Details of a Researcher - Osawa, Masanori

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Angiotensin-converting enzyme 2 (ACE2) 担持ビーズを用いた新型コロナウイルス感染症治療薬シードの探索

江川奏, 齋藤駿, 山本瑞生, 横川真梨子, 大澤匡範, 荻貴之, 荒井緑

日本薬学会第145年会,

2025.03

Poster presentation

新型コロナウイルス感染症（COVID-19）の原因となるSARS-CoV-2ウイルスは、そのスパイクタンパク質を用いてヒト細胞表面にあるアンジオテンシン変換酵素2（ACE2）受容体と相互作用することで細胞内に侵入する。また、ウイルスに変異が入りやすく、それによる変異株は免疫を逃避することが課題であるが、現状ウイルスの変異に対応できる治療薬は存在しない。したがって、宿主の受容体であるACE2を標的としてスパイクタンパク質との結合を阻害する化合物はウイルスの変異に対応可能な治療薬となり得る。そこで本研究では、当研究室で開発された「標的タンパク質指向型天然物単離法」としてACE2ビーズ法を構築し、沖縄県独自の植物エキスライブラリーからACE2に結合する天然物を探索することを目的とする。

Angiotensin-converting enzyme 2 (ACE2) 担持ビーズを用いた新型コロナウイルス感染症治療薬シードの探索

江川奏, 齋藤駿, 山本瑞生, 横川真梨子, 大澤匡範, 荻貴之, 荒井緑

第10回食品薬学シンポジウム ,

2024.10

Oral presentation (general)

新型コロナウイルス感染症（COVID-19）は、2019年暮れに中国・武漢で世界で初めて患者が確認されてから、2ヶ月あまりで世界中に拡散し、世界保健機構（WHO）も2020年3月にパンデミックを宣言した。COVID-19は季節性インフルエンザや新型インフルエンザに比べて感染力が高く1，2)、感染により肺などの呼吸器系だけではなく、特に心臓などの循環器系の合併症や様々な罹患後症状も引き起こす3， 4)。また、COVID-19の原因ウイルスであるSARS-CoV-2には変異が入りやすく、それによる変異株は自然感染やワクチンによって獲得した免疫を逃避することが報告されている5)。現在承認されているSARS-CoV-2の抗ウイルス薬はウイルスを標的としており、ウイルス変異によりその有効性が失われる可能性がある。そのため、未だにSARS-CoV-2の変異に対応できる治療薬の開発が求められている。

Development of novel anti-HBV drugs based on AI technology using bile acid derivatives to inhibit HBV and NTCP binding

Okumura A, Ikeda K, Arai J , Muraki Y , Nishimura S, Inden N, Yokogawa M, Yamashita Y, Iguchi Y, Ohashi H, Watashi K, Wakita T, Une M, Osawa M, Ito K

International HBV Meeting 2024,

2024.09

Oral presentation (general)

The development of safe and potent inhibitors of hepatitis B virus (HBV) entry is crucial to prevent new infections and achieve complete virus elimination in HBV carriers. Persistent HBV carriers have partial hepatocyte infection. If entry into uninfected hepatocytes could be completely inhibited, the number of infected cells would decrease over time. We previously reported that INT-767, a bile acid derivative, inhibits HBV entry by acting directly on the preS1 region. Due to safety concerns with INT-767, we aimed to develop a safer anti-HBV agent based on its mechanism of action.
Methods: A bile acid derivative library, based on the structure of INT-767, was created. We performed a preS1 binding assay to quantitatively evaluate the inhibitory effect of binding between the preS1 region and Sodium-taurocholate co-transporting polypeptide (NTCP) in library compounds. A three-dimensional quantitative structure-activity relationship (3D-QSAR) model was developed to predict anti-HBV activity from the structures of new candidate compounds.
Results: Analysis of the preS1 binding assay results revealed important structures for anti-HBV activity. Bile acid derivatives inhibiting preS1 and NTCP binding shared a common structure, including INT-767. Constructing a 3D-QSAR model using all assay data enabled more detailed predictions of anti-HBV efficacy. Further studies on various bile acid derivatives revealed novel structural features that enhance anti-HBV efficacy. Synthesizing newly designed lead compounds with these features and conducting in vitro HBV infection models demonstrated anti-HBV effects that exceeded those of existing lead compounds.
Conclusion: The 3D-QSAR model, constructed from data on the anti-HBV effects of bile acid derivatives, led to the development of novel bile acid derivatives with more potent anti-HBV activity. Future work with these derivatives will collect data on anti-HBV effects and cytotoxicity, enhancing predictive accuracy for further development.

