Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT),Chemistry - A European Journal

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Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT)
Chemistry - A European Journal ( IF 3.9 ) Pub Date : 2020-10-12 , DOI: 10.1002/chem.202003283
Doaa Samaha _{1,

2} , Housam H Hamdo ₁ , Xiaojing Cong ₃ , Fabian Schumacher _{4,

5} , Sebastian Banhart ₆ , Öznur Aglar ₇ , Heiko M Möller ₇ , Dagmar Heuer ₆ , Burkhard Kleuser ₄ , Essa M Saied _{1,

8} , Christoph Arenz ₁

Affiliation

Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.

中文翻译：

脂质体 FRET 测定鉴定神经酰胺转运蛋白 (CERT) 的有效药物样抑制剂

神经酰胺转移蛋白（CERT）介导神经酰胺从内质网到高尔基体的非囊泡转移，从而催化鞘磷脂生物合成的限速步骤。通常，CERT 配体在繁琐的结合测定或使用放射性标记神经酰胺的非均相转移测定中进行评估。本文提出了一种基于福斯特共振能量转移 (FRET) 的简单且灵敏的 CERT 检测方法。为此，我们混合了供体和受体囊泡，每个囊泡含有不同的荧光神经酰胺种类。通过 CERT 介导的荧光神经酰胺转移，建立了 FRET 系统，尽管组件具有高疏水性，但仍可以在 96 孔板格式中进行读数。对 2000 种化合物库的筛选产生了两种新的有效 CERT 抑制剂。一种被批准用于人类，另一种被批准用于动物。通过定量质谱和共聚焦显微镜对细胞活性的评估表明，神经酰胺运输和鞘磷脂生物合成受到抑制。

更新日期：2020-12-16

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