Click and count: specific detection of acid ceramidase activity in live cells

Casasampere, Mireia; Izquierdo, E.; Casas, Josefina; Abad, José Luís; Liu, Xiao; Xu, Ruijuan; Mao, Cungui; Chang, Young‐Tae; Delgado, Antonio; Fabriás, Gemma

doi:10.1039/d0sc03166f

Cited by 9 publications

(17 citation statements)

References 36 publications

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“…Moreover, some BODIPY-4 vesicles were bigger in size compared to the lysosomes or endosomes indicating they were clearly a different type of organelles. This pattern is in disagreement with the subcellular distribution of a similar BODIPY-labeled 1-deoxydhCer derivative described by Casasampere et al, which primarily accumulated in the lysosomes . However, it is worth noting that in that case the 1-deoxydhCer analogue was labeled in situ through a SPAAC reaction with the BODIPY fluorophore after cellular internalization, whereas in our case, the cellular uptake of the BODIPY-ceramide conjugates was directly studied.…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescently labeled SLs have found multiple applications in biology, including the visualization of cellular events involving SLs, the study of biophysical properties and dynamics of SLs in biological membranes, and the development of assays to monitor the activity of enzymes responsible for their metabolism . In this context, we have recently shown the potential of fluorescently labeled 1-deoxyCer and 1-deoxydhSo probes to study the activity of acid ceramidase (AC) and ceramide synthase (CerS) enzymes, respectively. , 1-DeoxySLs do not undergo the same metabolic reactions as canonical SLs; the lack of the C1-OH group prevents the 4,5-desaturation and avoids the formation of more complex SLs . The higher metabolic stability of 1-deoxySLs, compared to that of canonical SLs, has been shown to confer robustness to particular biological assays but could also be exploited for imaging applications that require longer observation times.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies

et al. 2022

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Ceramides (Cer) are bioactive sphingolipids that have been proposed as potential disease biomarkers since they are involved in several cellular stress responses, including apoptosis and senescence. 1-Deoxyceramides (1-deoxyCer), a particular subtype of noncanonical sphingolipids, have been linked to the pathogenesis of type II diabetes. To investigate the metabolism of these bioactive lipids, as well as to have a better understanding of the signaling processes where they participate, it is essential to expand the toolbox of fluorescent sphingolipid probes exhibiting complementary subcellular localization. Herein, we describe a series of new sphingolipid probes tagged with two different organic fluorophores, a far-red/NIR-emitting coumarin derivative (COUPY) and a green-emitting BODIPY. The assembly of the probes involved a combination of olefin cross metathesis and click chemistry reactions as key steps, and these fluorescent ceramide analogues exhibited excellent emission quantum yields, being the Stokes’ shifts of the COUPY derivatives much higher than those of the BODIPY counterparts. Confocal microscopy studies in HeLa cells confirmed an excellent cellular permeability for these sphingolipid probes and revealed that most of the vesicles stained by COUPY probes were either lysosomes or endosomes, whereas BODIPY probes accumulated either in Golgi apparatus or in nonlysosomal intracellular vesicles. The fact that the two sets of fluorescent Cer probes have such different staining patterns indicates that their subcellular distribution is not entirely defined by the sphingolipid moiety but rather influenced by the fluorophore.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies

et al. 2022

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“…Recently, we described a flow-cytometry-based assay that uses a deoxyceramide analog to monitor AC activity in intact cells. This assay could be potentially useful in the future for screening purposes, but it would require proper optimisation and the use of high-throughput flow cytometry platforms 24 . Herein, we report a fluorescence-based AC assay that has been adapted to a 384-well format.…”

Section: Introductionmentioning

confidence: 99%

High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase

Aseeri

Abad

Delgado

et al. 2022

Journal of Enzyme Inhibition and Medicinal Chemistry

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Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2–25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.

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“…Notably, the metabolic intermediates that have characterized during these studies have the potential to serve as potential biomarkers, not only for diseases such as HSAN1, but may also be highly predictive for the occurrence of neuropathy in diabetic patients [ 22 ]. Recently, the restricted metabolism of deoxysphingolipids was successfully employed for fluorescent “dead end” metabolic probes [ 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

Stereoselective Synthesis of Novel Sphingoid Bases Utilized for Exploring the Secrets of Sphinx

Saied

2021

IJMS

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Sphingolipids are ubiquitous in eukaryotic plasma membranes and play major roles in human and animal physiology and disease. This class of lipids is usually defined as being derivatives of sphingosine, a long-chain 1,3-dihydroxy-2-amino alcohol. Various pathological conditions such as diabetes or neuropathy have been associated with changes in the sphingolipidome and an increased biosynthesis of structurally altered non-canonical sphingolipid derivatives. These unusual or non-canonical sphingolipids hold great promise as potential diagnostic markers. However, due to their low concentrations and the unavailability of suitable standards, the research to explore the secret of this class of ‘Sphinx’ lipids is ultimately hampered. Therefore, the development of efficient and facile syntheses of standard compounds is a key endeavor. Here, we present various chemical approaches for stereoselective synthesis and in-depth chemical characterization of a set of novel sphingoid bases which were recently utilized as valuable tools to explore the metabolism and biophysical properties of sphingolipids, but also to develop efficient analytical methods for their detection and quantification.

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Click and count: specific detection of acid ceramidase activity in live cells

Abstract: The use of intact cells in medical research offers a number of advantages over employing cell-free systems. In diagnostic, cells isolated from liquid biopsies can be directly used, speeding up...

Cited by 9 publications

References 36 publications

Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies

Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies

High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase

Stereoselective Synthesis of Novel Sphingoid Bases Utilized for Exploring the Secrets of Sphinx

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