were visualized by TIRF microscopy. The agonist effect of the Cy3-3-acylcholine on nicotinic acetylcholine receptor (nAChR) was confirmed electrophysiologically. This newly synthesized fluorescent analogue will enable us to conduct more elaborate studies on single channel interaction processes between nAChR and ligands. (C) 2007 Elsevier Ltd. All rights reserved.
  • Kayoko Toshima; Makoto Ihara; Satoshi Kanaoka; Kiyoshi Tarumoto; Atsushi Yamada; David B. Sattelle; Kazuhiko Matsuda
    Effects of imidacloprid, clothianidin, thiacloprid and related compounds on the acetylcholine (ACh)-induced response of the recombinant, expressed chicken alpha 4 beta 2 nicotinic acetylcholine receptor (nAChR) were investigated using voltage-clamp electrophysiology. Imidacloprid and clothianidin enhanced the amplitude of the response to ACh of alpha 4 beta 2 nAChR. In complete contrast, thiacloprid attenuated the amplitude of the response to ACh of alpha 4 beta 2 nAChR. Replacing the nitro group of imidacloprid by a cyano group abolished the potentiating action, whereas exchanging the cyano group of thiacloprid for a nitro group conferred the ability to potentiate the ACh response. All three neonicotinoids shifted the ACh concentration-response curve without influencing the peak current amplitude of the ACh response. (C) Pesticide Science Society of Japan.
  • Makoto Ihara; Koichi Hirata; Chiharu Ishida; Shinzo Kagabu; Kazuhiko Matsuda
    Neuroscience letters ELSEVIER IRELAND LTD 425 (3) 137 - 40 0304-3940 2007/10 [Refereed]
    Neonicotinoid insecticides target nicotinic acetylcholine receptors (nAChRs), which, in both vertebrates and invertebrates, mediate fast-acting synaptic neurotransmission in the nervous system. Recently, Kagabu et al. synthesized bis-neonicotinoids. The neural activities of bis-neonicotinoids have been evaluated on the central nerve cord of American cockroaches. However, the action of bis-neonicotinoids on nAChRs expressed by dissociated insect neurons has not yet been studied. Thus, the actions of several alkylene-tethered bis-neonicotinoids on the terminal abdominal ganglion neurons of the American cockroach, Periplaneta americana, were investigated using whole-cell patch-clamp electrophysiology. All of the ligands tested did not induce membrane currents, but reduced the responses to ACh when bath applied prior to co-application with ACh. Of the compounds tested, HK- 13, which possesses two imidacloprid units linked with a hexamethylene bridge, had the highest antagonist potency. The antagonist action was reduced, not only by elongating, but also by shortening the linker. (C) 2007 Elsevier Ireland Ltd. All rights reserved.
  • Ikuya Ohno; Koichi Hirata; Chiharu Ishida; Makoto Ihara; Kazuhiko Matsuda; Shinzo Kagabu
    Bioorganic & medicinal chemistry letters PERGAMON-ELSEVIER SCIENCE LTD 17 (16) 4500 - 3 0960-894X 2007/08 [Refereed]
    Prod rugs of imidacloprid and the thiazolylmethyl analog masked with oxodioxolylniethyl group on the N3 site were prepared. The prodrugs decomposed in a buffer solution of pH 8.3 and in a physiological salt solution with half-lives of 10-15 h, releasing the parent insecticides. Being consistent with this, an inward current was evoked in dissociated cockroach neurons treated with the masked compound solutions, which were maintained for 24 h after preparation, as measured using patch-clamp electrophysiology, whereas no response was observed in neurons when the solutions were challenged immediately after preparation. The insecticidal test on the American cockroach showed that the minimum lethal dose for each compound at 24 h after injection was 6.4 x 10(-8) mol, which was similar to that for imidacloprid and the thiazolyl derivative. This result strongly suggested a regeneration of the active ingredients in vivo. (c) 2007 Elsevier Ltd. All rights reserved.
