von Itzstein Group

Supervisors: Prof Mark von Itzstein, Dr Patrice Guillon, Dr Ibrahim El-Deeb, Dr Larissa Dirr, Dr Thomas Ve, Dr Robin Thomson, & Dr Alpesh Malde

Molecular Modelling, Medicinal Chemistry, Molecular Biology, Biochemistry, Structural Biology

Human parainfluenza viruses (hPIV) are important respiratory tract pathogens, second only to respiratory syncytial virus. Infants, young children, the elderly and the immunocompromised are most severely infected, developing croup, pneumonia or bronchiolitis that may need patient hospitalisation. Currently there are neither vaccines nor specific antiviral therapy available to prevent or treat hPIV infections.

Among the hPIV proteins, the haemagglutinin-neuraminidase (HN) glycoprotein represents a promising target for new antiviral drug discoveries. The HN protein is crucial in several steps of the virus life cycle. Firstly, HN recognises and binds sialic acid exposed at the surface of the host cell. Moreover, HN binding is necessary for the activation of the hPIV fusion (F) protein that allows fusion of the cell and virus membranes. HN also has an important action during the viral budding process because it cleaves sialic acid from glycoconjugates to prevent the accumulation of virions at the cell surface and their auto-agglutination. Sialic acid recognition is the trigger of all these HN activities, and the research experiments of this project are focused on the development of high potency inhibitors of the HN–sialic acid interaction.¹

The X-ray crystal structures of the HN glycoprotein of hPIV types 3 and 5, and of Newcastle Disease Virus are available and can be used as homology models for the study of HN from other hPIV subtypes. While some characterisation of hPIV glycan receptor interaction has been undertaken, a complete systematic study is yet to be done. Furthermore, the combination of molecular modelling, structure-based design, fragment screening, and synthetic chemistry, may provide new inhibitors of viral replication. Using biochemistry and structural biology techniques on whole virus and recombinant HN glycoprotein, the effect of these new inhibitors on the virus/glycan interaction can be investigated.  A student working on this project may specialise in one particular aspect or be involved with a number of the different aspects of the project.

1. Reviewed in:  Chibanga V et al, Antiviral Res. 167: 89–97 (2019).  doi: 10.1016/j.antiviral.2019.04.001.

Techniques: Computational Chemistry including visualisation and molecular docking; Fragment screening using ¹⁹F NMR; Synthetic Chemistry; Protein expression and purification; Virology; Biological Assays; Advanced NMR techniques including STD-NMR, X-ray crystallography.

Supervisors: Prof Mark von Itzstein, Dr Robin Thomson, Dr Chih-Wei Chang, Dr Andrea Maggioni, Dr Benjamin Bailly, Dr Yun Shi, Dr Thomas Ve & Dr Xing Yu (Hunan Normal University, China)

Molecular Modelling, Medicinal Chemistry, Biochemistry, Molecular Microbiology, Virology, Structural Biology

Sialidases are involved in the infective cycles of a range of viruses, bacteria, and parasites. These include, for example, the causative agents of influenza, cholera, and African sleeping sickness.  The essential roles that the sialidases often play in the infection cycle make them interesting targets for drug design.  In the case of influenza virus sialidase, development of potent and selective inhibitors of the enzyme, based on knowledge of the enzyme structure, led to a new drug class to treat influenza.  In recent years, new structural and mechanistic characteristics of microbial sialidases have been discovered, presenting new opportunities for inhibitor design.

The von Itzstein group works on the development of new sialidase inhibitors against pathogenic organisms using a multidisciplinary approach that includes: computational chemistry and structure-based inhibitor design; synthetic chemistry, working on a range of inhibitor templates; expression and purification of recombinant enzymes; the use of whole virus particles or virus-like particles presenting the enzyme on a non-infectious particle; enzyme assays for evaluation of inhibitor affinity; cell-based evaluation of compounds, and; structural biology studies in solution phase (NMR) or through X-ray crystallography.  A student working on this project may specialise in one particular aspect or be involved with a number of the different interconnected aspects of the project.

Techniques: Computational Chemistry including visualisation and molecular docking; Synthetic carbohydrate chemistry; Protein expression and purification; Virology; Enzyme Assays; Cell-based assays; Advanced NMR techniques including STD-NMR; X-Ray crystallography.

