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Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783–92. ©2018 AACR.

Plots of tumor growth delay vs HF10; pO2; lac/pyr

Supplemental materials and methods

Median pO2 and HF10 of individual PDAC tumors in EPR oxygen imaging study.

Supplemental Figures

Supplemental Figure Legends

Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect.Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro. LDH activity was assessed in tumor homogenates.Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro. LDH activity correlated to FX-11 sensitivity.Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137–48. ©2018 AACR.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has a great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we for the first time report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Nuclear hyperpolarization has emerged as a method to dramatically enhance the sensitivity of NMR spectroscopy. By application of this powerful tool, small molecules with stable isotopes have been used for highly sensitive biomedical molecular imaging. The recent development of molecular probes for hyperpolarized in vivo analysis has demonstrated the ability of this technique to provide unique metabolic and physiological information. This review presents a brief introduction of hyperpolarization technology, approaches to the rational design of molecular probes for hyperpolarized analysis, and examples of molecules that have met with success in vitro or in vivo.

なし, DNP 研究会

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO

Nuclear hyperpolarization has emerged as a method to dramatically enhance the sensitivity of NMR. By applying this powerful tool, small molecules with stable isotopes have been subjected to highly sensitive biomedical molecular imaging. The recent development of molecular probes for hyperpolarized in vivo analysis has demonstrated the ability of this technique to provide unique metabolic and physiological information. This review presents a brief introduction of hyperpolarization technology, approaches to rational design of molecular probes for hyperpolarized analysis, and examples of molecules that have met with in vitro or in vivo success.

Introduction Our understanding of the mechanism of action of bioactive small molecules contributes to the research and development of new medical drugs, as well as elucidating the pathological mechanisms underlying various diseases. Researchers in this field are committed to a very ambitious goal: the discovery of novel therapeutic compounds along with their molecular targets. To achieve this goal, new methodological developments are indispensable. Areas covered This review gives an update on the advancements of phage display (PD) technology in the past decade (2011–2020) for determining the targets of the small molecule therapeutics. In particular, other than providing a brief overview of this field of research, we focus on reporting the research trends and the results solely obtained using this strategy. Expert opinion Despite the development of bioinformatics tools and artificial intelligence (AI)-mediated methods, affinity-guided information obtained experimentally are still indispensable to identify drug-protein interactions. By taking advantage of small-molecule-oriented PD methods and their improvements, the extension of the druggable proteome will be further expanded, providing new opportunities to generate small-molecule therapeutics.

Our understanding of the mechanism of action of bioactive small molecules contributes to the research and development of new medical drugs, as well as elucidating the pathological mechanisms underlying various diseases. Researchers in this field are committed to a very ambitious goal: the discovery of novel therapeutic compounds along with their molecular targets. To achieve this goal, new methodological developments are indispensable.This review gives an update on the advancements of phage display (PD) technology in the past decade (2011-2020) for determining the targets of the small molecule therapeutics. In particular, other than providing a brief overview of this field of research, we focus on reporting the research trends and the results solely obtained using this strategy.Despite the development of bioinformatics tools and artificial intelligence (AI)-mediated methods, affinity-guided information obtained experimentally are still indispensable to identify drug-protein interactions. By taking advantage of small-molecule-oriented PD methods and their improvements, the extension of the druggable proteome will be further expanded, providing new opportunities to generate small-molecule therapeutics.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1- 13 C]Gly- d 2 -NMe 2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that dramatically enhances the detection sensitivity of molecules using nuclear magnetic resonance imaging (NMR/MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging due to the lack of established design guidelines. Herein, we report Ala-[1-13C]Gly-d2-NMe2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N (APN) activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can dramatically enhance the detection sensitivity of nuclear magnetic resonance (NMR). DNP NMR has been applied to small molecules with stable isotopes and has been used to obtain metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium state, depending on the spin-lattice relaxation time (T1). This signal decay has remained a major problem associated with this technology. Therefore, DNP NMR molecular probes useful for in vivo analysis have been limited to naturally occurring small molecules that inherently show long T1. While peptides are promising targets for DNP NMR studies, because of the limitation in T1, DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Herein we propose a 13C-labeling strategy to utilize the C-terminal [1-13C]Gly-d2 residue for realizing long T1 in peptides. Structure-based T1 relaxation analysis of amino acids and peptides revealed that (1) T1 does not decrease monotonically with increasing molecular weight and (2) T1 is not significantly affected by a side chain on the neighboring amino acid residue. These findings suggest that the C-terminal [1-13C]Gly-d2 residue affords sufficiently long T1 for biological uses, even in oligopeptides, and allowed us to develop 13C-b- casomorphin-5 (Tyr-Pro-Phe-Pro-[1-13C]Gly-d2, T1 = 24 ± 4 s at 3 T in H2O) and 13C-glutathione (g-Glu-Cys-[1-13C]Gly-d2, T1 = 58 ± 3 s at 3 T in H2O) as DNP NMR probes with long T1. We succeeded in in vivo detection of enzymatic conversions of these two probes. These results demonstrate the utility of our strategy and would contribute to further expansion of the substrate scope for DNP applications.

Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can dramatically enhance the detection sensitivity of nuclear magnetic resonance (NMR). DNP NMR has been applied to small molecules with stable isotopes and has been used to obtain metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium state, depending on the spin-lattice relaxation time (T1). This signal decay has remained a major problem associated with this technology. Therefore, DNP NMR molecular probes useful for in vivo analysis have been limited to naturally occurring small molecules that inherently show long T1. While peptides are promising targets for DNP NMR studies, because of the limitation in T1, DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Herein we propose a 13C-labeling strategy to utilize the C-terminal [1-13C]Gly-d2 residue for realizing long T1 in peptides. Structure-based T1 relaxation analysis of amino acids and peptides revealed that (1) T1 does not decrease monotonically with increasing molecular weight and (2) T1 is not significantly affected by a side chain on the neighboring amino acid residue. These findings suggest that the C-terminal [1-13C]Gly-d2 residue affords sufficiently long T1 for biological uses, even in oligopeptides, and allowed us to develop 13C-β-casomorphin-5 (Tyr-Pro-Phe-Pro-[1-13C]Gly-d2, T1 = 24 ± 4 s at 3 T in H2O) and 13C-glutathione (γ-Glu-Cys-[1-13C]Gly-d2, T1 = 58 ± 3 s at 3 T in H2O) as DNP NMR probes with long T1. We succeeded in in vivo detection of enzymatic conversions of these two probes. These results demonstrate the utility of our strategy and would contribute to further expansion of the substrate scope for DNP applications.

Rapid cellular proliferation and incomplete neovascularization in solid tumors leads to spatially heterogeneous regions with relatively low oxygen concentrations, called hypoxia. Hypoxia in tumors causes significant changes in various physiological processes such as DNA repair pathways, anaerobic respiration, metabolic reprograming, unregulated angiogenesis, and the spread of cancer stem cells [Chem. Soc. Rev. 2019, 48, 771–813]. Consequently, the plasticity of these processes results in the acquisition of the malignant phenotype in tumors. Therefore, effective imaging methods that allow for the detailed analysis of hypoxia are needed to elucidate tumor pathophysiology. To date, a variety of imaging techniques for tumor hypoxia have been developed. Magnetic resonance imaging (MRI)- and positron emission tomography (PET)-based methods permit real-time monitoring and give three-dimensional (3D) information about tumor hypoxia without the limitation of imaging depth in vivo [Mol. Imaging Biol. 2010, 13, 399–410]. However, these imaging methods have poor resolution, restricting the detailed visualization of hypoxia-related biological events in cells or tissues. The imaging methods for tumor hypoxia with cellular resolution rely on tissue sectioning [Nat. Nanotechnol. 2016, 11, 724–730]. Although the reconstruction of serial sections theoretically enables 3D imaging of tumor hypoxia, it is impractical for multiple samples and difficult to obtain accurate data with the original tissue morphology. Recently, optical tissue-clearing techniques have been developed for depth-independent visualization. These techniques enable 3D imaging with fluorescent probes including genetically encoded proteins and chemical dyes in entire tissues without the preparation of tissue sections [Cell 2014, 157, 726–739]. However, the application of these techniques with conventional hypoxia probes suffers from some challenges. For example, a well-known hypoxia probe (pimonidazole) and hypoxia makers (e.g. HIF1-) need specific antibodies to be visualized [Nat. Commun. 2012, 3, 710–783]. Due to the low tissue permeability of antibodies [Mol. Cancer Ther. 2019, 18, 213–226], the existence of these probes deep inside tumors cannot be detected. Additionally, hypoxia probes lacking the ability to covalently bind to cellular components can be washed from tumors during tissue-clearing treatments. To achieve 3D and high-resolution imaging of tumor hypoxia throughout an entire tissue, we developed a fluorescent molecular probe for tumor hypoxia which is compatible with tissue-clearing. The fluorescent probe has low cytotoxicity, high fluorescence intensity in tissue-clearing solution, high tissue permeability, and the ability to covalently bind to cellular proteins only under hypoxic conditions. The designed probe was applied for the 3D imaging of spatially heterogeneous tumor hypoxia in combination with tissue-clearing., PACIFICHEM2021