  • Makoto Ihara; Masaru Shimomura; Chiharu Ishida; Hisashi Nishiwaki; Miki Akamatsu; David B Sattelle; Kazuhiko Matsuda
    Invertebrate neuroscience : IN 1 7 (1) 47 - 51 1354-2516 2007/03 [Refereed]
    The low mammalian toxicity of neonicotinoid insecticides has been shown to be attributable, at least in part, to their selective actions on insect nicotinic acetylcholine receptors (nAChRs). There are multiple nAChRs in insects and a wealth of neonicotinoid chemicals. Studies to date have discribed a wide range of effects on nAChRs, notably partial agonist, super agonist and antagonist actions. Both the diversity of the neonicotinoid actions and their selectivity for insect over vertebrate nAChRs are the result of physicochemical and steric interactions at their molecular targets (nAChRs). In such interactions, the formation and breakage of hydrogen bond (HB) networks plays a key role. Therefore the loss or gain of even a single HB resulting from either structural changes in neonicotinoids, or the amino acid sequence of a particular nAChR subunit, could result in a drastic modification of neonicotinoid actions. In addition to the amino acid residues, the backbone carbonyl of nAChRs may also be involved in the formation of HB networks with neonicotinoids. © 2007 Springer-Verlag.
  • Makoto Ihara
    JOURNAL OF PESTICIDE SCIENCE PESTICIDE SCI SOC JAPAN 32 (3) 278 - 280 1348-589X 2007 [Refereed]
    Ligand-gated ion channels (LGICs) mediate fast synaptic neurotransmission and are important targets for insecticides. Thus, the actions of several insecticides have been explored in electrophysiological studies on recombinant and native insect neuronal LGICs. I have shown that non-competitive antagonists of gamma-aminobutyric acid gated Cl- channels also act on glutamate-gated Cl- channels, albeit at higher concentrations. Neonicotinoids are more potent agonists on recombinant hybrid nicotinic acetylcholine receptors (nAChRs) consisting of Drosophila D alpha 2 and vertebrate beta 2 subunits than those consisting of only vertebrate nAChR subunits (alpha 4 beta 2). Using this hybrid nAChR, clothianidin and related compounds containing a acyclic guanidine moiety were found to be super-agonists. Similar super-agonist actions of neonicotinoids were also observed on cultured Drosophila cholinergic neurons. Single channel nAChR recordings show that a clothianidin analogue induces a high conductance state in channel opening more frequently than acetylcholine, thereby offering a possible explanation for its super-agonist action. Unlike the case for clothianidin, imidacloprid attenuates the acetylcholine-induced response of native neuronal nAChRs when co-applied with ACh. These new discoveries add to our understanding of both the selectivity and the diverse actions of insecticides targeting LGICs. (c) Pesticide Science Society of Japan.
  • Masaru Shimomura; Maiko Yokota; Makoto Ihara; Miki Akamatsu; David B Sattelle; Kazuhiko Matsuda
    Molecular pharmacology AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS 70 (4) 1255 - 63 0026-895X 2006/10 [Refereed]
    The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha 4 beta 2 nAChR and Drosophila melanogaster D alpha 2/chicken beta 2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta 2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha 4 beta 2 nAChR, T77R; E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K; E79R and T77N; E79R double mutations in the D alpha 2 beta 2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha 4 beta 2 and D alpha 2 beta 2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.
  • Laurence A Brown; Makoto Ihara; Steven D Buckingham; Kazuhiko Matsuda; David B Sattelle
    Journal of neurochemistry BLACKWELL PUBLISHING 99 (2) 608 - 15 0022-3042 2006/10 [Refereed]
    Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC50 value for ACh of 19 mu M. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 mu M ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.
  • Kumiko Minami; Toru Nakasugi; Han-Dong Sun; Ai-Jun Hou; Makoto Ihara; Masanori Morimoto; Koichiro Komai
    JOURNAL OF NATURAL MEDICINES SPRINGER TOKYO 60 (2) 138 - 140 1340-3443 2006 [Refereed]
    Seven histamine-release inhibitors were isolated from Pistacia weinmannifolia J. Pisson ex. Franch. They were identified as gallic acid, 3-O-galloylquinic acid, methyl gallate, ethyl gallate, penta-O-galloyl-beta-D-glucopyrano side, myricetin 3-O-alpha-L-rhamnopyranoside, and myricetin-3-O-(3"-O-gallOyl)-alpha-L-rhamnopyranoside. These compounds suppressed the compound 48/80-induced histamine release from rat peritoneal mast cells.
