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Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes such as energy utilisation and cell proliferation. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to ultimately produce pro-proliferative sphingosine-1-phosphate. Human ceramidases can be soluble proteins (acid and neutral ceramidase) or integral membrane proteins (alkaline ceramidases). Increasing ceramide levels to increase apoptosis has shown efficacy as a cancer treatment using small molecules inhibiting a soluble ceramidase. Due to the transmembrane nature of alkaline ceramidases, no specific small molecule inhibitors have been reported. Here, we report novel fluorescent substrates (FRETceramides) of ceramidases that can be used to monitor enzyme activity in real-time. We use FRETceramides to discover the first drug-like inhibitors of alkaline ceramidase 3 (ACER3) which are active in cell-based assays. Biophysical characterization of enzyme:inhibitor interactions reveal a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules.Table of contents summaryUse of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug-like small molecule inhibitors for alkaline ceramidase 3, an intra-membrane enzyme involved in sphingolipid metabolism in health and disease.

Sphingolipids including ceramides are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids are associated with various neurological pathologies, however, the entire spectrum of disorders affecting sphingolipid metabolism remains elusive. By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous system, we identified a homozygous variant p.(Ala280Val) inDEGS1,encoding an enzyme of the ceramide synthesis pathway. The blood sphingolipid profile and patient-derived fibroblasts both showed a significant shift from the unsaturated to the dihydro-forms of sphingolipids. Moreover, an atypical and potentially toxic sphingolipid metabolite is formed as consequence of the altered synthesis pathway. The changes in the sphingolipid profile were recapitulated in a CRISPR/Cas-basedDEGS1knockout HAP1-cell model and by chemical inhibition of DEGS1, suggesting a loss of DEGS1 function in the disease. DEGS1 insufficiency is thus a novel cause for a multisystem neurological disorder. A sphingolipid-rich diet may correct the metabolic profile and improve the clinical outcome of affected individuals and suggests that this heritable condition might be treatable.AbbreviationsSLSphingolipidsSPTserine-palmitoyltransferaseCerCeramidesdhCerdihydroceramideS1Psphingosine-1-phosphateSOsphingosineHSANhereditary sensory and autonomic neuropathy