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

抑制内质网腔内蛋白质上高甘露糖寡糖组装和修剪的化合物已被广泛用于生化研究，而特定化合物已在潜在的治疗应用中进行了试验（Aebi, 2013）。Bba 1833: 2430)。小分子抑制剂的使用已经被siRNA的使用所补充，siRNA主要作为一种载体来定义催化n -糖基化反应的寡糖基转移酶复合物的特定组分的作用（Roboti & High, 2012）。JCS 125: 3474)。该项目的主要目标是开发新的小分子甘露糖基转移酶抑制剂，并通过体外和纤维素读数确定其对n -链糖基化和o -链甘露糖基化的影响。该项目位于生物学和化学之间的界面，提供广泛的培训，范围从小分子设计，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。第一部分，抑制剂生产（Flitsch培训：Sardzik & Flitsch， J. Am）。化学。系统工程学报，2012,34 (4):13010；兰恩斯和弗莱奇，J. Am。化学。Soc, 2011, 33 (3): 842；约安诺和弗立奇，化学。普通。2011,47:11228)。该学生将通过从头合成、合作和购买相结合的方式，建立一个甘露糖类似物库，包括脱氧和氟脱氧版本。所得类似物也将转化为其gdp共轭或“激活”形式用于体外应用。第二部分，对蛋白质糖基化的影响（highandswanton的训练）。为了解决它们对蛋白质n -糖基化的影响，在经典的体外试验之前，将gdp共轭甘露糖类似物添加到内质网衍生的微粒体中（Wilson等人，2007年）。JCS 120: 648；Roboti & High, 2012。JCS 125: 3474)。将非共轭甘露糖类似物添加到培养的HeLa细胞中，并通过i)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 et al., 2013）。JCS 126: 2595)；ii)制备半透性细胞用于体外读数（Roboti & High, 2012）。同前)。第三部分，对糖蛋白质量控制的影响（由High和Swanton训练）。甘露糖修剪是通过内质网相关降解（ERAD）途径去除错误折叠糖蛋白的重要调节因子，甘露糖苷酶抑制剂阻止异常糖蛋白的清除导致其积累。我们将使用培养的表达明确的ERAD模型底物的哺乳动物细胞（Alcock & Swanton 2009, 2009）。JMB 385: 1032；Payapilly & High, 2014。127: 2898)来确定我们的甘露糖类似物是否抑制甘露糖修剪。第四部分，根据上述第1至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。因此，携带生物素亲和标记和/或荧光标记的类似物将（与Flitsch合作）制成，并用于基于生化和细胞生物学的应用（与High和Swanton合作）。如果使用点击化学是现实的，可以生成一个“可点击的”甘露糖类似物，允许添加一系列标签。与此同时，如果生物分析表明一个可能的细胞靶标，我们将尝试使用siRNA敲除它，从而允许与小分子抑制产生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.