title: A secondary assay for ceramide kinase inhibitors based on cell growth inhibition by short-chain ceramides.
abstract: We recently reported that ectopic expression of ceramide kinase (CerK) in various cell lines increases their sensitivity to cell death induced by the exogenous addition of short-chain (e.g., C2) ceramides (Cer). Here we show that this higher sensitivity results from CerK catalytic activity and production of C2-ceramide 1-phosphate (C2-C1P). If CerK activity is inhibited by the potent inhibitor NVP-231, C2-C1P is not produced and viability returns to control levels. The EC(50) of NVP-231 in this assay is in the low nanomolar range, consistent with the IC(50) determined in activity assays in vitro using purified CerK. NVP-995, a structurally related but inactive compound, does not protect against C2-Cer-induced cell death. This assay is robust and easy to implement and scale up, thereby providing a valuable secondary screen assay for CerK inhibitors.
authors.affiliation: Novartis Institutes for BioMedical Research, A-1235 Vienna, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Graf
authors.forename: Philipp
authors.initials: P
authors.lastname: Rovina
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bornancin
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000032:analysis* / Q000138:chemical synthesis; D001952:Bridged-Ring Compounds / Q000032:analysis* / Q000138:chemical synthesis; D019556:COS Cells; D016923:Cell Death; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000138:chemical synthesis; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000633:toxicity; D014162:Transfection
chemical_list: D052160:Benzothiazoles; D001952:Bridged-Ring Compounds; D002518:Ceramides; C064769:N-acetylsphingosine; C534511:NVP 231; C534512:NVP 995; D047428:Protein Kinase Inhibitors; C065576:ceramide 1-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D013110:Sphingosine