FRET probes for measuring sphingolipid metabolizing enzyme activity

Mohamed, Zainelabdeen H.; Rhein, Cosima; Saied, Essa M.; Kornhuber, Johannes

doi:10.1016/j.chemphyslip.2018.09.014

Cited by 21 publications

(13 citation statements)

References 55 publications

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“…The effect of phosphocholine which is a biologically important phosphate species and produced e.g. by enzymatic cleavage of sphingophospholipids,, was also studied, but the change of the luminescence intensity was comparable to AMP and not significant. The influence of citrate as a typical carboxy anion was also assessed (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Five‐, Four‐ and Three‐Dentate Europium Chelates for Anion Sensing and Their Applicability to Enzymatic Dephosphorylation Reactions

2018

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The design of anion-sensitive probes with sufficient sensitivity and selectivity is a demanding task in analytical sciences and chemical sensor technology. The reversible binding of anions to lanthanide centers is a possible approach for the development of molecular anion sensors, as reversibility is a prerequisite for continuous sensing and monitoring of enzymatic reactions. Some anion species lead to a strong increase of luminescence intensities and lifetimes by the replacement of luminescence quenching water molecules, though the selectivity of the luminescence response is still a major problem. We synthesized a series of positively charged pyridyl-based multidentate europium complexes (five-, four-and three dentate) including sensitizing chromophores and studied their luminescence intensity and lifetime responses to different polyphosphates, pyrophosphate, phosphate anions, and carboxyanions. The results revealed that the number and symmetry of the binding sites have a significant impact on the response. The five-dentate complex was used for the real-time monitoring of the activity of the ATP hydrolyzing enzyme apyrase.[a] Z.

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Section: Resultsmentioning

confidence: 99%

Five‐, Four‐ and Three‐Dentate Europium Chelates for Anion Sensing and Their Applicability to Enzymatic Dephosphorylation Reactions

2018

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“…Numerous reports suggest that inhibition of ASM could be a promising strategy for treating a variety of diseases like acute lung injury, major depression or metastasis of melanoma . However, despite its potential as a drug target, no potent drug‐like inhibitors are known so far …”

Section: Methodsmentioning

confidence: 99%

“…FRET probes, in contrast to quenched probes, turn‐on or turn‐off probes, offer the possibility of ratio measurements. The quotient of both, FRET donor and FRET acceptor fluorescence intensities reflects the degree of cleavage of the probe, independent of its concentration . In a previous work we have already synthesized a FRET substrate for the downstream enzyme acid ceramidase.…”

Section: Methodsmentioning

confidence: 99%

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A Novel Visible Range FRET Probe for Monitoring Acid Sphingomyelinase Activity in Living Cells

Kappe

Mohamed

Naser

et al. 2020

Chemistry A European J

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Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.Scheme1.Synthesis of probes 4 and 5.Reagents and conditions:a )H 4 N 2 ·H 2 O, MeOH,5 .5 h, r.t.,84%;b )5(6)-carboxyfluorescein, N-hydroxysuccinimide, DCC, CH 2 Cl 2 ,4h, r.t.,then NEt 3 ,DMF,2 0h,r .t.,6 6%;c )4m HCl dioxane/iso-PrOH,4h, 70 8C, then 1.3 equiv. BODIPY TR-X SE, NEt 3 ,pyridine/DMF,10h,r .t.,59% over 2s teps. d) 4 m HCl dioxane/iso-PrOH, 3.5 h, 70 8C, then 1.4 equiv.C 11 H 23 COOSu, NEt 3 ,iso-PrOH/THF,30h,r .t.,78% over2steps or 1.2 equiv.C 15 H 32 COCl, DIPEA, pyridine/CH 2 Cl 2 ,5 .5 h, 0 8C, 12 %o ver 2s teps.

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“…All enzyme assays were technically and experimentally duplicated. A Förster resonance energy transfer (FRET)-based assay 36 was performed using 20 µM substrate ES.173.cds, 36 50 ng of nCDase, 75 mM NaCl, 15 mM sodium phosphate (pH 7.4), and 0.3% Triton X-100 for 3 h in 96 black well plates. The excitation wavelength was 347 nm, the emission was measured at 430 nm and 530 nm, and a ratio of 430/530 nm was generated.…”

Section: Secondary and Tertiary Ncdase Assaysmentioning

confidence: 99%

Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening

et al. 2021

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There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC50 and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).

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FRET probes for measuring sphingolipid metabolizing enzyme activity

Cited by 21 publications

References 55 publications

Five‐, Four‐ and Three‐Dentate Europium Chelates for Anion Sensing and Their Applicability to Enzymatic Dephosphorylation Reactions

Five‐, Four‐ and Three‐Dentate Europium Chelates for Anion Sensing and Their Applicability to Enzymatic Dephosphorylation Reactions

A Novel Visible Range FRET Probe for Monitoring Acid Sphingomyelinase Activity in Living Cells

Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening

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