Osawa, Masanori

写真a

Affiliation

Faculty of Pharmacy, Department of Pharmaceutical Sciences Division of Physics for Life Functions (Shiba-Kyoritsu)

Position

Professor

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Research Areas 【 Display / hide 】

  • Life Science / Structural biochemistry

  • Life Science / Structural biochemistry

  • Life Science / Biophysics

  • Life Science / Biophysics

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Research Keywords 【 Display / hide 】

  • ion channel

  • ion channel

  • Signal transduction

  • Signal transduction

  • nuclear magnetic resonance

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Books 【 Display / hide 】

  • Peptide Toxins Targeting KV Channels

    Matsumura K, Yokogawa M, Osawa M, Springer Nature, 2021

    　View Summary

    A number of peptide toxins isolated from animals target potassium ion (K+) channels. Many of them are particularly known to inhibit voltage-gated K+ (KV) channels and are mainly classified into pore-blocking toxins or gating-modifier toxins. Pore-blocking toxins directly bind to the ion permeation pores of KV channels, thereby physically occluding them. In contrast, gating-modifier toxins bind to the voltage-sensor domains of KV channels, modulating their voltage-dependent conformational changes. These peptide toxins are useful molecular tools in revealing the structure-function relationship of KV channels and have potential for novel treatments for diseases related to KV channels. This review focuses on the inhibition mechanism of pore-blocking and gating-modifier toxins that target KV channels.

  • Nuclear magnetic resonance approaches for characterizing protein-protein interactions

    Toyama Y, Mase Y, Kano H, Yokogawa M, Osawa M, Shimada I, Methods in Molecular Biology, 2018

    　View Summary

    The gating of potassium ion (K+) channels is regulated by various kinds of protein-protein interactions (PPIs). Structural investigations of these PPIs provide useful information not only for understanding the gating mechanisms of K+ channels, but also for developing the pharmaceutical compounds targeting K+ channels. Here, we describe a nuclear magnetic resonance spectroscopic method, termed the cross saturation (CS) method, to accurately determine the binding surfaces of protein complexes, and its application to the investigation of the interaction between a G protein-coupled inwardly rectifying K+ channel and a G protein α subunit.

  • 【試料分析講座】「蛋白質の分析」

    OSAWA MASANORI, 分析化学会（丸善）, 2012

    Scope: NMR・CDスペクトル

  • 分子細胞生物学辞典 (第２版)

    OSAWA MASANORI, 東京化学同人, 2008

    Scope: 「等温滴定型カロリメトリー」,「滴定曲線」

  • 実験医学別冊「生命科学のための機器分析実験ハンドブック」

    OSAWA MASANORI, 羊土社, 2007

    Scope: 「等温滴定型カロリメトリー(ITC) ～熱量測定による相互作用の定量的解析～」

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Papers 【 Display / hide 】

  • KMN003 activates Nrf2 via disruption of the Keap1-Nrf2 interaction and p38-dependent transcriptional regulation

    Keijiro K., Kai T., Daisuke Y., Hanako I., Koujin K., Masanori O., Tomoya H., Tomoyuki O., Kenji T., Megumi F-T.

