Eine durch Lipid‐Wasser‐Trennung verstärkte FRET‐Sonde zur Detektion der Sauren Sphingomyelinase in lebenden Zellen

Chemical proteomics is widely applied in small‐molecule target identification. However, in general it does not identify non‐protein small‐molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image‐based morphological profiling in the cell painting assay. A compound‐induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

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Image‐Based Morphological Profiling Identifies a Lysosomotropic, Iron‐Sequestering Autophagy Inhibitor

Laraia

Garivet

Foley

et al. 2020

Angewandte Chemie

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Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases**

Healey,

Saied,

Cong

et al. 2021

Angewandte Chemie

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Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide,apro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate.Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases.H owever,d ue to their hydrophobic nature,nospecific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular,w ec hemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations.I n addition to revealing an ew paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules,o ur data lay the ground for targeting ACER3 in drug discovery efforts.

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Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

et al. 2022

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For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state nonradiatively, and are promising alternatives to fluorescent FRET acceptors. Small-molecule (dark) quenchers have been widely used as acceptors in FRET-based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small-molecule quenchers that are used in FRET-based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophorequencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

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Eine durch Lipid‐Wasser‐Trennung verstärkte FRET‐Sonde zur Detektion der Sauren Sphingomyelinase in lebenden Zellen

Cited by 8 publications

References 36 publications

Image‐Based Morphological Profiling Identifies a Lysosomotropic, Iron‐Sequestering Autophagy Inhibitor

Image‐Based Morphological Profiling Identifies a Lysosomotropic, Iron‐Sequestering Autophagy Inhibitor

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases**

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

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