Daisuke Asanuma
University of Tokyo
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Featured researches published by Daisuke Asanuma.
Science Translational Medicine | 2011
Yasuteru Urano; Masayo Sakabe; Nobuyuki Kosaka; Mikako Ogawa; Makoto Mitsunaga; Daisuke Asanuma; Mako Kamiya; Matthew Young; Tetsuo Nagano; Peter L. Choyke; Hisataka Kobayashi
A spirocyclic-caged, small-molecule imaging probe fluoresces upon cleavage by a cancer-specific enzyme and can be used during surgical or endoscopic tumor removal procedures. No Tumor Left Behind Although quick action with spray paint usually conjures images of a schoolboy prank, researchers now show that spray painting of tiny tumors might save lives by illuminating these troublemakers that are often overlooked by the naked eye. Ovarian cancer is a deadly gynecological disease, considering its propensity for invading the peritoneal cavity and depositing tumors throughout. Surgeons can miss these disseminated tumors during surgical removal of cancerous lesions, owing to their small size (~1 mm) and unclear borders. To help surgeons visualize and eliminate these clandestine killers, Urano et al. have developed a small-molecule aminopeptidase probe that fluoresces upon contact with cancer cells. The probe—γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG)—is intramolecularly caged, so that it is quenched (nonfluorescent) in its “off” state. When the probe encounters cancer cells, which overexpress the enzyme γ-glutamyltranspeptidase (GGT), the gGlu is cleaved, simultaneously turning “on” the fluorescent HMRG. Urano and colleagues first tested the probe in 11 human ovarian cancer cell lines in vitro and observed rapid fluorescence within 10 min after addition of the imaging agent to the cell cultures. They next moved into several mouse models of disseminated human peritoneal ovarian cancer, using a spray formulation of the probe that allowed the researchers to topically apply the probe during surgery or endoscopy. Within 1 min of spraying the tumors, gGlu-HMRG was enzymatically cleaved, revealing a bright fluorescent region of the peritoneal cavity in which the cancerous lesions were located. These small nodules were quickly and completely removed from living animals with forceps, demonstrating the power of rapid fluorescence-guided tumor resection. This gGlu-based fluorescent probe as well as several other aminopeptidase–based reagents identified by the authors could help surgeons to track down tiny tumors dispersed throughout body cavities, ensuring that no residual tumor is left behind. Complete obliteration of disseminated tumors should improve cancer outcomes after surgery. The ability of the unaided human eye to detect small cancer foci or accurate borders between cancer and normal tissue during surgery or endoscopy is limited. Fluorescent probes are useful for enhancing visualization of small tumors but are typically limited by either high background signal or the requirement for administration hours to days before use. We synthesized a rapidly activatable, cancer-selective fluorescence imaging probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), with intramolecular spirocyclic caging for complete quenching. Activation occurs by rapid one-step cleavage of glutamate with γ-glutamyltranspeptidase (GGT), which is not expressed in normal tissue, but is overexpressed on the cell membrane of various cancer cells, thus leading to complete uncaging and dequenching of the fluorescence probe. In vitro activation of gGlu-HMRG was evident in 11 human ovarian cancer cell lines tested. In vivo in mouse models of disseminated human peritoneal ovarian cancer, activation of gGlu-HMRG occurred within 1 min of topically spraying the tumor, creating high signal contrast between the tumor and the background. The gGlu-HMRG probe is practical for clinical application during surgical or endoscopic procedures because of its rapid and strong activation upon contact with GGT on the surface of cancer cells.
Journal of the American Chemical Society | 2011
Mako Kamiya; Daisuke Asanuma; Erina Kuranaga; Asuka Takeishi; Masayo Sakabe; Masayuki Miura; Tetsuo Nagano; Yasuteru Urano
We identified a rhodol bearing a hydroxymethyl group (HMDER) as a suitable scaffold for designing fluorescence probes for various hydrolases. HMDER shows strong fluorescence at physiological pH, but phenolic O-alkylation of HMDER results in a strong preference for the spirocyclic form, which has weak fluorescence. As a proof of concept, we utilized this finding to develop a new fluorescence probe for β-galactosidase. This probe has favorable characteristics for imaging in biological samples: it has good cellular permeability, and its hydrolysis product is well-retained intracellularly. It could rapidly and clearly visualize β-galactosidase activity in cultured cells and in Drosophila melanogaster tissue, which has rarely been achieved with previously reported fluorescence probes.
Journal of the American Chemical Society | 2013
Masayo Sakabe; Daisuke Asanuma; Mako Kamiya; Ryu J. Iwatate; Kenjiro Hanaoka; Takuya Terai; Tetsuo Nagano; Yasuteru Urano
We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations.
Nature Communications | 2015
Daisuke Asanuma; Masayo Sakabe; Mako Kamiya; Kyoko Yamamoto; Jun Hiratake; Mikako Ogawa; Nobuyuki Kosaka; Peter L. Choyke; Tetsuo Nagano; Hisataka Kobayashi; Yasuteru Urano
Fluorescence-guided diagnostics is one of the most promising approaches for facile detection of cancer in situ. Here we focus on β-galactosidase, which is overexpressed in primary ovarian cancers, as a molecular target for visualizing peritoneal metastases from ovarian cancers. As existing fluorescence probes are unsuitable, we have designed membrane-permeable HMRef-βGal, in which the optimized intramolecular spirocyclic function affords >1,400-fold fluorescence enhancement on activation. We confirm that HMRef-βGal sensitively detects intracellular β-galactosidase activity in several ovarian cancer lines. In vivo, this probe visualizes metastases as small as <1 mm in diameter in seven mouse models of disseminated human peritoneal ovarian cancer (SHIN3, SKOV3, OVK18, OVCAR3, OVCAR4, OVCAR5 and OVCAR8). Because of its high brightness, real-time detection of metastases with the naked eye is possible. Endoscopic fluorescence detection of metastases is also demonstrated. The results clearly indicate preclinical potential value of the probe for fluorescence-guided diagnosis of peritoneal metastases from ovarian cancers.