BTG2によるCaf1依存的ポリA分解の促進機構の解明

片岡奈緒, 横川真梨子, 石井裕一郎, 城えりか, 高嶋大翔, 沢崎綾一, 寒河江彪流, 尾上耕一, 星野真一, 大澤匡範

フィジカル・ファーマフォーラム2024,

2024.08

Oral presentation (general)

B-cell translocation gene2 (BTG2) は、発がん物質やDNA損傷などにより一過的に発現する細胞増殖抑制因子であり、mRNA 3′末端のポリA分解を促進することで翻訳を抑制し、細胞のがん化を抑える。ポリAには複数のポリA結合タンパク質C1 (PABPC1) が結合し、様々なタンパク質をリクルートすることにより翻訳を調節している。BTG2はポリA分解酵素であるCaf1とBTG2-Caf1複合体を形成し、複合体中のBTG2とポリAに結合したPABPC1が相互作用することでCaf1をリクルートし、効率的にポリAを分解すると考えられている (図1)。これまでにPABPC1の4個のRNA認識モチーフ (RRM1/2/3/4) のうち、RRM1とBTG2の結合様式が報告されているが、Caf1とポリAを含まない条件での実験に基づいているため、Caf1が機能している状態の分子間相互作用は分かっていない。そこで本研究では、Caf1やポリA共存下で解析を行いBTG2-Caf1複合体、PABPC1、ポリAの結合様式を明らかにすることで、BTG2によるCaf1依存的ポリA分解の促進機構を解明することを目的とした。

新規Keap1-Nrf2タンパク質間相互作用阻害剤とKeap1結合の構造基

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

フィジカル・ファーマフォーラム2024,

2024.08

Oral presentation (general)

Discovery of the Middle-sized Compounds Inhibiting the SARS-CoV-2 Viral Entry, Using in Silico Approach and NMR Analysis

Mariko Yokogawa, Shunki Kaneichi, Mahoro Horiuchi ,Taiga Otake, Tomoki Yonezawa,Yugo Shimizu, Kazuyoshi Ikeda, Yuichiro Yamamoto, Shota Sakai , Yoshimi Shimizu Kohji Noguchi, Masayoshi Fukasawa Mitsuhiko Ikura, Masanori Osawa

ICMRBS 2024,

2024.08

Poster presentation

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Although several treatments and vaccines are available, definitive treatment has yet to be established. Since the interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and the extracellular domain of hACE2 is important for viral entry, compounds that directly bind to either the RBD or hACE2 and inhibit their interaction should be a drug candidate for COVID-19. Here, we performed ELISA-based assays to find entry inhibitors targeting the interaction between the RBD and hACE2 using small-to-medium sized compounds from our PPI-inhibitor library, DLiP [1], and a number of compounds proposed by in silico approach. The assays discovered multiple hit candidates and most of them inhibited the SARS-CoV-2 infection confirmed by viral infection assay, however the estimated IC50 values were ~10-4 M. The NMR-derived information on the binding sites on the protein would be useful for drug design to improve binding affinity and thus inhibitory activity of the candidates. Our NMR analyses indicated that some compounds, possessing common chemical structure, directly bound to hACE2. To identify their binding site on hACE2 and design the compounds with higher affinity, assignments of the NMR signals of hACE2 are underway.

