A bioluminescent screen for inhibitors of N-linked glycosylation

描述(申请人提供)：N-连接糖基化(NLG)是受体酪氨酸激酶(RTK)以及大多数穿越分泌途径的蛋白质共同的共翻译蛋白质修饰。高效的NLG需要脂质连接的寡糖(LLO)的逐步生物合成，这一过程始于细胞质，在LLO通过寡糖转移酶转移之前在内质网上完成。我们利用NLG的序列特异性和荧光素酶检测的敏感性开发了一种报告，可以测量活细胞中NLG的抑制。在哺乳动物细胞中稳定表达该报告结构后，我们优化了一种在384个孔板格式中进行高通量筛选的化合物文库的方法。同时，我们还开发了第二种对NLG不敏感的荧光素酶结构，从而提供了一种对荧光素酶的非特异性激活剂进行分类的方法。第三个屏幕可以识别破坏NLG后糖蛋白(即EGFR)定位的特征变化，该屏幕也针对96个孔板的高含量成像进行了优化。在这项建议中，我们提出了一种顺序策略，使用这些分析从MLPCN化合物库中选择NLG抑制剂。我们还建议在中等吞吐量格式中执行额外的功能分析来区分NLG酶步骤，以确认化合物的活性，并确定NLG抑制剂的特定靶点和潜在的作用机制。我们提供了关于这一筛选策略可行性的初步数据，并预测拟议的研究将发现NLG生物合成机制的新抑制剂，为生物科学提供重要工具，并为治疗恶性疾病提供潜在的治疗方法。 公共卫生相关性：N-连接的糖基化是受体酪氨酸激酶和大多数穿越分泌途径的蛋白质共同的共翻译蛋白质修饰。我们已经确定并优化了一种对化合物文库进行高通量筛选的方法，以确定新的N-连接糖基化抑制剂。我们预测，拟议的研究将为生物科学提供重要工具，并为治疗恶性疾病提供潜在的治疗方法。

DESCRIPTION (provided by applicant): N-linked glycosylation (NLG) is a co-translational protein modification common to receptor tyrosine kinases (RTKs) as well as most proteins that traverse the secretory pathway. Efficient NLG requires the stepwise biosynthesis of lipid-linked oligosaccharides (LLO), a process initiated in the cytoplasm and completed in the endoplasmic reticulum prior to LLO transfer by oligosaccharyl transferase. We have taken advantage of the sequence specificity of NLG and the sensitivity of the luciferase detection to develop a reporter that measures NLG inhibition in living cells. Following stable expression of this reporter construct in mammalian cells, we optimized a method to perform high throughput screening of compound libraries in 384 well plate formats. In parallel we also developed a second luciferase construct that is insensitive to NLG and thus provides an assay to triage non-specific activators of luciferase. A third screen which identifies characteristic changes in glyco-protein (ie EGFR) localization after disrupting NLG has also been optimized for high content imaging in 96 well plates. In this proposal we set forth a sequential strategy to use these assays to select NLG inhibitors from the MLPCN compound libraries. We also propose additional functional assays performed in medium throughput formats to discriminate between NLG enzymatic steps in order to both confirm compound activity and to identify specific targets and potential mechanisms of action of NLG inhibitors. We provide preliminary data on the feasibility of this screening strategy and predict that the proposed research will identify novel inhibitors of the NLG biosynthetic machinery, providing important tools for the biological sciences as well as potential therapeutics for the treatment of malignant diseases. PUBLIC HEALTH RELEVANCE: N-linked glycosylation is a co-translational protein modification common to receptor tyrosine kinases and most proteins that traverse the secretory pathway. We have identified and optimized a method to perform high throughput screening of compound libraries to identify novel inhibitors of N-linked glycosylation. We predict that the proposed research will provide important tools for the biological sciences as well as potential therapeutics for the treatment of malignant diseases.