The exploitation of mannosyltransferase inhibitors to modulate glycoprotein biogenesis and quality control

抑制内质网腔中蛋白质附加的高甘露糖低聚糖的组装和修剪的化合物已被广泛用于生化研究，而特定的化合物已被试验用于潜在的治疗应用(Aebi，2013)。BBA 1833：2430)。小分子抑制剂的使用得到了siRNA的使用的补充，主要是作为一种工具来确定催化N-糖基化反应的寡糖转移酶复合体的特定成分的作用(Roboti&High，2012)。JCS 125：3474)。本项目的主要目标是开发新的甘露糖基转移酶小分子抑制剂，并在体外和细胞读数中确定它们对N-连接的糖基化和O-连接的甘露糖化的影响。该项目位于生物学和化学的交界处，提供从小分子设计、合成和碳水化合物化学、体外蛋白质合成和分析到哺乳动物细胞培养、Western blotting和免疫荧光显微镜的广泛培训。第1部分，抑制剂生产(Flitsch培训：Sardzik&Flitsch，J.Am化学。SoC。2012年，第134：13010；兰尼斯与弗利奇，J.Am化学。SOC，2011,133：8436；IoAnu&Flitsch，化学。交警。2011年，47：11228)。学生将通过从头合成、合作和购买的组合，组装一个包括脱氧和氟脱氧版本的甘露糖类似物库。由此产生的类似物也将被转化为其GDP结合或“激活”的形式，用于体外应用。第二部分，对蛋白质糖基化的影响，(由High和Swanton培训)。为了解决它们对蛋白质N-糖基化的影响，在经典的体外分析中使用之前，GDP-连接的甘露糖类似物将被添加到内质网衍生的微粒体内(Wilson等人，2007年)。JCS 120：648；Roboti&High，2012。JCS 125：3474)。非结合甘露糖类似物将被添加到HeLa细胞的培养中，并通过分析内源性糖蛋白的N-糖基化来确定对蛋白质N-糖基化的影响(Dumax-Vorzet等人，2013年)。JCS 126：2595)；ii)制备用于体外读数的半渗透细胞(Roboti&High，2012)。同上)。第三部分，对糖蛋白质量控制的影响(High和Swanton培训)。甘露糖修剪是通过内质网相关降解(ERAD)途径去除错误折叠的糖蛋白的重要调节因子，而甘露糖苷酶抑制剂可以阻止异常糖蛋白的清除，从而导致糖蛋白的积累。我们将使用培养的哺乳动物细胞表达明确的模型ERAD底物(Alcock&Swanton，2009,2009)。JMB 385：1032；Payapilly&High，2014。127：2898)以确定是否有我们的甘露糖类似物抑制甘露糖的修剪。第4部分，根据上面第1部分至第3部分的进展，博士生将创建特定先导化合物的其他变体，以进一步识别其细胞靶标(S)。因此，带有生物素亲和标记和/或荧光标记的类似物将被制造(与Flitsch一起)，并用于基于生物化学和细胞生物学的应用(与High和Swanton)。如果点击化学的使用是现实的，则可以产生允许附加一系列标签的“可点击的”甘露糖类似物。同时，如果生物检测表明一个可能的细胞靶点(S)，我们将尝试使用小干扰RNA将其击倒，从而与小分子抑制引起的表型进行直接比较。

Compounds that inhibit both the assembly and trimming of the high-mannose oligosaccharide appended to proteins in the ER lumen have been exploited extensively for biochemical studies, whilst specific compounds have been trialled in potential therapeutic applications (Aebi, 2013. BBA 1833: 2430). The use of small molecule inhibitors has been complemented by the use of siRNA, primarily as a vehicle to define the role of specific components of the oligosaccacharyltransferase complex that catalyses the N-glycosylation reaction (Roboti & High, 2012. JCS 125: 3474). The principal goal of this project is to develop novel small molecule inhibitors of mannosyltransferases and define their effect on N-linked glycosylation and O-linked mannosylation using both in vitro and in cellulo readouts. This project sits at the interface between biology and chemistry and provides extensive training that ranges from small molecule design, synthetic and carbohydrate chemistry, in vitro protein synthesis and analysis, to mammalian cell culture, western blotting and immunofluorescence microscopy. Part 1, inhibitor production (training by Flitsch: Sardzik & Flitsch, J. Am. Chem. Soc. 2012, 134: 13010; Rannes & Flitsch, J. Am. Chem. Soc, 2011, 133: 8436; Ioannou & Flitsch, Chem. Commun. 2011, 47: 11228). The student will assemble a library of mannose analogues including deoxy- and fluorodeoxy-versions, through a combination of de novo synthesis, collaboration and purchase. The resulting analogues will also be converted into their GDP-conjugated or "activated" forms for in vitro applications. Part 2, effects on protein glycosylation, (training by High and Swanton). To address their effect on protein N-glycosylation, GDP-conjugated mannose analogues will be added to ER derived microsomes prior to use in classical in vitro assays (Wilson et al., 2007. JCS 120: 648; Roboti & High, 2012. JCS 125: 3474). Non-conjugated mannose analogues will be added to HeLa cells in culture and the effects on protein N-glycosylation established by i) analysing the N-glycoylation of endogenous glycoproteins (Dumax-Vorzet et al., 2013. JCS 126: 2595); ii) preparing semi-permeabilised cells for use with in vitro readout (Roboti & High, 2012. Ibid). Part 3, effects on glycoprotein quality control (training by High and Swanton). Mannose trimming is an important regulator for the removal of misfolded glycoproteins via the pathway for endoplasmic reticulum associated degradation (ERAD), and mannosidase inhibitors prevent the clearance of aberrant glycoproteins leading to their accumulation. We will use cultured mammalian cells expressing well-defined model ERAD substrates (Alcock & Swanton 2009, 2009. JMB 385: 1032; Payapilly & High, 2014. 127: 2898) to establish whether any of our mannose analogues inhibit mannose trimming. Part 4, contingent on the progress of parts 1 to 3 above, the PhD student will create additional variants of a specific lead compound to further the identification of its cellular target(s). Hence, analogues that carry a biotin affinity label and or fluorescent tag will be made (with Flitsch) and used for biochemical and cell biology based application (with High and Swanton). Should the use of click chemistry be realistic, a "clickable" mannose analogue may be generated allowing a range of tags to be appended. In parallel, should biological assays indicate a likely cellular target(s) we will attempt to knock it down using siRNA , allowing a direct comparison to the phenotype resulting from small molecule inhibition.