  • M Ihara; LA Brown; C Ishida; H Okuda; DB Sattelle; K Matsuda
    JOURNAL OF PESTICIDE SCIENCE PESTICIDE SCI SOC JAPAN 31 (1) 35 - 40 1348-589X 2006 [Refereed]
    The actions of neonicotinoid insecticides on nicotinic acetylcholine receptors (nAChRs) in the terminal abdominal ganglion neurons of the American cockroach were investigated using whole-cell patch-clamp electrophysiology. Neonicotinoids possessing a nitromethylene group showed higher agonist affinity than the corresponding nitroimine analogues, whereas compounds with an acyclic guanidine moiety showed greater agonist efficacy than the corresponding cyclic compounds. Imidacloprid showed the lowest agonist efficacy of all neonicotinoids and low concentrations of imidacloprid attenuated acetylcholine-induced currents. However, such blocking actions were minimal with other neonicotinoids. The diverse actions of neonicotinoids on nAChRs, combined with target accessibility based on hydrophobicity, appears to account for their insecticidal potency on cockroaches measured in the presence of metabolic inhibitors. (c) Pesticide Science Society of Japan.
  • Makoto Ihara; Chiharu Ishida; Hiroshi Okuda; Yoshihisa Ozoe; Kazuhiko Matsuda
    Invertebrate neuroscience : IN Springer-Verlag 5 (3-4) 157 - 64 1354-2516 2005/11 [Refereed]
    4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl- channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC50 values under steady state condition. The IC50 values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH. © Springer-Verlag 2005.
  • Masaru Shimomura; Hitoshi Satoh; Maiko Yokota; Makoto Ihara; Kazuhiko Matsuda; David B Sattelle
    Neuroscience letters ELSEVIER IRELAND LTD 385 (2) 168 - 72 0304-3940 2005/09 [Refereed]
    A chimera based on the chicken alpha 4 nicotinic acetylcholine receptor (nAChR) subunit containing an insert from loop B to the N-terminus of the Drosophila melanogaster D alpha 2 (=SAD) subunit was constructed and co-expressed with the chicken beta 2 nAChR subunit in Xenopus laevis oocytes. The actions of the neonicotinoid insecticide imidacloprid were examined. Replacement of the region loop B to the N-terminus of the alpha 4 subunit by the corTesponding region of the D alpha 2 subunit had little effect on the concentration-response curve for imidacloprid. However, replacement of Glu219 by proline in the YXCC motif in loop C of the chimeric alpha 4 subunit resulted in a marked displacement to the left of the concentration-response curve for imidacloprid not seen when an equivalent mutation was made in the alpha 4 beta 2 nAChR. The results suggest that the region loop B to the N-terminus in the D alpha 2 subunit contributes to the high imidacloprid sensitivity of the hybrid D alpha 2 beta 2 nAChR. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
  • Kazuhiko Matsuda; Masaru Shimomura; Makoto Ihara; Miki Akamatsu; David B Sattelle
    Bioscience, biotechnology, and biochemistry TAYLOR & FRANCIS LTD 69 (8) 1442 - 52 0916-8451 2005/08 [Refereed]
    Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.
  • Makoto Ihara; Kazuhiko Matsuda; Masaru Shimomura; David B Sattelle; Koichiro Komai
    Bioscience, biotechnology, and biochemistry TAYLOR & FRANCIS LTD 68 (3) 761 - 3 0916-8451 2004/03 [Refereed]
    To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SADbeta2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.
  • Valérie Raymond Delpech; Makoto Ihara; Claudio Coddou; Kazuhiko Matsuda; David B Sattelle
    Invertebrate neuroscience : IN SPRINGER HEIDELBERG 5 (1) 29 - 35 1354-2516 2003/11 [Refereed]
    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha 7, chicken alpha 4 beta 2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta 2 and ALS/beta 2. No agonist action of NTX (0.1-100 mu M) was observed on chicken alpha 7, chicken alpha 4 beta 2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha 7) or heteromeric (alpha 4 beta 2) nicotinic AChRs. Block by NTX of the chicken a7, chicken alpha 4 beta 2 and the SAD/beta 2 and ALS/beta 2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.
