1,721,194 research outputs found
Native functionality and therapeutic targeting of arenaviral glycoproteins
Full text linkSurface glycoproteins direct cellular targeting, attachment, and membrane fusion of arenaviruses and are the primary target for neutralising antibodies. Despite significant conservation of the glycoprotein architecture across the arenavirus family, there is considerable variation in the molecular recognition mechanisms used during host cell entry. We review recent progress in dissecting these infection events and describe how arenaviral glycoproteins can be targeted by small-molecule antivirals, the natural immune response and immunoglobulin-based therapeutics. Arenaviral glycoprotein-mediated assembly and infection pathways present numerous opportunities and challenges for therapeutic intervention
Iminosugars as dengue virus therapeutics: Molecular mechanisms of action of a drug entering clinical trials
Full text linkIminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy
Characterization of endoplasmic reticulum targeting liposomes as drug delivery vesicles
Full text linkDue to rapid viral and drug resistance mutations, there is a great need for broad-spectrum antiviral therapies that target the host rather than viral processes. This research builds upon previous studies developing α-glucosidase inhibitors known as iminosugars as broad-spectrum antiviral drugs. Initially pH-sensitive liposomes were designed and developed as drug delivery vehicles to enhance cellular internalization for α-glucosidase inhibitors for HIV-1 antiviral treatment. Recent studies by Pollock et al. have shown that polyunsaturated endoplasmic reticulum targeting liposomes known as PERLs, traffic to the ER and are antiviral as a stand-alone therapy against hepatitis B, hepatitis C, and HIV. In addition, α-glucosidase inhibitors formulated and delivered by 'ER-targeting' liposomes have shown potential in recent preclinical studies in vitro and are candidates for future clinical trials as antiviral treatments. There are two main purposes of this research. Firstly, it aims to develop crucial quantitative analytical methods that detect the α-glucosidase inhibitor drugs when formulated in 'ER-targeting' liposomes. The analytical methods will help elucidate how the chemical properties of small molecules, such as hydrophobicity or polarity, affect the encapsulation efficiency into 'ER-targeting' liposomes, which will help select appropriate formulation processes and dosing regimes for potential future clinical trials. Secondly, this research intends to dissect the possible cellular internalization and trafficking pathways that the 'ER-targeting' liposomes, such as PERLs, utilize to get to the ER in vitro. A large screening assay was conducted to determine what influence each type of phospholipid has on the cellular entry and intracellular movement of 'ER-targeting' liposomes. Several key observations were drawn from this research that will hopefully further the knowledge of 'ER-targeting' liposomes' capability to deliver iminosugars and the pathways involved with the cellular internalization and trafficking of the liposomes in vitro
The mechanism of action of iminosugars as antiretrovirals
Full text linkIminosugars are a class of molecules that resemble sugars but for the substitution of a nitrogen for the oxygen in the hemiacetal ring. Several iminosugars are inhibitors of cellular glycosidase enzymes in the N-linked glycan processing pathway. Iminosugars inhibiting endoplasmic reticulum (ER) α-glucosidases are known to be antiviral against a broad range of enveloped viruses including human immunodeficiency virus (HIV), hepatitis C virus, dengue virus and influenza virus. This antiviral effect is believed to be due to the indispensability of the calnexin/calreticulin pathway, entry into which is dependent on the trimming of glucoses from N-linked glycans, for the correct folding of viral glycoproteins. Thus cells treated with these drugs are unable to correctly fold the envelope glycoproteins of viruses with the result that the secreted virions have diminished infectivity. A long standing question arising from this hypothesis is how such a mechanism can prove to be damaging to such a wide range of viral glycoproteins yet not show any significant cytotoxicity. This thesis answers that question by demonstrating that, at antiviral concentrations, only a small proportion of viral glycoproteins are misfolded suggesting an amplification effect from protomer oligomerisation and clustering. This thesis also shows how α-glucosidase inhibition results in abnormal disulphide bond formation and isomerisation of the HIV envelope protein, gp120, during folding, as well as faster transit times through the secretory pathway, both potentially explaining the observed misfolding. A specific glycan, N241, is identified as the potential key for gp120/calnexin interaction. α-glucosidase inhibitors have two effects on glycoproteins; inhibition of calnexin mediated folding but also on the structure of the resultant glycan. This thesis uses mannosidase inhibiting iminosugars to examine the comparative significance of each of these to the antiviral effects of iminosugars. This thesis confirms that failure to process glycans beyond glucose trimming can be substantially antiviral in at least one HIV isolate. Finally this thesis looks at the potential for using α-glucosidase inhibiting iminosugars as antivirals against Ebola virus in a guinea pig model. These experiments show that iminosugars are safe, even at very high doses, when given intravenously in animals and that one of them, NB-DNJ, may offer partial protection against Ebola virus, allowing 1/4 guinea pigs to survive a lethal dose of the virus. Although preliminary, this is the first time that a licensed drug has been shown to be antiviral against Ebola virus and shows the potential utility of these drugs as protection against emerging viral infections for which specific therapies are not yet available
Hepatocellular lipid metabolism in Hepatitis C virus infection
