| Summary: | <p>Glycosylation is the most common protein co- and post-translational modification. It has crucial functions for glycoprotein folding, structure, trafficking, localisation and stability. Glycosylation is not only critical for eukaryotic cell function but vital for many enveloped viruses which have evolved to exploit the host cell glycosylation pathway in order to fold their proteins correctly and coat many of their surface proteins with glycans that can contribute to viral pathogenesis. Vaccines often focus on the viral fusion glycoproteins that protrude from the virion envelope as they are among the most immunogenic biomolecules and can elicit a humoral immune response. A robust vaccine antigen needs to mimic viral glycosylation to produce an appropriate and strong B-cell response, as many neutralising antibodies incorporate glycans as part of their binding epitopes, and other epitopes are shielded by sugars and inducing antibodies against those would lead to a less relevant antibody response.</p>
<p>The current COVID-19 pandemic has highlighted the urgent need for vaccines and antiviral drugs. The principal topic of my DPhil centres on addressing these needs for viral infection. Chapter 4 focuses on the site-specific glycosylation occupancy, and N- and O-glycan structures attached to virion derived SARS-CoV-2 spike glycoproteins. This glycan signature was compared to a stabilised prefusion trimeric spike, to a monomeric subunit of spike (S1) and to an antigen of a non-stabilised vaccine candidate, revealing subunit shedding of the S1 glycoprotein for the vaccine candidate. Whether or not monomeric S1 shedding has an effect on the vaccine antibody response or could be implicated in the observed rare side effects, remains to be investigated but evidence from other studies implicates this occurrence as detrimental in animal models.</p>
<p>The COVID-19 pandemic highlighted again the lack of broad-spectrum antivirals available to patients before thorough investigation of an emergent virus, such as its genome or target host receptor. Chapter 5 discusses the establishment of a medium-throughput drug screening platform against SARS-CoV-2. FDA-approved compounds were prioritised and triaged within the screen for clinical committees such as the UK steering group for therapeutics for COVID-19. Compounds of a global anti-COVID-19 effort called the “Moonshot project” targeting the main protease of SARS-CoV-2 were also screened and promising compounds were progressed further to produce a specific direct-acting SARS-CoV-2 antiviral compound. Iminosugars, which inhibit N-linked glycan processing enzymes such as endoplasmic reticulum alpha-glucosidase I and II of the glycoprotein quality control pathway, constituted one of the two most promising groups of drugs in this antiviral cellular screen that have not entered clinical trials yet.</p>
<p>The second part of this thesis focuses on ER alpha-glucosidase I as a key antiviral target to develop a broad-spectrum antiviral. Previously, it had been observed that a single high-dose treatment with the iminosugar MON-DNJ led to long-lasting antiviral effects in lethal dengue mouse models via alpha-glucosidase I inhibition. In Chapter 6 the mechanism of action was investigated in an in cellulo model recapitulating the in vivo data. This led to preliminary results and the hypothesis that the unfolded protein response might play a fundamental role in activating the apoptosis pathway in dengue-infected and high-dose treated cells alone. This warrants further investigation in a more relevant cell type, precipitating the development of the human induced pluripotent stem cell knockouts of the unfolded protein response constituents. This chapter also describes how I produced these cells.</p>
<p>An important factor that warrants consideration is that inhibition of the alpha-glucosidases in the ER by iminosugars as glucose mimetics can lead to unintended side-effects as many host enzymes can recognise glucose molecules. In Chapter 7, I describe attempts towards identification of a specific alpha-glucosidase I inhibitor. X-ray crystallisation of ER alpha-glucosidase I with the iminosugar NB-DNJ bound in the active site, led to one crystal which was analysed, but the crystallisation process was not reproducible enough to be useful for fragment or drug screening. Further methods such as thermal shift assay screening of 1600 FDA-approved compounds did not reveal any compounds that bound to the soluble enzyme. Screening of a library of 2.9 billion DNA-tagged molecules against the soluble enzyme found 937 potential binders. This needs further validation to establish the binding mode and inhibitory activity.</p>
<p>Overall, this thesis describes the unique influence of glycan analysis in the rational design of vaccines and how subtle changes in glycosylation help interpret large-scale changes in the structure of an antigen, or the foundation for a totally new type of broad-spectrum antiviral that functions through inhibiting the host glycosylation pathway – collectively these outputs will help preparedness for future pandemics.</p>
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