A biologically relevant ceramide fluorescent probe to assess the binding of potential ligands to the CERT transfer protein

Abstract

The conversion of ceramide into sphingomyelin (SM) was only recently considered in relation to cancer.Overexpression of the CERT protein, responsible for a highly specific inter-organelle ceramide transfer step along the de novo SM synthesis pathway, has been associated with multi-drug resistance of cancer cells.Identification of new CERT antagonists may therefore lead to potential resensitizing agents. This work describes the first attempt to design a ceramide-based fluorescent probe optimised to evaluate the binding of potential CERT ligands. A prototypical structure with an v-labelled sphingosine backbone was selected. Its possible recognition mode by CERT was first evaluated by means of a precursory molecular modelling study. Three derivatives with various amide chain lengths were prepared and tested. The binding efficiency was shown to be proportional to the lipophilicity of the acyl moiety. The best compound bearing a C16 amide fragment was used to implement a practical binding experiment. Facile assessment of the recognition by the CERT START domain of various structures was thus ensured. Metabolism and imaging experiments were also used to illustrate the capacity of the proposed fluorescent ceramide analogue to mimic the natural ceramide cellular behaviour. This work led to the synthesis and evaluation as an efficient CERT START domain ligand of a v-biotinylated ceramide, a potential probe to develop the screening of new CERT antagonists.

Development of a CERT START Domain–Ceramide HTRF Binding Assay and Application to Pharmacological Studies and Screening

“…This heterogeneous binding assay (Fluorescence INtensity Test; FLINT) includes numerous steps, such as centrifugation, which makes extensive pharmacological studies and automation difficult. 11 Moreover, the high amount of the purified START protein domain required to carry out FLINT experiments renders it inappropriate for large screening experiments.…”

“…1A). To discover antagonists of the START-Cer interaction, a biologically relevant Cer fluorescent probe was developed 11 (Fig. 1B, left).…”

“…15,16 Here, TR-FRET was applied for the first time to monitor a protein-lipid, namely, START-Cer, interaction. Thanks to a specifically designed ω-biotinylated Cer (BioCer) probe, 11 we were able to measure a TR-FRET between an europium kryptate linked to an anti-histidine antibody, as the donor, and d2 carried by streptavidine, as the acceptor. The performance of this miniaturized assay was carefully analyzed and shown to allow large screening experiments.…”

“…After centrifugation, the supernatant was collected as the bound fraction. For more details, see Combemale et al 11 …”

“…10 We recently developed a START domain binding assay for the identification of Cer binding competitors as potential inhibitors of Cer trafficking by CERT. 11 However, this procedure, requiring high reagent amounts and phase separation steps, was not suitable for extensive pharmacological analysis and screening, limiting the mechanistic analysis of START interaction with Cer as well as the identification of new Cer competitors.…”