Gut | 2013
Makoto Mitsunaga; Nobuyuki Kosaka; Peter L. Choyke; Matthew R. Young; Christopher R. Dextras; Shakir M. Saud; Nancy H. Colburn; Masayo Sakabe; Tetsuo Nagano; Daisuke Asanuma; Yasuteru Urano; Hisataka Kobayashi
Objectives Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer. Methods Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry. A mouse model (azoxymethane/dextran sulphate sodium) of CAC was used and mice were examined with white light and fluorescence colonoscopy before and after topical gGlu-HMRG administration. Results Expression of GGT, although variable, was higher in human colon cancer cells than normal human colon cells. Using fluorescence colonoscopy in mice, gGlu-HMRG fluorescent lesions were detected 5 min after topical administration and fluorescence persisted for at least 30 min. Fluorescence guided biopsy revealed all fluorescent lesions that contained cancer or dysplasia (n=16), whereas three out of 12 non-fluorescent lesions contained low grade dysplasia and others did not contain neoplastic histology. Microscopic inflammatory infiltration also had variable fluorescence but in general was much lower (∼10-fold) in signal than cancer. Repeat fluorescence endoscopy allowed individual tumours to be monitored. Conclusion These results suggest that gGlu-HMRG can improve endoscopic detection of CAC with a higher target to background ratio than conventional white light colonoscopy. This could be of benefit to patients with long-standing colitis who must undergo repeated screening colonoscopies.
Angewandte Chemie | 2014
Daisuke Asanuma; Yousuke Takaoka; Shigeyuki Namiki; Kenji Takikawa; Mako Kamiya; Tetsuo Nagano; Yasuteru Urano; Kenzo Hirose
Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.
Nature Communications | 2014
Rikiya Watanabe; Naoki Soga; Daishi Fujita; Kazuhito V. Tabata; Lisa Yamauchi; Soo Hyeon Kim; Daisuke Asanuma; Mako Kamiya; Yasuteru Urano; Hiroaki Suga; Hiroyuki Noji
Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function.
Nature Neuroscience | 2018
Hirokazu Sakamoto; Tetsuroh Ariyoshi; Naoya Kimpara; Kohtaroh Sugao; Isamu Taiko; Kenji Takikawa; Daisuke Asanuma; Shigeyuki Namiki; Kenzo Hirose
The weight of synaptic connections, which is controlled not only postsynaptically but also presynaptically, is a key determinant in neuronal network dynamics. The mechanisms controlling synaptic weight, especially on the presynaptic side, remain elusive. Using single-synapse imaging of the neurotransmitter glutamate combined with super-resolution imaging of presynaptic proteins, we identify a presynaptic mechanism for setting weight in central glutamatergic synapses. In the presynaptic terminal, Munc13-1 molecules form multiple and discrete supramolecular self-assemblies that serve as independent vesicular release sites by recruiting syntaxin-1, a soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein essential for synaptic vesicle exocytosis. The multiplicity of these Munc13-1 assemblies affords multiple stable states conferring presynaptic weight, potentially encoding several bits of information at individual synapses. Supramolecular assembling enables a stable synaptic weight, which confers robustness of synaptic computation on neuronal circuits and may be a general mechanism by which biological processes operate despite the presence of molecular noise.The authors show that Munc13-1 molecules form multiple supramolecular self-assemblies that serve as vesicular release sites. Having multiple Munc13-1 assemblies affords a stable synaptic weight, which confers robustness of synaptic computation.
Angewandte Chemie | 2014
Kenji Takikawa; Daisuke Asanuma; Shigeyuki Namiki; Hirokazu Sakamoto; Tetsuro Ariyoshi; Naoya Kimpara; Kenzo Hirose
Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high-throughput screening system, designated hybrid-type fluorescence indicator development (HyFInD), to identify optimal position-specific fluorophore labeling in hybrid-type sensors consisting of combinations of ligand-binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine-reactive fluorescence probes with a set of cysteine-scanning mutants of the 274 amino acid S1S2 domain of AMPA-type glutamate receptor GluA2 subunit. HyFInD identified a glutamate-responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400 %, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse-to-synapse heterogeneity of short-term plasticity.
Bioconjugate Chemistry | 2016
Hiroyuki Matsuzaki; Mako Kamiya; Ryu J. Iwatate; Daisuke Asanuma; Toshiaki Watanabe; Yasuteru Urano
Precise tumor diagnosis and evaluation of disease extent are crucial for treatment of solid cancers. In order to complement the limited ability of the unaided human eye to discriminate tumor tissue and normal tissue, we have developed a series of fluorescence probes activatable specifically in cancer tissues. Here, we describe the design, synthesis, and application of a new fluorescence probe targeting hexosaminidase (HMRef-βGlcNAc), which is located in lysosomes and is overexpressed in several carcinomas, including colorectal cancer. This probe could sensitively detect intracellular hexosaminidase activity in human colorectal cancer cell lines, and could visualize tiny metastatic nodules (smaller than 1 mm) in a mouse model of disseminated human peritoneal colorectal cancer (HCT116). In human colorectal cancer specimens obtained at surgery, the probe showed high tumor sensitivity/specificity, together with a high tumor-to-normal signal ratio. HMRef-βGlcNAc is a promising candidate for clinical application during surgical or endoscopic procedures to treat colorectal cancer.