Detecting activities of peptidases in vivo is an attractive challenge in chemical biology because peptidase-activities are correlated strongly with various biological phenomena and diseases. Nuclear magnetic resonance (NMR) is a powerful technique for this purpose due to its advantages in high tissue penetration and low invasiveness, allowing a molecular detection at deep area. However, the sensitivity of NMR is severely low for in vivo application. To overcome this problem, dynamic nuclear polarization (DNP) has emerged. NMR signals are dramatically enhanced by using this method. However, there are few molecular probes to detect peptidase-activity by using DNP technique to date. Herein, we have developed a practical molecular probe for DNP-NMR to detect the activity of Aminopeptidase N (APN), which is a representative peptidase, in vivo through rational molecular design. It is demonstrated that this probe has many advantages for in vivo application and enables detection of APN-activity in xenograft tumors bearing mice. Starting with this success, we developed some DNP-NMR molecular probes for detecting peptidase-activities based on chemical design., PACIFICHEM2021

なし, 第 49 回日本磁気共鳴医学会大会スタディーグループ

生物個体の表現系として現れる生命現象は、細胞、組織、臓器、個体という多階層に張り巡らされた複雑な分子ネットワークの産物である。その分子の動きを、細胞・組織のレベルを越え、臓器・個体全身の中で包括的に捉えることができれば、様々な未知未踏の発見が得られると考えられる。 近年めざましい発展を遂げた生体組織の透明化技術は、生物個体全身・全臓器における蛍光標識された分子を、１細胞解像度で包括的に定量解析するための有用なツールである1。組織透明化の分野では、標的分子のラベリングに蛍光タンパクが広く用いられるが、生体内反応の経時変化観察や内在性分子の蛍光標識には制限がある。また、蛍光色素を結合させた抗体も活用されているが、抗体は分子サイズが大きいため、再現性良く全組織に浸透させることが困難である。組織透明化の実用に際したこれらの問題点は、内在性分子の活性や局在の直接的かつ経時的な検出が可能で、分子サイズの小ささゆえ再現性良く全身の標的を蛍光標識することが期待される低分子量の「蛍光分子プローブ」を活用することで解決できる。そこで本研究では、組織透明化に応用可能な蛍光分子プローブの開発、および開発した蛍光分子プローブを用いた透明化組織の高解像度三次元イメージングの実証を目的とした。 本研究ではまず、組織透明化に応用可能な蛍光色素の検討を行った。そして、イメージング対象として生体内低酸素環境に着目し、標的を検出し結合を形成する反応部位を蛍光色素に組み込んだ蛍光分子プローブの合成を行った。さらに、培養細胞、およびモデル動物を用いて蛍光分子プローブの機能評価を行った後、蛍光ラベリングされた組織を実際に透明化し、三次元イメージングに挑戦した。本発表では、これらの成果を報告する。, 第 11 回 CSJ 化学フェスタ 2021

Hyperpolarized-nuclear magnetic resonance and imaging (HP-NMR/MRI) is a promising technique that allows direct monitoring of metabolic reactions in vitro and in vivo. Given the 13C- or 15N-labeled molecular probes whose NMR signal is amplified by the dynamic nuclear polarization (DNP) under a cryogenic magnetic field (3.35T, 1.4K), the sample is rapidly dissolved with a superheated solvent (~458 K), and then quickly reacted with enzymes or cells, or administered in animals put into the magnet. The metabolic reaction is readout onto the NMR spectra or MR image based on the >10,000-fold enhanced signals. By elucidating the extent of the metabolic reaction of the probes, early detection of disease-specific elevation of enzymatic activity, metabolic reprogramming, and alteration of their flux upon the treatment is possible. The development of various molecular probes will be a key factor to expand the utility of this approach. In this presentation, we will concisely introduce the basics of the dis-DNP, design and application of our recent new molecular probes that allow in vitro and in vivo HP-NMR/MRI., The 24th International Spin Symposium (SPIN2021)

