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

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.