研究者詳細 - 堂浦　智裕

Intratumour oxidative hotspots provide a niche for cancer cell dissemination 査読有り国際誌

Ueda, Y; Kiyonaka, S; Selfors, LM; Inoue, K; Harada, H; Doura, T; Onuma, K; Uchiyama, M; Kurogi, R; Yamada, Y; Sun, JH; Sakaguchi, R; Tado, Y; Omatsu, H; Suzuki, H; Aoun, M; Nakayama, T; Kajimoto, T; Yano, T; Holmdahl, R; Hamachi, I; Inoue, M; Mori, Y; Takahashi, N

NATURE CELL BIOLOGY 27 巻 ( 3 ) 頁： 530 - 543 2025年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Cell Biology

Intratumour heterogeneity represents the hierarchical integration of genetic, phenotypic and microenvironmental heterogeneity. Although single-cell sequencing has clarified genetic and phenotypic variability, the heterogeneity of nongenetic, microenvironmental factors remains elusive. Here, we developed T-AP1, a tumour-targeted probe tracking extracellular H2O2, which allows the visualization and characterization of tumour cells exposed to oxidative stress, a hallmark of cancer. T-AP1 identified actively budding intratumour regions as H2O2-rich microenvironments (H2O2 hotspots), which were primarily established by neutrophils. Mechanistically, tumour cells exposed to H2O2 underwent partial epithelial–mesenchymal transition through p38–MYC axis activation and migrated away from H2O2 hotspots. This escape mechanism was absent in normal epithelial cells but prevalent in most cancers except NRF2-hyperactivated tumours, which exhibited abrogated p38 responses and enhanced antioxidant programmes, thus revealing an intrinsic stress defence programme in cancers. Together, T-AP1 enabled the identification of H2O2 hotspots that provide a niche for cancer cell dissemination, offering insights into metastasis initiation.

DOI： 10.1038/s41556-025-01617-w

特集高速分子動画:動的構造からタンパク質分子制御へ Ⅶ.ケミカルバイオロジー:タンパク質の分子制御へ向けて動的構造情報に基づく受容体タンパク質活性制御法の開発

原隆史, 堂浦智裕, 清中茂樹

生体の科学 75 巻 ( 3 ) 頁： 269 - 274 2024年6月

Hijacking endogenous mRNA for genetic code expansion 招待有り査読有り国際誌

Doura, T; Matsuoka, Y; Kiyonaka, S

NATURE CHEMICAL BIOLOGY 20 巻 ( 6 ) 頁： 660 - 661 2024年6月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Chemical Biology

Labeling of endogenous proteins with chemical probes is highly desirable for life science studies. The combination of RNA base editing and site-specific incorporation of non-canonical amino acids allows the introduction of small chemical tags into endogenous proteins in living cells.

DOI： 10.1038/s41589-024-01574-9

Crystal structure reveals the binding mode and selectivity of a photoswitchable ligand for the adenosine A2A receptor 査読有り国際誌 Open Access

Araya, T; Matsuba, Y; Suzuki, H; Doura, T; Nuemket, N; Nango, E; Yamamoto, M; Asada, H; Kiyonaka, S; Iwata, S

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 695 巻頁： 149393 2024年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.

DOI： 10.1016/j.bbrc.2023.149393

高次脳機能の理解に向けた分子標的ケモジェネティクス法の開発招待有り

清中茂樹, 堂浦智裕

MEDCHEM NEWS 33 巻 ( 3 ) 頁： 136 - 141 2023年8月

記述言語：日本語掲載種別：論文集(書籍)内論文出版者・発行元：公益社団法人日本薬学会

記憶や学習に代表される高次脳機能は、数多くの神経細胞とグリア細胞が複雑に相互作用して神経回路を形成することにより生み出される。高次脳機能を理解するために、動物個体レベルで神経回路を構成する特定の細胞種の活動を制御可能な光遺伝学（オプトジェネティクス）や化学遺伝学（ケモジェネティクス）などの細胞操作技術が非常に有用である。しかし、既存の手法では、観察対象となる細胞に本来存在しない人工受容体を発現させて細胞機能を制御するため、細胞が有する本来の機能を観察できていない可能性がある。本稿では、筆者らが取り組んでいる特定の神経細胞やグリア細胞に内在的に発現する受容体の細胞種選択的な制御を可能にする「分子標的ケモジェネティクス」の構築について述べる。