  • Makoto Ihara; Kazuhiko Matsuda; Maiko Otake; Morihiko Kuwamura; Masaru Shimomura; Koichiro Komai; Miki Akamatsu; Valérie Raymond; David B Sattelle
    Neuropharmacology PERGAMON-ELSEVIER SCIENCE LTD 45 (1) 133 - 44 0028-3908 2003/07 [Refereed]
    The 2-nitroimino-imidazolidine and related moieties are structural features of neonicotinoid insecticides acting on nicotinic acetylcholine receptors (nicotinic AChRs). To evaluate these moieties in neonicotinoid interactions with nicotinic AChR alpha subunits, the actions of imidacloprid and related compounds on the chicken alpha7, alpha4beta2 and Drosophila melanogaster-chicken hybrid (SADbeta2 and ALSbeta2) receptors expressed in Xenopus laevis oocytes were studied by voltage-clamp electrophysiology. Imidacloprid and nitenpyram were partial agonists and a nitromethylene analog of imidacloprid (CH-IMI) was a full agonist of the beta receptor, whereas their agonist actions on the alpha4beta2 receptor were very weak, contrasting with full agonist actions of DN-IMI, a desnitro derivative of imidacloprid. The neonicotinoids and DN-IMI were either full or partial agonists of the SADbeta2 receptors. Nitenpyram and DN-IMI were partial agonists of the ALSbeta2 receptor, whereas imidacloprid and CH-IMI scarcely activated the ALSbeta2 receptor. Imidacloprid and CH-IMI in fact suppressed ACh-induced responses of the ALSbeta2 receptor, whereas imidacloprid potentiated and CH-IMI suppressed ACh-induced responses of the alpha4beta2 receptor. These results suggest that interactions with alpha subunits of the 2-nitroimino-imidazolidine moiety of imidacloprid play a role in determining not only agonist and antagonist actions on all four receptors, but also the potentiation of ACh-induced responses of the alpha4beta2 receptor. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • K Matsuda; M Ihara; K Nishimura; DB Sattelle; K Komai
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 65 (7) 1534 - 1541 0916-8451 2001/07 [Refereed]
    Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken beta2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.
  • K Matsuda; M Shimomura; Y Kondo; M Ihara; K Hashigami; N Yoshida; Raymond, V; NP Mongan; JC Freeman; K Komai; DB Sattelle
    BRITISH JOURNAL OF PHARMACOLOGY NATURE PUBLISHING GROUP 130 (5) 981 - 986 0007-1188 2000/07 [Refereed]
    1 The nitroguanidine insecticide imidacloprid along with a second generation of related compounds including nitenpyram, all nicotinic acetylcholine (ACh) receptor ligands, are used increasingly in many countries. Site-directed mutagenesis and heterologous expression in Xenopus laevis oocytes have been deployed to investigate mutants (G189D and G189E) of the chicken alpha 7 homomer-forming nicotinic receptor subunit which are predicted to enhance the negative charge at the negative subsite (loop D) of the ACh binding site. 2 Xenopus oocytes expressing wild-type a7 nicotinic receptors respond to imidacloprid with rapid inward currents. Imidacloprid and nitenpyram are partial agonists, whereas ACh, (-)-nicotine and (+)-epibatidine are full agonists. 3 Compared to wild-type alpha 7, the mutant G189D and G189E receptors are much less sensitive to the insecticides, whereas their sensitivity to (-)-nicotine, ACh and (+)-epibatidine is only slightly reduced. In contrast, G189N and G189Q mutants are sensitive not only to ACh, (-)-nicotine and (+)-epibatidine, but also to the two insecticides. Thus reduction of the insecticide-sensitivity by the mutations G189D and G189E are attributed to an increase in negativity of loop D. Desnitro-imidacloprid (DN-IMI), an imidacloprid derivative lacking the nitro group is a potent agonist on the G189D and G189E mutants suggesting an important role of loop D in nicotinic receptor interactions with the nitro group of nitroguanidine insecticides.


Lectures, oral presentations, etc.