    Cellular Signalling  2026.01

    Research paper (scientific journal), Joint Work, Accepted

    　View Summary

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a crucial role in cellular defenses against oxidative stress and inflammation. Under normal conditions, Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase adaptor, binds to Nrf2, facilitating its ubiquitination and subsequent degradation via the proteasome. In this study, we investigated the properties of KMN003, a novel Nrf2 activator specifically designed to stabilize Nrf2 by disrupting its interaction with Keap1. X-ray crystallographic analysis revealed that KMN003 binds to the DGR-Cul3 (DC) domain of Keap1, occupying the Nrf2 interaction site. An AlphaScreen assay further showed that KMN003 effectively inhibits the binding between the Keap1 DC domain and the DLG motif of Nrf2 (IC₅₀ = 300 nM). We also investigated the mechanism of Nrf2 activation by KMN003 and its anti-inflammatory properties using murine macrophage-like RAW264.7 cells. KMN003 significantly reduced the lipopolysaccharide (LPS)-induced production of nitric oxide, CCL2, and tumor necrosis factor-alpha (TNFα) as well as the mRNA expression of inducible nitric oxide synthase, CCL2, and TNFα, which are essential inflammatory markers. KMN003 strongly inhibited nuclear translocation and transcriptional activation of nuclear factor-kappa B (NF-κB), a central regulator of inflammatory gene expression. KMN003 did not affect the LPS-induced phosphorylation of ERK or JNK, but strongly induced p38 phosphorylation in the absence of the LPS stimulation. The inhibition of p38 with SB203580 blocked KMN003-induced Nrf2 transcriptional activation despite promoting Nrf2 accumulation. These results highlight KMN003 as a promising anti-inflammatory drug that selectively stabilizes and activates Nrf2 via the p38 pathway.

  • Development of Keap1-Nrf2 Protein–Protein Interaction Inhibitor Activating Intracellular Nrf2 Based on the Naphthalene-2-acetamide Scaffold, and its Anti-Inflammatory Effects

    Daisuke Y., Kai T., Koujin K., Hanako I., Kazuma K., Riyo I., Kayoko K., Hirotatsu K., Megumi F-T., Masanori O., Tomoyuki O., Tomoya H.

    ChemMedChem   2025.09

    Research paper (scientific journal), Joint Work, Accepted

    　View Summary

    Nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) axis is an attractive therapeutic target for various intractable diseases. Although protein–protein interaction inhibitors against Keap1-Nrf2 have been developed over the past decade, more structural expansion is needed to improve efficacy. In this article, several candidate compounds are designed and synthesized as novel Nrf2 activators and their intracellular Nrf2-activating effects are evaluated. Among the synthesized compounds, a novel naphthalene-1,4-(4-ethoxybenzensulfonamide) bearing a tertiary acetamide side chain at the 2-position strongly activated intracellular Nrf2. Particularly, the pyrrolidine-type acetamide compound showed the strongest intracellular Nrf2 activation. X-ray cocrystallography revealed that this compound can bind to the DC domain of Keap1. Additionally, the pyrrolidine-type acetamide compound induced the mRNA expression of the representative Nrf2 target genes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Moreover, the compound exhibited anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage cell line. Conclusively, these results suggest that the pyrrolidine-type naphthalene-2-acetamide is a promising compound for the development of Nrf2 activators that can be applied to treat inflammatory diseases.

  • NMR <sup>1</sup>H, <sup>13</sup>C, <sup>15</sup>N backbone resonance assignments of 14-3-3ζ binding region of human FOXO3a (residues 1-284)

    Enomoto S., Nakatsuka S., Kuwayama T., Kawatsu K., Yokogawa M., Osawa M.

    Biomolecular NMR Assignments 18 ( 2 ) 275 - 283 2024.12

    ISSN  18742718

    　View Summary

    In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

  • “NMR 1H, 13C, 15N backbone resonance assignments of 14-3-3ζ binding region of human FOXO3a (residues 1-284)

    Enomoto S, Nakatsuka S, Kuwayama T, Kawatsu K, Yokogawa M, and Osawa M.

    Biomolecular NMR Assignments   2024.09

    Research paper (scientific journal), Corresponding author, Accepted

    　View Summary

    In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

  • Ribonuclease inhibitor and angiogenin system regulates cell type-specific global translation

    Stillinovic M, Sarangdhar M. A., Andina N, Tardivel A, Greub F, Bombaci G, Ansermet C, Zatti M, Saha D, Xiong J, Sagae T, Yokogawa M, Osawa M, Heller M, Keogh A, Keller I, Angelillo-Scherrer A, and Allam R.

    Science Advances (American Association for the Advancement of Science)  10 ( 22 )  2024.05

    Research paper (scientific journal), Joint Work, Accepted

    　View Summary

    Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type–specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type–specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non–hematopoietic-origin cells. RNH1-mediated cell type–specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type–specific global translation regulators and highlights the complex translation regulation in vertebrates.

