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Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro‐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. However, due to their hydrophobic nature, no specific 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, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non‐lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts. [ABSTRACT FROM AUTHOR]

Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis. [ABSTRACT FROM AUTHOR]

1‐Deoxysphingolipids are a recently described class of sphingolipids that have been shown to be associated with several disease states including diabetic and hereditary neuropathy. The identification and characterization of 1‐deoxysphingolipids and their metabolites is therefore highly important. However, exact structure determination requires a combination of sophisticated analytical techniques due to the presence of various isomers, such as ketone/alkenol isomers, carbon–carbon double‐bond (C=C) isomers and hydroxylation regioisomers. Here we demonstrate that cryogenic gas‐phase infrared (IR) spectroscopy of ionized 1‐deoxysphingolipids enables the identification and differentiation of isomers by their unique spectroscopic fingerprints. In particular, C=C bond positions and stereochemical configurations can be distinguished by specific interactions between the charged amine and the double bond. The results demonstrate the power of gas‐phase IR spectroscopy to overcome the challenge of isomer resolution in conventional mass spectrometry and pave the way for deeper analysis of the lipidome. [ABSTRACT FROM AUTHOR]

1‐Deoxysphingolipide sind eine erst kürzlich beschriebene Klasse von Sphingolipiden, die in Zusammenhang mit verschiedenen Krankheiten wie diabetischer und erblicher Neuropathie gebracht werden. Die Identifizierung und Charakterisierung von 1‐Deoxysphingolipiden und deren Metaboliten ist in diesem Kontext sehr wichtig. Eine exakte Strukturbestimmung erfordert jedoch die Kombination von verschiedenen anspruchsvollen analytischen Techniken, da unterschiedliche Isomerentypen vorliegen können: Keton/Alkenol‐Isomere, Kohlenstoff‐Kohlenstoff‐Doppelbindungs(C=C)‐Isomere und Hydroxyl‐Regioisomere. Wir zeigen, dass kryogene Gasphasen‐Infrarot(IR)‐Spektroskopie von ionisierten 1‐Deoxysphingolipiden die Identifizierung und Unterscheidung von Isomeren anhand ihres einzigartigen spektroskopischen Profils ermöglicht. Insbesondere die Position und Konfiguration von C=C‐Bindungen können aufgrund von spezifischen Interaktionen zwischen dem geladenen Amin und der Doppelbindung bestimmt werden. Die Ergebnisse verdeutlichen das Potential der Gasphasen‐IR‐Spektroskopie, die Herausforderungen der Isomerenunterscheidung in der konventionellen Massenspektrometrie zu überwinden und den Weg für eine umfangreiche Analyse des Lipidoms zu ebnen. [ABSTRACT FROM AUTHOR]

HPA-12 is an inhibitor of CERT-mediated non-vesicular transport of ceramide from the ER membranes to the Golgi apparatus. The inhibitor effectively blocks the synthesis of the membrane lipid sphingomyelin and may represent a novel drug prototype. Previous syntheses relied on non-commercial catalysts or specialized chemistries. Here we present a straightforward and effective method to synthesize HPA-12 from commercially available protected L-serinol in four steps. Some new analogues were synthesized and evaluated for their CERT-binding activity. [ABSTRACT FROM AUTHOR]