な し, 量子生命科学会 第 3 回大会

なし, 第 49 回日本磁気共鳴医学会大会スタディーグループ

超偏極−核磁気共鳴代謝イメージングが幕を開けて 15 年以上が経過する。その間、超偏極技術は MRI とともに発展を遂げ、[1-13C]ピルビン酸を主役に脳、心臓、肝臓、膵臓、腎臓、前立腺などの機能や、がん・老化・炎症・レドックス・遺伝子変異などの関連疾患や早期治療応答を検出するための画像技術としてその可能性が示されてきた。2010 年に開始された前立腺がんを対象とする初の臨床試験を皮切りに、世界では臨床研究拠点の形成と臨床研究が進行中であるが、基礎研究についてはややもすると停滞気味の様相を呈してきている。大型装置を取り揃えた拠点形成の難しさや、装置の開発・導入・維持に高額な費用がかかるといった根本的事情に加え、2000 年代後半の市販機 HyperSenseTM の登場・事業停止から 10 年以上が経過した現在、装置の経年劣化の問題も無視できなくなりつつある。また、超偏極ピルビン酸の応用研究に底が見え始めていることや、根源的な弱点とも言える偏極寿命の短さとこれに起因するプローブ開発の制限など、その原因は多岐に亘る。特に、新しい分子プローブの創出は本技術の将来を左右しうる重要な要素であり、現状を打破しその応用範囲を拡げるためのブレイクスルーが今なお切望されている。 本講演では、超偏極−核磁気共鳴代謝イメージングについて [1-13C]ピルビン酸の応用を中心に概説し、本技術の基礎や開発動向、国内外の現状について説明しながらこれまでの約 20 年を振り返る。本技術が抱える現状の課題にまで理解を深めていただくとともに、それらの克服を目指した演者らの最近の取り組みについても紹介する。最新の分子プローブが切り拓く新たな研究展開や、本技術ならではのユニークな画像診断の可能性について触れながら、超偏極-核磁気共鳴代謝イメージングの未来予想図を描いてみたい。 Vaeggemose M. et al. Metabolites 11, 219, 2021 (Review) Kondo Y. et al. Angew Chem Int Ed Engl 60, 14779, 2021 (Review), 第 49 回日本磁気共鳴医学会大会

Serine hydroxymethyltransferase (SHMT) is an enzyme that catalyzes the reaction that converts serine to glycine. It plays an important role in one-carbon metabolism. Recently, SHMT has been shown to be associated with various diseases. Therefore, SHMT has attracted attention as a biomarker and drug target. However, the development of molecular probes responsive to SHMT has not yet been realized. This is because SHMT catalyzes an essential yet simple reaction; thus, the substrates that can be accepted into the active site of SHMT are limited. Here, we focus on the SHMT-catalyzed retro-aldol reaction rather than the canonical serine–glycine conversion and succeed in developing fluorescent and 19F NMR molecular probes. Taking advantage of the facile and direct detection of SHMT, the developed fluorescent probe is used in the high-throughput screening for human SHMT inhibitors, and two hit compounds are obtained., The enzyme serine hydroxymethyltransferase (SHMT) has been implicated in several diseases, however is hard to investigate. Here, the authors used a design strategy based on the retro-aldol-type reaction catalyzed by SHMT to develop SHMT-responsive fluorescence and 19F NMR molecular probes.