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Papers, etc., Registered in KOARA 【 Display / hide 】

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Reviews, Commentaries, etc. 【 Display / hide 】

  • PoSSuM v.3: A Major Expansion of the PoSSuM Database for Finding Similar Binding Sites of Proteins

    Journal of Chemical Information and Modeling  2023.11

  • Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization.

    BMC molecular and cell biology  2021.01

    　View Summary

    <h4>Background</h4>Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state.<h4>Results</h4>We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis.<h4>Conclusions</h4>The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of K<sub>V</sub> channels.

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Presentations 【 Display / hide 】

  • 新型コロナウイルスの侵入を阻害するタンパク質タンパク質相互作用阻害化合物の創製

    横川真梨子、金一駿希、堀内まほろ、大竹帝河、米澤朋起、清水祐吾、池田和由、山本雄一朗、酒井祥太、清水芳実、野口耕司、深澤征義、伊倉光彦、大澤匡範

    NMR討論会2025, 

    2025.11
    , 

    Oral presentation (general)

  • ALS関連タンパク質TDP-43の凝集と核酸による抑制機構の解明

    西村 錬、西田 優理華、八城 立樹、坂上 史佳、三浦 元輝、横田 隆徳、石田 英子、横川 真梨子、大澤 匡範

    NMR討論会2025, 

    2025.11
    , 

    Poster presentation

  • NMR化学シフト変化に基づく低親和性リガンドの結合様式推定手法の構築

    小島行人、石田英子、米澤朋起、原田彩佳、前川こひろ、横川真梨子、清水祐吾、池田和由、大澤匡範

    NMR討論会2025, 

    2025.11
    , 

    Poster presentation

  • 14-3-3ζ interacts with DNA-binding domain of FOXO3a and competitively dissociates DNA by dual-motif tethering

    Shota Enomoto, Tomoya Kuwayama, Shoichi Nakatsuka, Mariko Yokogawa, Kosaku Kawatsu, Risa Nakamura, Tomomi Kimura, Mikio Tanabe, Toshiya Senda, Jun Saito, Hideyuki Saya, Masanori Osawa

    NMR討論会2025, 

    2025.11
    , 

    Poster presentation

  • Enhancing the Predictive Performance of PPI Inhibitory Activity Models through Data Imbalance Correction

    Yixuan SUI, Yugo Shimizu, Masanori Osawa, Kazuyoshi Ikeda

    CBI学会2025大会, 

    2025.10
    , 

    Poster presentation

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Research Projects of Competitive Funds, etc. 【 Display / hide 】

  • Mechanism of antagonistic action for intermolecular interactions between etiological biopolymers, and novel strategies for drug discovery

    2024.04
    -
    2027.03

    Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Principal investigator

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    本研究では、「がん関連転写因子FOXO3aをめぐるDNAと14-3-3ζの拮抗メカニズムの解明」、「孤発性ALSの原因タンパク質であるTDP-43の細胞内凝集・沈殿と、RNA結合によるその阻害のメカニズムの解明」を実施例として、複合体の立体構造や解離定数だけからでは説明のつかない効率性の高い拮抗メカニズムを解明することを目的とする。さらに明らかにしたメカニズムに基づき、これらの相互作用系が密接に関わるがんとALSに対する創薬戦略を構築し、新規薬剤の創製に貢献していく。

  • Elucidation of the mechanism of hepatitis B virus entry and control of infection by entry inhibitors

    2022.04
    -
    2025.03

    Grants-in-Aid for Scientific Research, 伊藤 清顕, 宇根 瑞穂, 大澤 匡範, 梅澤 一夫, Grant-in-Aid for Scientific Research (B), No Setting