  • セイヨウミツバチのニコチン性アセチルコリン受容体の機能的発現と薬理特性に対する補助因子の効果  [Not invited]
    伊原 誠; 高山 浩一; 伊藤 稜; 森 澄海人; 武林 真由花; 江川 瑠夏; 山本 新菜; 木村 裕紀; 松田 一彦
    日本農芸化学会2024年度大会  2024/03
  • Paraherquamide A の C. elegans ニコチン性アセチルコリン受容体サブタイプ選 択性の分子機構  [Not invited]
    伊原誠; 大坪柊也; 高山浩一; 松田一彦
    2023 年度 日本農芸化学会 中部・関西支部合同大会 (中部支部:第 196 回例会)(関西支部:第 527 回講演会)  2023/10
  • Makoto Ihara
    Control of Human Disease Vectors, Pests and Parasites  2023/09
  • Paraherquamide A のC. elegans ニコチン性アセチルコリン受容体に対するサブタイプ 選択性の分子メカニズム  [Not invited]
    伊原誠; 大坪柊也; 高山浩一; 松田一彦
    日本農薬学会2023年度大会  2023/03
  • キイロショウジョウバエのニコチン性アセチルコリン受容体機能発現におけるRIC3の関与  [Not invited]
    伊原 誠; 高山 浩一; 小森 勇磨; 神谷 昌輝; 西浦 凜; 松田 一彦
    日本農芸化学会2022年度大会  2022/03
  • 殺虫性メロテルペン化合物chrodrimanin Bのラット脳膜画分に対する作用  [Not invited]
    伊原誠; 田中啓司; 松田一彦
    日本農薬学会2022年度大会  2022/03
  • Game-changing, co-factor aided, robust functional expression of insect nAChRs in Xenopus laevis oocytes revealed nanomolar and picomolar target-site actions of neonicotinoids  [Not invited]
    Makoto Ihara; David B. Sattelle; Kazuhiko Matsuda
    ACS Fall 2021  2021/08
  • 昆虫ニコチン性アセチルコリン受容体の再構築とネオニコチノイドの活性
    伊原 誠; 小森 勇磨; 神谷 昌輝; 小泉 航; 吉成 祐人; 丹羽 隆介; SATTELLE David B; 松田 一彦
    日本農芸化学2021年度大会  2021/03
  • ピレスリン生合成酵素TcGLIPに対するホスホン酸エステル型阻害剤の絶対配置の解明と構造活性相関
    伊原 誠; 小椋 明生; 泊口 佳史; 松尾 憲忠; 田辺 陽; 松田 一彦
    日本農芸化学2020年度大会  2020/03
  • Makoto Ihara; David B. Sattelle; Kazuhiko Matsuda
    IUPAC2019 GHENT 14th IUPAC International Congress of Crop Protection Chemistry  2019/05
  • Neonicotinoids: molecular mechanisms of action, insights into resistance and impact on pollinators  [Invited]
    Makoto Ihara
    Pyrethrum Workshop 2018  2018/09
  • 線虫C. elegansの筋肉型および神経型ニコチン性アセチルコリン受容体に対するParaherquamide Aの阻害活性発現機構
    伊原 誠; 小鑓 亮平; 古谷 章悟; 甲斐 建次; 林 英夫; David; B. SATTELLE; 松田 一彦
    日本農薬学会2017年度大会  2017/03
  • ネオニコチノイド研究によって明らかとなったニコチン性アセチルコリン受容体のアゴニスト認識機構  [Invited]
    伊原 誠
    日本農芸化学会2015年度大会 シンポジウム  2015/03
  • ネオニコチノイドの選択毒性に寄与するニコチン性アセチルコリン受容体の新奇構造因子
    伊原 誠; 岡島 俊英; 山下 敦子; 尾田 拓麻; 浅野 拓也; 松井 美佳奈; David B. Sattell; 松田 一彦
    日本農芸化学会2015年度大会  2015/03
  • ピレスリン生合成に関わる TcGLIP の機能および構造の解明(3)−阻害活性と阻害の様式−  [Not invited]
    竹内孝幸; 明石拓也; 宇都宮麻衣; 伊原 誠; 平竹 潤; 松田一彦
    日本農芸化学会2015年度大会  2015/03
  • ピレスリン生合成に関わる TcGLIP の機能および構造の解明(2)−ホスホン酸型反応機構依存的阻害剤の設計と合成−  [Not invited]
    明石拓也; 竹内孝幸; 宇都宮麻衣; 伊原 誠; 松田一彦; 平竹 潤
    日本農芸化学会2015年度大会  2015/03
  • Modulation by chloride-channel-targeting pesticides of proton-sensitive chloride channels expressed in the silkworm Bombyx mori  [Not invited]
    Yuri Nakatani; Shogo Furutani; Makoto Ihara; Kazuhiko Matsuda
  • ニコチン性アセチルコリン受容体の多型によって生じるネオニコチノイド感受性変化の分子機構
    伊原 誠; 島津 直弥; 宇都宮 麻衣; 赤松 美紀; David B. SATTELL; 松田 一彦
    日本農芸化学会2014年度大会  2014/03
  • 農薬とイオンチャネル  [Invited]
    新学術領域研究「天然物ケミカルバイオロジー:分子標的と活性制御」ミニシンポジウム  2014/03
  • Molecular Bases of the Multimodal Regulation of Fungal TRP Channels  [Invited]
    Makoto Ihara
    アグロバイオシンポジウム2013  2013/11
  • 島津直弥; 伊原誠; 松田一彦
    日本農芸化学会関西支部講演会講演要旨集  2013/09
  • Electrophysiological actions of neonicotinoids on the identified Kenyon cell from Drosophila melanogaster
    IHARA Makoto; MATSUDA Kazuhiko; KOMAI Koichiro
    講演要旨集  2005/03  Pesticide Science Society of Japan
