Insitute for Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
Department of Pharmaceutical Chemistry, College of Pharmacy, Helwan University, Cairo, 11795, Egypt.
Chemistry. 2020 Dec 15;26(70):16616-16621. doi: 10.1002/chem.202003283. Epub 2020 Nov 9.
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.
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运，从而催化鞘磷脂生物合成的限速步骤。通常，使用放射性标记的神经酰胺在繁琐的结合测定或非均相转移测定中评估 CERT 配体。在此，提出了一种基于Förster 共振能量转移（FRET）的简单灵敏的 CERT 测定法。为此，我们混合了供体和受体囊泡，每个囊泡都包含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，尽管该系统成分具有高疏水性，但仍可在 96 孔板格式中进行读出。对 2000 种化合物文库的筛选产生了两种新的强效 CERT 抑制剂。其中一种已获准在人类中使用，另一种已获准在动物中使用。通过定量质谱法和共聚焦显微镜评估细胞活性表明，神经酰胺运输和鞘磷脂生物合成受到抑制。