    　View Summary

    我々は、胆汁酸誘導体の一つであるINT-767がHBVのエンベロープ蛋白質の疎水性領域に結合してHBVの感染を強力に阻害することを発見した（Ito K et al. Hepatology. 2021）。本研究によりHBVの肝細胞内への侵入機構を分子レベルで解明し、胆汁酸誘導体や低分子化合物を利用して、より有効性および安全性が高い新たな感染阻害剤を開発する。HBVと同様に新型コロナウイルスやヒト免疫不全ウイルスなどのエンベロープウイルスもエンベロープ蛋白質の疎水性領域を利用して細胞膜やエンドソーム膜と融合して感染が成立するため、本研究成果は他のエンベロープウイルスに対する治療薬開発にも繋がる。

  • 電位依存性イオンチャネルの機能構造と構造間遷移機構の解析による動作機構解明

    2021.04
    -
    2024.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, 大澤 匡範, 横川 真梨子, Grant-in-Aid for Scientific Research (B), Principal investigator

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    電位依存性イオンチャネル(VGIC)は、神経伝達や心臓の拍動を担う膜タンパク質であり、創薬の標的としても重要である。VGICは一般に、膜電位に応じた構造変化によりイオン透過ゲートを開閉し、特定のイオンを膜透過させることで膜電位を制御する。しかしながら、これまではその構造遷移メカニズムは不明であった。
    そこで本研究では、リガンドの結合や化学修飾により未解明である機能構造を安定化して構造解析を実現可能とする手法を確立し、その立体構造をX線結晶構造解析あるいは電子顕微鏡により原子レベルで明らかにするとともに、NMRによりそれらの間の構造遷移を解析することにより、VGICの動作メカニズムを解明する。

  • B型肝炎ウイルスの肝細胞侵入・増殖機構の構造基盤と立体構造に基づく創薬

    2021.04
    -
    2024.03

    Keio University, Grants-in-Aid for Scientific Research, 横川 真梨子, 大澤 匡範, Grant-in-Aid for Scientific Research (C), No Setting

    　View Summary

    B型肝炎ウイルス(HBV) 外殻膜の表面抗原タンパク質（LHBs）は、肝細胞への感染時には肝細胞膜上のタンパク質であるNTCPと結合し、宿主内での増殖時にはHBVのキャプシドを形成するタンパク質であるCpと結合する。そこで本研究は、LHBs―NTCPおよびLHBs―Cpの相互作用様式を原子分解能で明らかにすることで、HBVの肝細胞侵入・増殖メカニズムを解明する。明らかにした立体構造に基づくin silicoスクリーニング、および得られた候補化合物のin vitroアッセイを行うことで、これらのタンパク質－タンパク質相互作用を阻害する中分子合成化合物を探索し、新規作用機序の抗HBV薬を創製する。

  • 14-3-3タンパク質によるリン酸化シグナル経路の熱力学的・構造生物学的基盤

    2019.04
    -
    2021.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, 大澤 匡範, Grant-in-Aid for Scientific Research on Innovative Areas, Principal investigator

