Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells

Yanagi, Kouichi; Komatsu, Toshimitsu; Ogihara, Shinji; Okabe, Takayoshi; Kojima, Hirotatsu; Nagano, Tetsuo; Ueno, Takahiro; Hanaoka, Kenjiro; Urano, Yasuteru

doi:10.1016/j.celrep.2021.109311

Cited by 5 publications

(9 citation statements)

References 43 publications

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“…The key factors for developing metabolic pathway-oriented screenings are: (1) selection of proper input and output metabolites, and (2) development of fluorogenic probes to detect the target metabolites exclusively in the extracellular space. Selective detection of output metabolites is realizable by coupled assays using NAD(P)H 20 . For live cell-based coupled assays, the assay system needs to be separated from the intracellular space that contain high concentration of NAD(P)H, so the probe should be retained in extracellular media throughout the assay.…”

Section: Resultsmentioning

confidence: 99%

“…We recently established the concept of live cell-based coupled assays for monitoring targeted metabolic pathways for live cellbased HTS 20,21 . In this metabolic pathway-oriented screening methodology, we specify the input and output metabolites, and the in situ detection of the extracellular metabolite (output) that is generated and released from cells can be used as a readout of the activity of the targeted metabolic pathways.…”

mentioning

confidence: 99%

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Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells

Yanagi

Komatsu²,

Fujikawa³

et al. 2023

Commun Chem

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Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate d-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses d-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells

Yanagi

Komatsu²,

Fujikawa³

et al. 2023

Commun Chem

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“…Importantly, real‐time metabolites levels can also be monitored in live cells using fluorescence resonance energy transfer (FRET)‐based biosensor techniques 209,210 . Yanagi et al monitored the real‐time activity of metabolic enzymes by using fluorescent substrates in cancer cells treated with various drugs 211 . These new imaging methods can provide insights into the interplay between the cell cycle and metabolism in living cells.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

“…209,210 Yanagi et al monitored the real-time activity of metabolic enzymes by using fluorescent substrates in cancer cells treated with various drugs. 211 These new imaging methods can provide insights into the interplay between the cell cycle and metabolism in living cells.…”

Section: Cell Cycle Inhibitors Impact Beyond the Cell Cyclementioning

confidence: 99%

Out of the cycle: Impact of cell cycle aberrations on cancer metabolism and metastasis

Cheung

Hui

Wong

et al. 2022

Intl Journal of Cancer

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The use of cell cycle inhibitors has necessitated a better understanding of the cell cycle in tumor biology to optimize the therapeutic approach. Cell cycle aberrations are common in cancers, and it is increasingly acknowledged that these aberrations exert oncogenic effects beyond the cell cycle. Multiple facets such as cancer metabolism, immunity and metastasis are also affected, all of which are beyond the effect of cell proliferation alone. This review comprehensively summarized the important recent findings and advances in these interrelated processes. In cancer metabolism, cell cycle regulators can modulate various pathways in aerobic glycolysis, glucose uptake and gluconeogenesis, mainly through transcriptional regulation and kinase activities. Amino acid metabolism is also regulated through cell cycle progression. On cancer metastasis, metabolic plasticity, immune evasion, tumor microenvironment adaptation and metastatic site colonization are intricately related to the cell cycle, with distinct regulatory mechanisms at each step of invasion and dissemination. Throughout the synthesis of current understanding, knowledge gaps and limitations in the literature are also highlighted, as are new therapeutic approaches such as combinational therapy and challenges in tackling emerging targeted therapy resistance. A greater understanding of how the cell cycle modulates diverse aspects of cancer biology can hopefully shed light on identifying new molecular targets by harnessing the vast potential of the cell cycle.

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“…However, where the biological systems are concerned, the greater complexity and redundancy have made it difficult to conduct complete individual studies of natural biological molecules and the pathways they control. Therefore, attempts have been made to design small-molecule tools or chemical probes to study the roles of target molecules [ 3 , 4 , 5 ]. Compared with biomolecule (such as protein, peptide, and nucleic acids)-based imaging tools, the features of small-molecule fluorescent probes are obvious: low molecular weight, simple and non-natural structure.…”

Section: Introductionmentioning

confidence: 99%

Development of Small-Molecule Fluorescent Probes Targeting Enzymes

Xie

Yang

et al. 2022

Molecules

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As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.

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Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells

Abstract: Highlights d Live-cell-based HTS systems for specific metabolic pathways are established d 6-BIO and regorafenib inhibit glycolysis and glutaminolysis of tumor cells d Regorafenib reduces cellular glutaminase activity by modifying its PTM state

Cited by 5 publications

References 43 publications

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells

Out of the cycle: Impact of cell cycle aberrations on cancer metabolism and metastasis

Development of Small-Molecule Fluorescent Probes Targeting Enzymes

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