Identification of Novel CERT Ligands as Potential Ceramide Trafficking Inhibitors

Abstract

A highly compartmentalized enzymatic network regulates the pro-apoptotic and proliferative effects of sphingolipids. Over-conversion of ceramide (Cer) correlates with insensitivity to apoptosis signaling (in response to chemotherapy) and to drug resistance of cancer cells. De novo sphingomyelin biosynthesis relies on non-vesicular ceramide trafficking by the CERT (CERamide Transfer) protein. Therefore, blocking CERT transfer, thus leading to increased intracellular ceramide availability, represents a potential anticancer strategy. Our study is based on the implementation of an in vitro binding assay, supported by in silico molecular docking. It constitutes the first attempt to explore at the molecular level for the identification of novel CERT ligands. This approach is the first step toward in silico design and optimization of CERT inhibitor candidates, potentially relevant as innovative ceramide-transfer-targeting therapeutic agents.

The CERT antagonist HPA-12: First practical synthesis and individual binding evaluation of the four stereoisomers

“…The (1R,3R) and (1R,3S) isomers gave comparable calculated complexes. 17 A convergent interaction trend was observed for the two 1S stereoisomers. The four HPA-12 stereoisomers were thus found to fit into the START cavity in an overall disposition reminiscent to that of the Cer natural ligand (see ESI, Fig.…”

“…14a This route notably led to the revision of the initially reported (1R,3R)-anti stereochemical assignment for HPA-12 to the (1R,3S)-syn configuration. 14a Prompted by our preliminary in vitro/in silico protein interaction study of CERT ligands, 17 we described here an in-depth optimization of this route demonstrating the effectiveness of Crystallisation-Induced Asymmetric Transformations (CIATs) 18 in delivering multi-gram quantities of all four stereoisomers of HPA-12.…”

“…We also demonstrated that it could be used, in conjunction with molecular modelling, for the identification of new CERT ligands. 17 We applied this approach to the comparative evaluation of the four HPA-12 stereoisomers. The extent of CERT START domain binding is correlated to the diminution of fluorescence in the fraction of the ceramide probe bound to the protein, compared to the control value in the absence competitor.…”

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

“…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. [21,35] Arenz and co-workers described n-a nd tert-butyld erivatives that showed as trongly reduced binding to the CERT protein in an in vitro fluorescent intensity assay.…”

“…In silico studies were used to rationalize these trends, leading to the first model of protein recognition coherent with the stronger bindingo f( 1 R,3S)-HPAs. [21,35]…”