14-3-3ζ Interacts with FOXO3a-DBD and Competitively Dissociates DNA through the Tethering Effects via Two Binding Motifs.

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

ICMRBS 2024,

2024.08

Poster presentation

FOXO3a is a transcription activator of apoptosis-related genes [1]. In cancer cells, FOXO3a is phosphorylated by a Ser/Thr kinase AKT, and then recognized by an adopter protein 14-3-3ζ [1]. The 14-3-3ζ binding dissociates FOXO3a from DNA, which suppresses the transcriptional activity [1, 2]. Consequently, apoptosis of the cancer cells is impaired, resulting in the cancer exacerbation. Although the interaction between 14-3-3ζ and FOXO3a is a potential target for anti-cancer drugs, it remains unknown how 14-3-3ζ binds to FOXO3a and dissociates it from DNA. To reveal this mechanism, we investigated the interaction FOXO3a with DNA and 14-3-3ζ. We prepared human 14-3-3ζ as a functional dimer, and human FOXO3a residues 1-284 containing a DNA binding domain (DBD) as well as two 14-3-3ζ-binding motifs, Np and Cp, each of which possess a phosphorylation site, T32 and S253, respectively. Di-phosphorylated form of FOXO3a (dpFOXO3a) was prepared by phosphorylation using AKT. Isothermal titration calorimetry (ITC) analyses showed that the dissociation constant (Kd) for the dpFOXO3a-14-3-3ζ binding was 59 nM and that for the dpFOXO3a-DNA binding was 131 nM. Although their Kd values differed only 2-fold, fluorescence-detection size exclusion chromatography (FSEC) indicated that 95% of DNA bound to dpFOXO3a was substituted by 14-3-3ζ, when 1 equivalent of 14-3-3ζ dimer was added. NMR analyses demonstrated that, while 14-3-3ζ mainly interacts with Np and Cp of dpFOXO3a, it directly interacts with the DNA-binding surface on DBD of dpFOXO3a, indicating that the replacement of DNA with 14-3-3ζ is the direct competition. However, the binding affinity of 14-3-3ζ for the isolated DBD is 700-fold weaker than that of DNA, suggesting that the tethering via Np and Cp should plays an important role in the replacement of DNA. FSEC analyses with mono-phosphorylated form of the FOXO3a mutants demonstrated that 14-3-3ζ binding to N-pFOXO3a (S253A) – DNA complex did not dissociate DNA, whereas 14-3-3ζ binding to C-pFOXO3a (T32A) – DNA complex dissociates 42% of DNA from C-pFOXO3a (T32A). These results together with the FSEC results of dpFOXO3a indicated that the 14-3-3ζ binding to Cp is more necessary to dissociate DNA, and both Np and Cp are required for the complete DNA dissociation. Detailed analysis of the NMR spectra revealed that the DBD of dpFOXO3a bound and traveled to each protomer of the 14-3-3ζ dimer. These results suggest that efficient substitution of DNA with14-3-3ζ occurs by their competition on the DBD, in which direct but weak binding of 14-3-3ζ to the DBD is significantly enhanced by increase in the accessibility and the local concentration of 14-3-3ζ around the DBD through tethering via the Np and Cp.

INT-767の作用機序に基づいた胆汁酸誘導体による新規抗HBV薬の開発

奥村彰規, 荒井潤, 池田和由, 山下ユキコ, 井口裕介, 横川真梨子, 梅澤一夫, 宇根瑞穂, 大澤匡範, 伊藤清顕

第110回日本消化器病学会総会,

2024.05

Symposium, workshop panel (public)

Structural insights into the inhibitory mechanism of a transcription factor FOXO3a by 14-3-3ζ

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

第46回分子生物学会年会 (神戸) ,

2023.12

Poster presentation, 日本分子生物学会

Structural mechanism for the acceleration of the Caf1-dependent deadenylation of mRNA by BTG2

