title: Development of a CERT START Domain-Ceramide HTRF Binding Assay and Application to Pharmacological Studies and Screening.
abstract: Sphingomyelin (SM) metabolism deregulation was recently associated with cell metastasis and chemoresistance, and several pharmacological strategies targeting SM metabolism have emerged. The ceramide (Cer) generated in the endoplasmic reticulum (ER) is transferred to the Golgi apparatus to be transformed into SM. CERamide Transfer (CERT) protein is responsible for the nonvesicular trafficking of Cer to Golgi. Blocking the CERT-mediated ER-to-Golgi Cer transfer is an interesting antioncogenic therapeutic approach. Here, we developed a protein-lipid interaction assay for the identification of new CERT-Cer interaction inhibitors. Frequently used for protein-protein interaction by enzymatic and analyte dosage assays, homogeneous time-resolved fluorescence technology was adapted for the first time to a lipid-protein binding assay. This test was developed for high-throughput screening, and a library of 672 molecules was screened. Seven hits were identified, and their inhibitory effect quantified by EC50 measurements showed binding inhibition three orders of magnitude more potent than that of HPA12, the unique known CERT antagonist to date. Each compound was tested on an independent test, confirming its high affinity and pharmacological potential.
authors.affiliation: Unité de Service et de Recherche CNRS, Toulouse, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Fleury
authors.affiliation: Unité de Service et de Recherche CNRS, Toulouse, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Faux
authors.affiliation: LSPCMIB, CNRS-Université Paul Sabatier-Toulouse III, Toulouse, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Santos
authors.affiliation: LSPCMIB, CNRS-Université Paul Sabatier-Toulouse III, Toulouse, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Ballereau
authors.affiliation: LSPCMIB, CNRS-Université Paul Sabatier-Toulouse III, Toulouse, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Génisson
authors.affiliation: Unité de Service et de Recherche CNRS, Toulouse, France frederic.ausseil@pierre-fabre.com.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ausseil
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: D002352:Carrier Proteins; D002518:Ceramides; D054852:Small Molecule Libraries; D017346:Protein Serine-Threonine Kinases
keywords: CERT (CERamide Transfer); HTRF; assay development; ceramide; high-throughput screening; homogenous time-resolved fluorescence (TR-FRET)