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    多くのがん細胞においては、Ras タンパク質の活性化を起源として、その下流にあるキナーゼを活性化し、そのキナーゼは連鎖的に別のキナーゼをリン酸化・活性化する、というリン酸化シグナルが亢進している。そのシグナルを持続させる機能をもつタンパク質が14-3-3タンパク質である。本研究では、この14-3-3タンパク質がいかにしてリン酸化シグナルを持続させるのかを定量的に、かつ、タンパク質の立体構造の観点より解明することにより、この過程を数理モデル化して理解することを目的としている。このメカニズムが解明できれば、リン酸化シグナルを抑制する革新的な制がん剤の創製につながると考えている。
    多くのがん細胞においては、遺伝子変異によるRas タンパク質の活性化を起源として、その下流にあるキナーゼを活性化し、そのキナーゼは連鎖的に別のキナーゼをリン酸化・活性化する。このようなリン酸化シグナルの連鎖と増幅が、がん細胞の増殖能や浸潤能を高める。 Ser/Thr のリン酸化によるキナーゼの活性化は、14-3-3ζ の結合により持続・亢進し、14-3-3ζ の結合阻害により抑制されることが分かってきた。したがって、がんにおけるリン酸化シグナルの数理モデルを確立するためには、14-3-3ζ と各キナーゼの結合親和性や、その基盤となる複合体の立体構造を明らかにする必要がある。そこで本研究は、14-3-3ζの結合により活性化されるキナーゼや転写因子など（14-3-3ζのclientタンパク質）の全長、あるいは、14-3-3ζとの相互作用部位全体を用いた結合親和性の定量的解析、両者の複合体のX線結晶構造解析を行うことにより、リン酸化シグナルの数理モデルを構築する上での熱力学的・構造生物学的基盤を確立することを目的とする。
    本年度は、14-3-3ζのclientタンパク質のうち、キナーゼ3種類、転写因子2種類の大量発現系を構築し、大腸菌をホストとした大量発現・精製を試みた。これまでに、キナーゼおよび転写因子の各1種類について、大量発現・精製法確立に成功した。キナーゼについてはサイズ排除クロマトグラフィーにより多量体状態の解析を行い、単量体～2量体で存在することが分かった。現在、酵素処理によるリン酸化条件を検討している。転写因子については、これを基質とする別のリン酸化酵素によるリン酸化に成功した。GST融合14-3-3ζを用いたグルタチオンセファロースによるプルダウンアッセイにより、14-3-3ζとリン酸化した転写因子の高親和性結合を確認した。
    当初の計画は、以下の通りであった。
    (1) 14-3-3ζのclient タンパク質の大量調製法の確立、性状解析・活性確認 (2) 14-3-3ζとclient タンパク質全長（相互作用部位全体）の結合親和性の定量解析 (3) 14-3-3ζとclient タンパク質全長（相互作用部位全体）の複合体の結晶化 (4) 14-3-3ζと阻害剤BA およびUTKO1 との相互作用解析、複合体の立体構造解析
    <BR>
    これまでに、(1)については、転写因子およびタンパク質キナーゼの大量調製に成功し、特に転写因子についてはリン酸化方法を確立し、プルダウンアッセイによりリン酸化の有無による14-3-3ζとの結合親和性の変化の検出に成功した。(2)、(3)への取り組みを開始したところである。(4)については、未着手である。
    当初の計画の(2)、(3)については、結晶化に成功し次第、X線結晶構造解析を行う。
    また、大量調製法が確立できていない転写因子とタンパク質リン酸化酵素については、発現条件の検討を行う。

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Awards 【 Display / hide 】

  • Best Poster Award

    Masanori Osawa, 2016.07, The organizing committee of International and Interdisciplinary Symposium 2016, Structural Basis for the Inhibition of Voltage-dependent K+ Channel by Gating Modifier Toxin

    Type of Award： Other

  • 長瀬研究振興賞

    2016.04, NAGASE Science Technology Foundation, Development of novel nanodisc that enables solution NMR analyses of membrane protein interactions in the lipid bilayer

    Type of Award： Other

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Courses Taught 【 Display / hide 】

  • STUDY OF MAJOR FIELD:(PHYSICS FOR LIFE FUNCTIONS)SPRING

    2025

  • STUDY OF MAJOR FIELD:(PHYSICS FOR LIFE FUNCTIONS)AUTUMN

    2025

  • STUDY OF MAJOR FIELD:(PHYSICS FOR LIFE FUNCTIONS)

    2025

  • SEMINAR:(PHYSICS FOR LIFE FUNCTIONS)SPRING

    2025

  • SEMINAR:(PHYSICS FOR LIFE FUNCTIONS)AUTUMN

    2025

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Courses Previously Taught 【 Display / hide 】

  • STUDY OF MAJOR FIELD:(PHYSICS FOR LIFE FUNCTIONS)AUTUMN

    Keio University

    2025.04
    -
    2026.03

  • STUDY OF MAJOR FIELD:(PHYSICS FOR LIFE FUNCTIONS)SPRING

    Keio University

    2025.04
    -
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