Full text linkThe work described in this thesis investigates the lipid metabolism of human hepatocytes in the context of Hepatitis C Virus (HCV) infection. This includes lipoprotein signalling and cholesterol metabolism targeted analysis of gene expression as well as the influence of polyunsaturated ER targeting liposomes (PERLs) on infection. These analyses indicate that HCV suppresses the expression of key regulators throughout the cholesterol biosynthesis pathway. This effect was quantified and the influence of liposome treatment evaluated. The latter resulted in the formulation of the hypothesis that PERL treatment interfers with virus-induced abberations of the cholesterol biosynthesis pathway and normalises the expression of four genes directly involved in cholesterol regulation. In addition, the lipidome of isolated lipid droplet was analysed by mass spectrometry. These data, combined with microscopy data suggest that PERLs interfere with S-palmitoylation of the HCV core protein resulting in dissociation of core from lipid droplets. This is likely to interrupt the viral assembly process, leading to inhibition of the production of infectious viral particles. Further described here are two different yet unsuccessful approaches to fluorescently label HCV RNA for live cell microscopy studies, namely an MS2 coat protein mediated approach, and Alexa®UTP labelling
Structural and electrophysiological analysis of Hepatitis C Virus p7
Full text linkInfection with the hepatitis C virus (HCV) has a big impact on global health. It is estimated that approximately 3 % of the world’s population carry HCV, putting more than 200 million people at risk of developing severe liver disease, including chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The HCV encoded viroporin p7 forms ion channels that are crucial for the assembly and secretion of infectious viruses, making it a potential drug target. Its hydrophobic nature makes p7 notoriously difficult to investigate in an untagged native form. A previously determined 16 Å electron microscopy single-particle reconstruction in detergent showed a hexameric, flower-shaped p7 protein. In conjunction with one hexameric and several monomeric p7 solution state NMR structures published, this constitutes the currently available structural information framework. An E. coli expression system is introduced, which is especially adapted to express isotopically labeled p7. For the first time, suitable solution-state NMR conditions at physiological pH and temperature were identified that gave rise to high quality spectra suitable to interrogate iminosugar drug interactions with untagged isotopically labeled J4 p7 (C27S) solubilised in detergent. A novel secondary structure topology was observed and preliminary iminosugar binding sites were determined. Further, a DIB (droplet interface bilayer) system to analyse p7 ion channel function was established, which is suitable to elucidate how inhibitors act on p7 genotypes and how different lipids influence the ion channel function of p7. The p7 oligomeric state was further investigated using native gel analysis, showing that isolates representing HCV genotypes 1 - 6 form oligomeric complexes. An ion channel defective dibasic mutant implicated in severely compromising viral fitness is also shown for the first time to form an oligomer, implicating that it is not an assembly problem that leads to the abrogated function
Emerging principles for the therapeutic exploitation of glycosylation.
No full textGlycosylation plays a key role in a wide range of biological processes. Specific modification to a glycan's structure can directly modulate its biological function. Glycans are not only essential to glycoprotein folding, cellular homeostasis, and immune regulation but are involved in multiple disease conditions. An increased molecular and structural understanding of the mechanistic role that glycans play in these pathological processes has driven the development of therapeutics and illuminated novel targets for drug design. This knowledge has enabled the treatment of metabolic disorders and the development of antivirals and shaped cancer and viral vaccine strategies. Furthermore, an understanding of glycosylation has led to the development of specific drug glycoforms, for example, monoclonal antibodies, with enhanced potency
Mechanisms of immunoglobulin deactivation by Streptococcus pyogenes
Full text linkThe bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved N-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications
Antiviral and immunomodulatory activities of iminosugars: candidate therapeutics for arbovirus infections
Full text linkThe antiviral efficacy of iminosugars with glucose-stereochemistry has been demonstrated against a broad range of enveloped viruses possessing N-glycoproteins. This antiviral activity is primarily understood to be mediated by competitive inhibition of endoplasmic reticulum (ER) α-glucosidases, leading to a reduction in functional viral glycoprotein folding and infectious virion production.
Dengue virus (DENV) is one such virus for which iminosugar efficacy has previously been established. Here, the antiviral efficacy of N-8â-2ââ-tetrahydrofuranyl-octyl-deoxynojirimycin (2THO-DNJ) is evaluated against DENV infection of primary human monocyte-derived macrophages (MDMΦs). The potential for iminosugars to impact stages of the DENV infection cycle in addition to virion secretion is considered, and DNJ-derivative iminosugars are demonstrated to inhibit DENV replication in primary human immune cells for the first time. Alongside DENV, iminosugar efficacy against Zika virus (ZIKV) is investigated, finding important differences in the iminosugar susceptibility and ER α-glucosidase dependence of the two viruses.
Moving away from flaviviruses, the potential for iminosugars to be employed against the emerging Crimean-Congo haemorrhagic fever virus is considered, through use of the little-characterised âsurrogateâ Hazara virus (HAZV). Despite possession of N-glycoproteins, a lack of iminosugar antiviral activity is observed in HAZV-infected SW13 cells.
Common to viral haemorrhagic fevers and certain other disease states is a strong pro-inflammatory, pathological immune response. Immunomodulatory activity of iminosugars has previously been reported, but with a limited range of stimuli used. Here, the impact of iminosugars on cytokine responses to both viral infection and stimulation of MDMΦs with a spectrum of pattern-associated molecular patterns (PAMPs) is investigated, with secretion of biologically functional TNF-α the major read-out. The suppressive effects of DNJ-derivative iminosugars on TNF-α secretion are broadly conserved across stimuli, supporting previous findings that iminosugar immunomodulatory activity is not dependent on viral replication or restricted to PAMPs of a viral origin. In contrast, the galactostereochemistry iminosugar N-nonyl-deoxygalactonojirimycin had more selective effects on cytokine production than the corresponding DNJ-derivative. Preliminary mechanistic investigations considering potential effects of iminosugars on TLR signalling pathways are conducted.
Overall, this thesis explores exciting new features and nuances of iminosugar activities relevant for treating viral infections and combatting a wider range of inflammatory conditions.</p
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