DOI： 10.14894/medchem.33.3_136

CiNii Research

高次脳機能の理解に向けた次世代ケモジェネティクス法の開発招待有り

Matsuoka Y, Kashiwa S, Doura T, Kiyonaka S

Brain and nerve = Shinkei kenkyu no shinpo 75 巻 ( 4 ) 頁： 367 - 374 2023年4月

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue 査読有り国際誌 Open Access

Ojima, K; Kakegawa, W; Yamasaki, T; Miura, Y; Itoh, M; Michibata, Y; Kubota, R; Doura, T; Miura, E; Nonaka, H; Mizuno, S; Takahashi, S; Yuzaki, M; Hamachi, I; Kiyonaka, S

NATURE COMMUNICATIONS 13 巻 ( 1 ) 頁： 3167 - 3167 2022年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

DOI： 10.1038/s41467-022-30828-0

Chemogenetics of cell surface receptors: beyond genetic and pharmacological approaches 査読有り

Miura Yuta, Senoo Akinobu, Doura Tomohiro, Kiyonaka Shigeki

RSC CHEMICAL BIOLOGY 3 巻 ( 3 ) 頁： 269 - 287 2022年3月

Orthogonal Activation of Metabotropic Glutamate Receptor Using Coordination Chemogenetics 査読有り国際誌 Open Access

Senoo, A; Yamada, Y; Ojima, K; Doura, T; Hamachi, I; Kiyonaka, S

FRONTIERS IN CHEMISTRY 9 巻頁： 825669 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Chemistry

Cell-surface receptors play a pivotal role as transducers of extracellular input. Although different cell types express the same receptor, the physiological roles of the receptor are highly dependent on cell type. To understand each role, tactics for cell-specific activation of the target receptor are in high demand. Herein, we developed an orthogonal activation method targeting metabotropic glutamate receptor 1 (mGlu1), a G-protein coupled receptor. In this method, direct activation via coordination-based chemogenetics (dA-CBC) was adopted, where activation of mGlu1 was artificially induced by a protein conformational change in response to the coordination of a metal ion or metal-ion complex. Our structure-based protein design and screening approach identified mGlu1 mutants that were directly activated by the coordination of Cu2+ or Zn2+, in addition to our previous Pd-complex-sensitive mGlu1 mutant. Notably, the activation of the mutants was mutually orthogonal, resulting in cell-type selective activation in a model system using HEK293 cells.

DOI： 10.3389/fchem.2021.825669

Tethering-based chemogenetic approaches for the modulation of protein function in live cells 査読有り国際共著国際誌

Tsai, YH; Doura, T; Kiyonaka, S

CHEMICAL SOCIETY REVIEWS 50 巻 ( 14 ) 頁： 7909 - 7923 2021年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Society Reviews

Proteins are the workhorse molecules performing various tasks to sustain life. To investigate the roles of a protein under physiological conditions, the rapid modulation of the protein with high specificity in a living system would be ideal, but achieving this is often challenging. To address this challenge, researchers have developed chemogenetic strategies for the rapid and selective modulation of protein function in live cells. Here, the target protein is modified genetically to become sensitive to a designer molecule that otherwise has no effect on other cellular biomolecules. One powerful chemogenetic strategy is to introduce a tethering point into the target protein, allowing covalent or non-covalent attachment of the designer molecule. In this tutorial review, we focus on tethering-based chemogenetic approaches for modulating protein function in live cells. We first describe genetic, optogenetic and chemical means to study protein function. These means lay the basis for the chemogenetic concept, which is explained in detail. The next section gives an overview, including advantages and limitations, of tethering tactics that have been employed for modulating cellular protein function. The third section provides examples of the modulation of cell-surface proteins using tethering-based chemogenetics through non-covalent tethering and covalent tethering for irreversible modulation or functional switching. The fourth section presents intracellular examples. The last section summarizes key considerations in implementing tethering-based chemogenetics and shows perspectives highlighting future directions and other applications of this burgeoning research field.

DOI： 10.1039/d1cs00059d

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking 査読有り国際誌 Open Access

Ojima, K; Shiraiwa, K; Soga, K; Doura, T; Takato, M; Komatsu, K; Yuzaki, M; Hamachi, I; Kiyonaka, S

NATURE COMMUNICATIONS 12 巻 ( 1 ) 頁： 831 2021年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

DOI： 10.1038/s41467-021-21082-x

その他リンク： http://www.nature.com/articles/s41467-021-21082-x

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene 査読有り国際誌

Aoyama, H; Doura, T

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 30 巻 ( 4 ) 頁： 126888 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioorganic and Medicinal Chemistry Letters

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC50 value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.

