A bioluminescent screen for inhibitors of N-linked glycosylation

描述（由申请人提供）：N-连接糖基化（NLG）是受体酪氨酸激酶（RTK）以及大多数穿过分泌途径的蛋白质常见的共翻译蛋白质修饰。高效的NLG需要逐步生物合成脂联寡糖（LLO），这是一个在细胞质中起始并在内质网中完成的过程，然后通过寡糖基转移酶转移LLO。我们利用NLG的序列特异性和荧光素酶检测的灵敏度来开发测量活细胞中NLG抑制的报告基因。在哺乳动物细胞中稳定表达该报告构建体之后，我们优化了在384孔板形式中进行化合物文库的高通量筛选的方法。同时，我们还开发了第二种荧光素酶构建体，其对NLG不敏感，因此提供了一种对荧光素酶的非特异性激活剂进行分类的测定。第三次筛选，其鉴定了破坏NLG后糖蛋白（即EGFR）定位的特征性变化，也已优化用于96孔板中的高含量成像。在该提案中，我们提出了一种顺序策略，使用这些测定从MLPCN化合物文库中选择NLG抑制剂。我们还提出了以中等通量形式进行的其他功能测定，以区分NLG酶促步骤，以确认化合物活性并鉴定NLG抑制剂的特定靶标和潜在作用机制。我们提供了关于这种筛选策略可行性的初步数据，并预测拟议的研究将确定NLG生物合成机制的新型抑制剂，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-连接糖基化是受体酪氨酸激酶和大多数穿过分泌途径的蛋白质常见的共翻译蛋白质修饰。我们已经确定并优化了一种方法来进行高通量筛选的化合物库，以确定新的抑制剂的N-连接的糖基化。我们预测，拟议的研究将提供重要的工具，生物科学以及潜在的治疗恶性疾病的治疗。

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.