  • IHARA Makoto; MATSUDA Kazuhiko; KOMAI Koichiro
    講演要旨集  2004/03  Pesticide Science Society of Japan
  • Actions of clothianidin and related neonicotinoids on recombinant and native nicotinic acetylcholine receptors.  [Not invited]
    Makoto Ihara; Kazuhiko Matsuda; David B. Sattelle; Koichiro Komai
    XXII International Congress of Entomology, Brisbane, Australia  2004
  • Structural changes of aromatic and imino moieties in imidacloprid on the agonist actions on recombinant nicotinic acetylcholine receptors
    講演要旨集  2001/02  Pesticide Science Society of Japan
  • Actions of clothianidin and related compounds on recombinant nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes  [Not invited]
    Makoto Ihara
    Neurotox 2003, Nottingham, UK.

Affiliated academic society


Research Themes

  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    Date (from‐to) : 2022/04 -2026/03 
    Author : 伊原 誠
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2016/04 -2018/03 
    Author : IHARA Makoto; HIKIDA Mai; YUKUTA Yoshie
    Neonicotinoids are widely used for crop protection and veterinary use worldwilde. Recently the adverse effects of neonicotinoids have been focused, and some of them were banned from EU. In order to elucidate the molecular mechanism of such adverse effects of neonicotinoids, X-ray crystallography of acetylcholine binding protein, a surrogate protein of ligand binding domain of nicotinic acetylcholine receptors, has been employed and the X-ray crystal structures of AChBP-neonicotinoids complexes were solved. Using those structures, homology models were built and evaluated their interactions. These results suggested that LOOPs D, E and G cooperatively form neonicotinoid-fovored environment in insect nAChRs. Physiological evaluation confirmed that even single mutation at the Loop D, E and G strikingly weakened the neonicotinoid actions on insect nAChRs. These results suggest the adverse effects of neonicotinoids might be controlled through structure guided approaches.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2013/04 -2016/03 
    Author : MATSUDA Kazuhiko; HIRATAKE Jun; MATSUI Kenji; YAMASHITA Atsuko; IHARA Makoto
    Insecticide pyrethrins containing an ester bond are biosynthesized by catalysis of a GDSL lipase TcGLIP using the acyl and the alcohol substrates. The aim of this study was to elucidate the molecular mechanism of TcGLIP in pyrethrin synthesis and regulation in terms of gene expression and catalysis. First, TcGLIP was expressed in E. coli and amino acids essential for the catalysis were identified. Next, a method to obtain recombinant TcGLIP with high yield and purity was established and solutions to crystallize the enzyme were found. On the other hand, phosphonates were synthesized as TcGLIP inhibitor candidates and tested for their inhibitory activity. As a result, several compounds were found to block the pyrethrin-synthesizing activity of TcGLIP at nanomolar concentrations. On the other hand, a TcGLIP homolog gene was knocked out in Arabidopsis, resulting in reduced potency of exudates to activate the defensin gene promoter.