Development of Fluorogenic Substrates of α-<scp>l</scp>-Fucosidase Useful for Inhibitor Screening and Gene-expression Profiling

Miura, Kazuki; Tsukagoshi, Takumi; Hirano, Tetsufumi; Nishio, Takeshi; Hakamata, Wataru

doi:10.1021/acsmedchemlett.9b00259

Cited by 12 publications

(20 citation statements)

References 27 publications

(53 reference statements)

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“…Such a finding is consistent with previous binding studies involving α-fucopyranosides. 45,46 Moreover, QM-NHαfuc proved essentially non-toxic in both control and senescent cells even at concentrations up to 100 μM (Fig. S11 † ).…”

Section: Resultsmentioning

confidence: 97%

Harnessing α-l-fucosidase for in vivo cellular senescence imaging

Koo

Won

et al. 2021

Chem. Sci.

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Section: Resultsmentioning

confidence: 97%

Harnessing α-l-fucosidase for in vivo cellular senescence imaging

Koo

Won

et al. 2021

Chem. Sci.

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“…Inhibitor Screening The targeted Golgi β-galactosidase activity was previously observed in three human cell lines, HeLa cells, HT-1080 cells, and SK-N-SH cells, using QMC platform-based fluorogenic substrates. 12) To construct the cellbased inhibitor HTS system in a 96-well format to identify inhibitors, we used HeLa cells and our developed fluorogenic substrate 1 incorporating 2-methyl TokyoGreen (2MeTG) as the fluorophore. Substrate 1 has an acetyl modification that provides cell-membrane permeability.…”

Section: Resultsmentioning

confidence: 99%

“…9) Previously, we developed fluorogenic substrates for a β-galactosidase based on the quinone methide cleavage (QMC)-substrate design platform. [10][11][12][13][14] and discovered unreported β-galactosidase activity in the Golgi apparatus in three human cell lines. 12) This newly discovered β-galactosidase activity is entirely different from that of GLB1 isoform 1 and galactocerebrosidase; however, we could not identify the protein responsible for this activity.…”

Section: Introductionmentioning

confidence: 99%

“…[10][11][12][13][14] and discovered unreported β-galactosidase activity in the Golgi apparatus in three human cell lines. 12) This newly discovered β-galactosidase activity is entirely different from that of GLB1 isoform 1 and galactocerebrosidase; however, we could not identify the protein responsible for this activity. To identify the responsible protein, we focused on the protein-maturation processes of GLB1 isoform 1 and galactocerebrosidase.…”

Section: Introductionmentioning

confidence: 99%

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Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase

Miura

Onodera

Takagi

et al. 2020

CHEMICAL & PHARMACEUTICAL BULLETIN

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The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit β-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its β-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated β-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel β-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi β-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi β-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against β-galactosidase, with an IC 50 value of 14.8 µM and competitive inhibition with K i value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi β-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.

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“…We first observed that, the red fluorescence of To demonstrate the scope of the AIE-based glycocluster for glycosidase sensing at the cellular level, we further used TD-Fuc6 for probing the endogenous AFU activity in 293T (human embryonic kidneys) cells. 68 Cells pre-treated with a known AFU inhibitor (1deoxyfuconojirimycin) 69 were used as control. To our delight, an intensive fluorescence emission corresponding to the residual AIEgen of TD-Fuc6 after removal of the fucosyl epitopes was observed in the cytoplasm of 293T cells (Fig.…”

Section: Fluorescence Imaging Of Glycosidases In Cells With the Hexavalent Glycoclustersmentioning

confidence: 99%

A General Strategy to the Intracellular Sensing of Glycosidases using AIE-Based Glycoclusters

Dong

Zhang

Han

et al. 2021

Preprint

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Glycosidases, which are the enzymes responsible for removal of residual monosaccharides from glycoconjugates, are implicated in a myriad of biological and pathological events. The ability to detect sensitively the activity and spatiotemporal distribution of glycosidases in cells may facilitate the study of glycobiology, and add useful diagnostic tools. However, the currently developed fluorogenic probes for glycosidases are generally based on glycosylation of a phenol group of a donor-acceptor type fluorogen. This general molecular scaffold has potential drawbacks in terms of substrate scope, sensitivity because of aggregation-caused quenching (ACQ), and the inability for long-term cell tracking. Here, we developed glycoclusters characterized by aggregation-induced emission (AIE) properties as a general platform for the sensing of a variety of glycosidases. To overcome the low chemical reactivity during phenol glycosylation, we synthesized an AIE-based dye composed of tetraphenylethylene conjugated with dicyanomethylene-4H-pyran (TPE-DCM) with a red fluorescence emission. Subsequently, a pair of dendritic linkages was introduced to both sides of the fluorophore, to which six copies of different monosaccharides (D-glucose, D-galactose and L-fucose) were conjugated through azide-alkyne click chemistry. The resulting AIE-active glycoclusters were shown to be capable of (1) fluorogenic sensing of a diverse range of glycosidases including β-D-galactosidase, β-D-glucosidase and α-L-fucosidase through the AIE mechanism, (2) fluorescence imaging of the endogenous glycosidase activities in healthy and cancer cells, and during cell senescence, and (3) glycosidase-activated, long-term imaging of cells. The present study provides a general strategy to the functional imaging of glycosidase activities through the multivalent display of sugar epitopes of interest onto AIE-active dyes.

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Development of Fluorogenic Substrates of α-l-Fucosidase Useful for Inhibitor Screening and Gene-expression Profiling

Cited by 12 publications

References 27 publications

Harnessing α-l-fucosidase for in vivo cellular senescence imaging

Harnessing α-l-fucosidase for in vivo cellular senescence imaging

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase

A General Strategy to the Intracellular Sensing of Glycosidases using AIE-Based Glycoclusters

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