Nao Kataoka, Mariko Yokogawa, Yuichiro Ishii, Erika Shiro, Hiroka Takashima, Ryoichi Sawazaki, Takeru Sagae, Koichi Ogami, Shin-ichi Hoshino, Masanori Osawa

第46回分子生物学会年会 (神戸) ,

2023.12

Poster presentation, 日本分子生物学会

タンパク質-タンパク質相互作用をターゲットとした感染阻害大環状物質の探索と構造活性相関解析

米澤朋起, 清水祐吾, 池田和由, 山本雄一朗, 野口耕司, 酒井祥太, 深澤征義, 浅見仁太, 清水敏之, 大戸梅治, 横川真梨子, 大澤匡範

第51回構造活性相関シンポジウム (東京) ,

2023.11

Oral presentation (general), 日本薬学会構造活性相関部会

Inhibitory mechanism of transcription factor FOXO3a by 14-3-3ζ

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

第62回NMR討論会 (横須賀) ,

2023.11

Oral presentation (general), 日本核磁気共鳴学会

NMR-based drug discovery of the entry inhibitor of SARS-CoV-2 that directly binds to hACE2

Mariko Yokogawa, Mahoro Horiuchi, Shunki Kaneichi, Taiga Otake, Tomoki Yonezawa, Yugo Shimizu, Kazuyoshi Ikeda, Yuichiro Yamamoto, Shota Sakai, Kohji Noguchi, Masayoshi Fukasawa, Masanori Osawa

第62回NMR討論会 (横須賀) ,

2023.11

Poster presentation, 日本核磁気共鳴学会

NMR-based drug discovery of the entry inhibitor of SARS-CoV-2 that directly binds to spike protein of SARS-CoV-2

Shunki Kaneichi, Mariko Yokogawa, Mahoro Horiuchi, Taiga Otake, Tomoki Yonezawa, Yugo Shimizu, Kazuyoshi Ikeda, Yuichiro Yamamoto, Shota Sakai, Kohji Noguchi, Masayoshi Fukasawa, Masanori Osawa

第62回NMR討論会 (横須賀) ,

2023.11

Poster presentation, 日本核磁気共鳴学会

Structural basis for a novel PPI inhibitor – Keap1 binding obtained by NMR interaction analyses

Koujin Kojima, Hanako Ishida, Ayaka Harada, Tomoki Yonezawa, Yugo Shimizu, Kazuyoshi Ikeda, Mariko Yokogawa, Masanori Osawa

第62回NMR討論会 (横須賀) ,

2023.11

Poster presentation, 日本核磁気共鳴学会

Discovery of the middle-sized compounds inhibiting the SARS-CoV-2 viral entry, using in silico approach and NMR analysis

Mariko YOKOGAWA, Mahoro HORIUCHI, Shunki KANEICHI, Taiga OTAKE, Tomoki YONEZAWA, Yugo SHIMIZU, Kazuyoshi IKEDA, Yuichiro YAMAMOTO, Shota SAKAI, Kohji NOGUCHI, Masayoshi FUKASAWA, Masanori OSAWA

CBI学会2023年大会 (船堀東京) ,

2023.10

Poster presentation, CBI学会

Structural basis of the inhibition of the Keap1-Nrf2 interaction by novel PPI inhibitors discovered by machine learning

Kojima Koujin, Ishida Hanako, Harada Ayaka, Yonezawa Tomoki, Shimizu Yugo, Ikeda Kazuyoshi, Yokogawa Mariko, Osawa Masanori

CBI学会2023年大会 (船堀東京) ,

2023.10

Poster presentation, CBI学会

ALSの原因となるTDP-43の沈殿形成を抑制するRNAの探索

西田優理華, 西村錬, 横川真梨子, 坂上史佳, 横田隆徳, 大澤匡範

第６７回日本薬学会関東支部大会 (東京) ,

2023.09