【背景】超偏極技術は、核磁気共鳴の根本的な弱点である「感度の低さ」を克服するための革新的技術であり、その信号強度を数万倍に増幅可能である。がんをはじめとする疾患の画像診断や治療応答の超早期検出法としての応用が期待されており、超偏極 [1-13C]ピルビン酸からの [1-13C]乳酸生成 (Warburg 効果) を指標に、ヒト前立腺がんの悪性度評価を目的とした臨床試験が進められている (Sci Transl Med 2013)。本研究では、PSA 陰性のヒト前立腺がん細胞株を対象に、超偏極 [1-13C]ピルビン酸核磁気共鳴分光法及び代謝イメージングを実施し、代謝能の比較や薬物治療効果との相関を調べることで、その悪性度診断や治療効果予測法としての応用性を検討した。 【方法】2 種類のヒト前立腺がん細胞株 (DU145, PC-3) を、通常培養、自発的細胞凝集を誘発するスフェロイド容器による静置培養、ヌードマウスへの接種にて各々生育させた。必要に応じて病理染色や治療試験を実施し、組織性状や感受性を調べた。市販の偏極機 (HyperSenseTM) を用いて [1-13C]ピルビン酸 (170.2 ppm) を偏極し、10-mm 径の NMR 管に入れた細胞やスフェロイドと速やかに反応させることにより、[1-13C]乳酸 (182.4 ppm) の生成度を NMR スペクトル上で観測した。また、担がんマウスをイソフルラン麻酔下で実験動物用 MRI に保定し、超偏極-[1-13C]ピルビン酸を装置外部より尾静注後、担がんからの高感度化 13C 信号を速やかに受信した。 【結果】超偏極-[1-13C]ピルビン酸の代謝反応によって生成した [1-13C]乳酸の信号の検出に成功し、がん細胞の組織形成に伴う乳酸脱水素酵素 (LDH) 活性の上昇が認められた。また、生育が早くかつ転移能の高い DU145 においてより高い乳酸生成能が確認され [Lac/Pyr 比 = 7.07 (DU145), 2.59 (PC-3)]、LDH 阻害剤である FX-11 の奏効性とも相関していることが示唆された。 【結論】超偏極技術を応用することにより、非侵襲代謝解析を指標とした PSA 陰性前立腺がんの悪性度診断や治療効果予測が可能であることを示した。 1. Scroggins B T et al. Clin Cancer Res. 2018 2. Takakusagi Y et al. Proc ISMRM 3720, 2018, 第 15 回 日本分子イメージング学会総会・学術集会

近年めざましい発展を遂げた生体組織の透明化技術により、生体内の三次元構造を高解像度で観察することが可能となった。透明化組織のイメージングには主に蛍光タンパク質が用いられるが、生体内反応の経時変化観察や、内因性分子の蛍光標識には向いていない。今回我々は、イメージング対象として生体内低酸素環境に着目し、低酸素細胞を検出する蛍光分子プローブの合成と機能評価、および組織透明化技術との適合性を検討した。, 日本化学会 第101春季年会 (2021)

ペプチドをN末端アミノ酸残基特異的に切断するアミノペプチダーゼは、タンパク質成熟や血圧制御などの多様な生命現象に関わり、特にがんや腎疾患のバイオマーカーとして知られる。本研究では、非侵襲的に生体深部の標的分子を検出できる核磁気共鳴法と、その感度を向上させる動的核偏極法に着目し、複数種のアミノペプチダーゼ活性の生体内検出を指向した超偏極分子プローブの開発を行った。, 日本化学会 第101春季年会 (2021)

