Dataset Information

Ratiometric Fluorescence Assay for Nitroreductase Activity: Locked-Flavylium Fluorophore as a NTR-Sensitive Molecular Probe.

ABSTRACT: Nitroreductases belong to a member of flavin-containing enzymes that can reduce nitroaromatic compounds to amino derivatives with NADH as an electron donor. NTR activity is known to be elevated in the cancerous environment and is considered an advantageous target in therapeutic prodrugs for the treatment of cancer. Here, we developed a ratiometric fluorescent molecule for observing NTR activity in living cells. This can provide a selective and sensitive response to NTR with a distinct increase in fluorescence ratio (FI₅₃₀/FI₆₃₀) as well as color changes. We also found a significant increase in NTR activity in cervical cancer HeLa and lung cancer A549 cells compared to non-cancerous NIH3T3. We proposed that this new ratiometric fluorescent molecule could potentially be used as a NTR-sensitive molecular probe in the field of cancer diagnosis and treatment development related to NTR activity.

SUBMITTER: Kim SJ

PROVIDER: S-EPMC7923055 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Ratiometric Fluorescence Assay for Nitroreductase Activity: Locked-Flavylium Fluorophore as a NTR-Sensitive Molecular Probe.

Kim Su Jung SJ Yoon Jung Won JW Yoon Shin A SA Lee Min Hee MH

Molecules (Basel, Switzerland) 20210219 4

Nitroreductases belong to a member of flavin-containing enzymes that can reduce nitroaromatic compounds to amino derivatives with NADH as an electron donor. NTR activity is known to be elevated in the cancerous environment and is considered an advantageous target in therapeutic prodrugs for the treatment of cancer. Here, we developed a ratiometric fluorescent molecule for observing NTR activity in living cells. This can provide a selective and sensitive response to NTR with a distinct increase i ...[more]

PMID: 33669590

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Project description:Potassium is the most abundant intracellular metal in the body, playing vital roles in regulating intracellular fluid volume, nutrient transport, and cell-to-cell communication through nerve and muscle contraction. On the other hand, aberrant alterations in K+ homeostasis contribute to a diverse array of diseases spanning cardiovascular and neurological disorders to diabetes to kidney disease to cancer. There is an unmet need for studies of K+ physiology and pathology owing to the large differences in intracellular versus extracellular K+ concentrations ([K+]intra = 150 mM, [K+]extra = 3-5 mM). With a relative dearth of methods to reliably measure dynamic changes in intracellular K+ in biological specimens that meet the dual challenges of low affinity and high selectivity for K+, particularly over Na+, currently available fluorescent K+ sensors are largely optimized with high-affinity receptors that are more amenable for extracellular K+ detection. We report the design, synthesis, and biological evaluation of Ratiometric Potassium Sensor 1 (RPS-1), a dual-fluorophore sensor that enables ratiometric fluorescence imaging of intracellular potassium in living systems. RPS-1 links a potassium-responsive fluorescent sensor fragment (PS525) with a low-affinity, high-selectivity crown ether receptor for K+ to a potassium-insensitive reference fluorophore (Coumarin 343) as an internal calibration standard through ester bonds. Upon intracellular delivery, esterase-directed cleavage splits these two dyes into separate fragments to enable ratiometric detection of K+. RPS-1 responds to K+ in aqueous buffer with high selectivity over competing metal ions and is sensitive to potassium ions at steady-state intracellular levels and can respond to decreases or increases from that basal set point. Moreover, RPS-1 was applied for comparative screening of K+ pools across a panel of different cancer cell lines, revealing elevations in basal intracellular K+ in metastatic breast cancer cell lines vs. normal breast cells. This work provides a unique chemical tool for the study of intracellular potassium dynamics and a starting point for the design of other ratiometric fluorescent sensors based on two-fluorophore approaches that do not rely on FRET or related energy transfer designs.

Dataset Information

Ratiometric Fluorescence Assay for Nitroreductase Activity: Locked-Flavylium Fluorophore as a NTR-Sensitive Molecular Probe.

Publications

Ratiometric Fluorescence Assay for Nitroreductase Activity: Locked-Flavylium Fluorophore as a NTR-Sensitive Molecular Probe.

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