Activity-Based Imaging of Acid Ceramidase in Living Cells

Yadira F. Ordóñez; José Luís Abad; Mazen Aseeri; Josefina Casas; Virginie Garcia; Mireia Casasampere; Edward H. Schuchman; Thierry Levade; Antonio Delgado; Gemma Triola; Gemma Fabrias

doi:10.1021/jacs.8b11687

Activity-Based Imaging of Acid Ceramidase in Living Cells

Yadira F. Ordóñez, José Luís Abad, Mazen Aseeri, Josefina Casas, Virginie Garcia, Mireia Casasampere, Edward H. Schuchman, Thierry Levade, Antonio Delgado, Gemma Triola, Gemma Fabrias

Genetics and Genomic Sciences

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

Original language	English
Pages (from-to)	7736-7742
Number of pages	7
Journal	Journal of the American Chemical Society
Volume	141
Issue number	19
DOIs	https://doi.org/10.1021/jacs.8b11687
State	Published - 15 May 2019

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title = "Activity-Based Imaging of Acid Ceramidase in Living Cells",

abstract = "Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.",

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T1 - Activity-Based Imaging of Acid Ceramidase in Living Cells

AU - Ordóñez, Yadira F.

AU - Abad, José Luís

AU - Aseeri, Mazen

AU - Casas, Josefina

AU - Garcia, Virginie

AU - Casasampere, Mireia

AU - Schuchman, Edward H.

AU - Levade, Thierry

AU - Delgado, Antonio

AU - Triola, Gemma

AU - Fabrias, Gemma

PY - 2019/5/15

Y1 - 2019/5/15

N2 - Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

AB - Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

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Activity-Based Imaging of Acid Ceramidase in Living Cells

Abstract

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