なし, バイオインダストリー奨励賞受賞者企画講演会 ~生命現象の根本的解明を目指す量子生命科学について~

後に記載, 量子生命科学会第 2 回大会

核磁気共鳴法（NMR）は生体深部の標的分子を非侵襲的に検出することができる手法である。しかし、NMRは他の検出法と比較して格段に感度が低いという欠点を持つため、生体内に大量に存在する水以外の分子にNMRを応用することは依然として困難である。この欠点を克服するために、我々はNMRの感度を劇的に向上させる手法である動的核偏極法（DNP）に着目した。一方、ペプチドを配列特異的に切断するペプチダーゼは、シグナル伝達タンパク質の代謝、翻訳後修飾、細胞外マトリックスの分解といった様々な生命現象に関わっている。それ故、多くの疾患においてペプチダーゼの発現量や活性に異常が認められており、重要なバイオマーカーとして認識されている。特にアミノペプチダーゼは、がん、アルツハイマー病、高血圧症などの多様な疾患に関わることが報告されている。 ペプチドN末端の疎水性残基を特異的に切断する酵素であるアミノペプチダーゼN（APN）の活性を検出するDNP-NMR分子プローブとしてAla-NH2が報告されているが、この分子プローブは、酵素反応速度が小さく、酵素反応前後の化学シフト変化が小さいという二つの問題点により生体応用には至らなかった。最近、当研究室においてAla-NH2から構造展開することで、これら二つの問題点を改善した新たなDNP-NMR分子プローブを開発し、ヒト腫瘍担癌マウス中でAPNの活性を良好な感度で検出することに成功した。この新たな分子プローブは、i) 水溶性が高い、ii) 縦緩和時間が十分に長い、iii) 酵素反応速度が大きい、iv) 酵素反応前後の化学シフト変化が大きいというDNP-NMR分子プローブに必要な4条件を備えている。本研究では、この分子プローブのAPN認識部位を改変することで、様々なアミノペプチダーゼの活性を生体内で検出することを目的とした超偏極分子プローブの開発を行った。現在までに、ペプチドN末端の酸性残基を特異的に切断する酵素であるアミノペプチダーゼA（APA）に対するDNP-NMR分子プローブ候補の開発に成功した。さらに我々は、NMRが化学シフトによって複数の化学種を同時に分離して検出可能であることに注目し、複数のアミノペプチダーゼ活性を生体内で同時に評価することを目指している。これまでに、i)~iv)の4条件を損なうことなく、2種類の分子プローブを化学シフトによって同時に分離して検出するための分子設計を見出した。, 量子生命科学会第 2 回大会

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as "broken relics", of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

ペプチドを配列特異的に切断するペプチダーゼは、シグナル伝達タンパク質の代謝や翻訳後修飾などの多様な生命現象に関わっている。それ故、様々な疾患においてペプチダーゼの発現量や活性に異常が認められており、重要なバイオマーカーとして認識されている。一方、核磁気共鳴法（NMR）は非侵襲的な手法であり、生体深部での標的分子の検出を可能にする。NMRは感度が低いという弱点を有するが、動的核偏極法（DNP）によりNMRの感度を劇的に向上させることができる。 ペプチドN末端の疎水性残基を認識して切断する酵素であるアミノペプチダーゼN（APN）の活性を検出するDNP-NMR分子プローブとしてAla-NH2が報告されているが、この分子プローブは、i) 酵素反応速度が小さく、ii) 酵素反応前後の化学シフト変化が小さいという二つの問題点により生体応用には至らなかった。そこで、我々はAla-NH2から構造展開することで、これら二つの問題点を改善した新たなDNP-NMR分子プローブを開発した。今回、この新たな分子プローブを用いて、ヒト腫瘍担癌マウス中でAPNの活性を良好な感度で検出することに初めて成功した。さらに、この新たな分子プローブのAPN認識部位を改変することで、様々なアミノペプチダーゼの活性を生体内で検出することを目的とした超偏極分子プローブの開発を行った。, 第 10 回 CSJ 化学フェスタ 2020

本発表では、令和 2 年 8/1 より発足した QST 量子生命科学領域/量子超偏極 MRI グループについて紹介し、量子技術イノベーション戦略を踏まえて、その発足経緯や活動内容、最新のトピックスについて説明する。, 第 48 回日本磁気共鳴医学会大会 超偏極による他核MRSIの高感度計測スタディーグループ

近年注目を集めている「超偏極ー核磁気共鳴代謝イメージング」に関して、その生命科学や医療への応用展開について概説する。

The enhanced permeability and retention (EPR) effect is variable depending on nanoparticle properties and tumor/vessel conditions. Thus, intratumoral evaluations of the vasculature and nanoparticle distribution are important for predicting the therapeutic efficacy and the intractability of tumors. We aimed to develop a tumor vasculature evaluation method and high-resolution nanoparticle delivery imaging using magnetic resonance (MR) micro-imaging technology with a gadolinium (Gd)-dendron assembled liposomal contrast agent. Using the Gd-liposome and a cryogenic receiving coil, we achieved 50-μm isotropic MR angiography with clear visualization of tumor micro-vessel structure. The Gd-liposome-enhanced MR micro-imaging revealed differences in the vascular structures between Colon26- and SU-DHL6-grafted mice models. The vessel volumes and diameters measured for both tumors were significantly correlated with histological observations. The MR micro-imaging methods facilitate the evaluation of intratumoral vascularization patterns, the quantitative assessment of vascular-properties that alter tumor malignancy, particle retentivity, and the effects of treatment.