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 EC 50 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.

Chemistry and Biology of HPAs: A Family of Ceramide Trafficking Inhibitors

“…[31] The configuration of the two stereocenters was early shown to strongly influence the cellular inhibitory activity,t he most potent of the four stereoisomers being assigned as the 1R,3S isomer.H owever,a mong the two diastereoisomeric pairs of HPA-12, only the syn-(1R*,3S*) enantiomers, initially described as anti-(1R*,3R*), were evaluated individually as inhibitors of de novo SM production in living cells. [32] Not only the 1R,3S led to the strongest binding, but both 1R and 3S stereocenters were shown to similarly contributet ot he extent of recognition. [21] AT R-FRET homogeneousphase assay was specifically developed, allowing complete determination of HPA-12 EC 50 .…”

“…[21] AT R-FRET homogeneousphase assay was specifically developed, allowing complete determination of HPA-12 EC 50 . [32] Not only the 1R,3S led to the strongest binding, but both 1R and 3S stereocenters were shown to similarly contributet ot he extent of recognition. Crystals tructures of as eries of (1R,3R)-HPAs with C13 to C16 acyl chains in complex with the CERT START domain were reported,i ndicating ar ecognition mode very similart ot hat of the naturalc argo lipid Cer.…”

“…Kobayashi and co-workers [31] Replacement of the phenyl ring by an alkyl group Aza-Michael-based crystallization-induced asymmet- Binding assay by competition versus NBD-ceramide-C16 bound to CERT Arenz and coworkers [23] HPA-12 stereoisomers Aza-Michael-based crystallization-induced asymmetric transformation EC 50 determination by TR-FRET binding assay by competition versus biotinylated-ceramide bound to STARTd omain of CERT [32] BerkeŠ,Ballereau, GØnisson and coworkers [21] Meta-orpara-substituents on phenyl ring Chelation-controlled reduction of N-protected bamino ketones…”

“…Ramapanicker and co-workers [26] Long alkyl chain in meta-o rpara-position on aromatic ring Aza-Michael-based crystallization-induced asymmetric transformation followed by Sonogashira coupling and triple bond reduction EC 50 determination by TR-FRET binding assay by competition versus biotinylated-ceramide bound to the STARTd omain of CERT [32] Daïch, GØnisson, Berkeš and coworkers [33] Replacement of the phenyl ring by long alkyl chains Aza-Michael-based crystallization-induced asymmetric transformation followed by hydrodesulfurization of thiophene ring EC 50 determination by TR-FRET binding assay by competition versus biotinylated-ceramide bound to STARTd omain of CERT [32] Daïch, GØnisson, Berkeš and coworkers [33] Not evaluated…”

Asymmetric Synthesis and Binding Study of New Long‐Chain HPA‐12 Analogues as Potent Ligands of the Ceramide Transfer Protein CERT

“…[28] In line with our precursory study on the identificationo ft he SM biosynthesis inhibitor jaspineB as ap otentialC ERT antagonist, [29] we recently described the first time-resolved FRET-based homogeneous (HTR-FRET) binding assay for the CERT START domain that proved amenablet oh igh throughput screening (HTS). [30] This in vitro protocol also allowed us to quantify the ligand-protein bindingt hrough the determination of EC 50 values.…”