DOI： 10.1016/j.bmcl.2019.126888

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene 査読有り

Hiroshi Aoyama, Tomohiro Doura

Bioorganic and Medicinal Chemistry Letters 30 巻 ( 4 ) 頁： 126888 2020年2月

内在性のGタンパク質共役型受容体を光によって制御する招待有り査読有り

堂浦智裕

ファルマシア 56 巻 ( 5 ) 頁： 441 - 441 2020年

記述言語：日本語掲載種別：論文集(書籍)内論文出版者・発行元：公益社団法人日本薬学会

生体中には800種類以上のGタンパク質共役型受容体(GPCRs)が存在している．個々のGPCRの生物学的・病理学的機能を解明することは生命機能や病態の理解，治療法の開発に結びつくため重要である．代表的なGPCRの化学遺伝学的活性制御法はdesigner receptors exclusively activated by designer drugs(DREADDs)であるが，本手法で使用されるムスカリン受容体は人工リガンドにのみ結合するように改変されているため，本来の機能を喪失している． そこで，標的GPCRの機能を維持したままGPCRの機能を解明する化学遺伝学的手法として，Broichhagen J.らは光刺激によってGPCRの活性を制御するphotoswitchable orthogonal remotely tethered ligand(PORTL)を開発した．細胞表層にSNAP-tag融合代謝型グルタミン酸受容体2(SNAP-mGluR2)を発現させ，mGluR2の天然アゴニストであるグルタミン酸・光スイッチ部位・SNAP-tag基質を適切なリンカーで連結した分子(BGAG)でラベル化して作製したPORTL(SNAG-mGluR2)に光照射することによりSNAG-mGluR2の活性制御に成功している．しかし，PORTLは原理的に内在性GPCRの活性を制御できない． 本稿では， Donthamsetti P. C.らが開発した内在性GPCRの活性を光制御可能なPORTLの発展型であるmaPORTLについて紹介する． なお，本稿は下記の文献に基づいて，その研究成果を紹介するものである. 1) Urban D. J. et al., Annu. Rev. Pharmacol. Toxicol., 55, 399-417(2015)． 2) Broichhagen J. et al., ACS Cent. Sci., 1, 383-393(2015)． 3) Donthamsetti P. C. et al., J. Am. Chem. Soc., 141, 11522-11530(2019)．

DOI： 10.14894/faruawpsj.56.5_441

CiNii Research

Relationship between the glutathione-responsive degradability of thiol-organosilica nanoparticles and the chemical structures 査読有り国際共著国際誌

T. Doura, T. Nishio, F. Tamanoi, M. Nakamura

J. Mater. Res. 34 巻 ( 7 ) 頁： 1266 - 1278 2019年2月

Biodegradability of disulfide-organosilica nanoparticles evaluated by soft X-ray photoelectron spectroscopy: cancer therapy implications 査読有り

H. Mekaru, A. Yoshigoe, M. Nakamura, T. Doura, F. Tamanoi

ACS Appl. Nano Mater. 2 巻 ( 1 ) 頁： 479 - 488 2018年12月

Miniaturization of thiol-organosilica nanoparticles induced by an anionic surfactant 査読有り国際共著国際誌

Tomohiro Doura, Fuyuhiko Tamanoi, Michihiro Nakamura

Journal of Colloid and Interface Science 526 巻頁： 51 - 62 2018年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Academic Press Inc.

Thiol-organosilica nanoparticles are a promising nanomaterial for biomedical applications. The enhanced permeability and retention (EPR) effect is useful for tumor targeting within the biomedical applications of nanomaterials, and nanomaterials with a size of less than 200 nm exhibit the maximum EPR effect. However, the synthesis of thiol-organosilica nanoparticles with a diameter of less than 200 nm is not efficient for the yield using the present conventional synthetic methods. Herein, we report the development of an efficient synthetic method of thiol-organosilica nanoparticles with a diameter of less than 200 nm using an anionic surfactant and discuss its mechanism. Compared with the conventional synthetic methods, a greater than 10-fold miniaturization of thiol-organosilica nanoparticles and an approximately 40-fold increase in the production efficiency of small thiol-organosilica nanoparticles were achieved using the sodium dodecyl sulfate (SDS)-addition synthetic method or sodium dodecylbenzenesulfonate (SDBS)-addition synthetic method. This is the first report about the miniaturization of organosilica nanoparticles induced by an anionic surfactant. The SDS-addition synthetic method or SDBS-addition synthetic method will accelerate the biomedical applications of thiol-organosilica nanoparticles.

DOI： 10.1016/j.jcis.2018.04.090