Supervisors: Dr Benjamin Bailly, Dr Crystall Swarbrick & Prof Mark von Itzstein

Virology, Structural Biology, Cell Biology

The hand, foot and mouth disease causing agent enterovirus 71 engages a variety of receptors on the surface of host-cells prior to entry. These receptors include the P-selectin glycoprotein ligand-1 (PSGL-1), the scavenger receptor class B member 2 (SCARB2), glycosaminoglycans (GAG) and sialylated glycans. The interplay between these receptors is still poorly understood. The types of GAGs and sialylated glycans the virus binds to have not been fully investigated, and we believe that given our progress with GAG-like binding inhibitors they may be more important than previously reported. Furthermore, in our experience different cell-types have different susceptibilities to glycan-based binding inhibitors, suggesting that cell binding events may be more complicated than previously characterised.

This multidisciplinary research project involves the differentiation of various cell types and subsequent functional assays to investigate virus-cell binding events, glycan-array experiments, cell-based chemical combination assays using glycans, competition STD-NMR experiments and crystallography using purified virus particles.

Techniques: Virology; Cell biology; Crystallography; NMR techniques including STD-NMR; Glycan-Array.

Supervisors: Dr Mehfuz Zaman & Prof Mark von Itzstein

Vaccinology, liposome design and formulation, drug delivery, immunology, virology, microbiology

The upper respiratory tract (URT) is the major entry site for multiple pathogens including Influenza A-B, Streptococcus pyogenes (group A streptococci, GAS) and coronaviruses. We are establishing a ‘modular’ multi-pathogen vaccine platform using liposomes (phospholipid vesicles). The liposomal delivery system allows the incorporation of both viral and bacterial peptide epitopes (a part of a protein recognised by antibodies and cells of the immune system) to prevent URT infection. The liposomal formulation can be stored as a lyophilised powder and reconstituted prior to immunisation, yielding a stable product that potentially does not require a cold-chain from production to needle-free administration. Incorporating lipid-linked sugars (glycolipids) enhances secretory immunoglobulin A (IgA)-mediated mucosal immunity that may reduce infectivity of human secretions and transmission.

This multidisciplinary research project involves liposome formulation, testing in pre-clinical models and immunological and functional assays to examine the mechanisms of protection.

Techniques: Vaccine design, Enzyme Linked Immunosorbent Assays (ELISA), in-vitro cell culture assays such as viral propagation and plaque forming assays, in-vivo techniques such as viral and bacterial challenges, immunization and sample collection from pre-clinical models.

Supervisors: Dr Thomas Ve, Dr Yun Shi, Dr Andrea Maggioni, Prof Johnson Mak & Prof Mark von Itzstein

Structural Biology, Biochemistry, Virology

Nipah virus (NiV) is a highly lethal (risk group 4) zoonotic paramyxovirus causing severe, rapidly progressive encephalitis in humans with the case fatality rate ranging from 40-70%. NiV is closely related to Hendra virus (HeV), another risk group 4 paramyxovirus that is native to Australia and infects both horses and humans. NiV is widely distributed in Southeast Asia, India, and Africa. WHO has earmarked NiV on a priority list of eight pathogens that is expected to cause severe outbreaks in the near future. While a one-health approach of vaccinating the intermediate host (horse in the case of Hendra) is able to block the transmission of Hendra virus, the limited uptake of the Hendra vaccine by horse owners could potentially make such approach ineffective. Furthermore, transmission of NiV to humans may occur after direct contact with infected bats, infected pigs, or from other NiV infected people making a one-health preventive approach not practical to NiV, due to the lack of reliance of an intermediate host.

The NiV envelope proteins, glycoprotein G and fusion protein F, are the determinants of viral entry. G and F achieve this via their recognition of the host- cell surface proteins Ephrin-B2 and -B3, and the glycosaminoglycan heparan sulfate (HS). Although detailed structural information is available for the G/Ephrin-B2/B3 interactions, the structural basis for how the G protein coordinates selective binding to specific glycans, such as HS is completely unknown. Furthermore, the mechanistic details of how any of the host-cell receptors trigger viral fusion are poorly defined.

This project will involve a combination of biophysical and cell-biology approaches and aims to define the molecular basis of NiV/HeV interaction with host-cell glycans (glycointeractome), define the molecular mechanisms underlying fusion activation and identify inhibitors targeting these processes. A student working on this project may specialise in one particular aspect or be involved with a number of the different aspects of the project.

Techniques: X-ray crystallography, cryo electron microscopy (cryo-EM), saturation transfer difference nuclear magnetic resonance (STD NMR), library screening, surface plasmon resonance (SPR), glycan arrays, and viral infection assays using pseudotyped particles.

Supervisors: Dr Andrea Maggioni & Prof Mark von Itzstein

Cancer Biology, Biochemistry

Exosomes are vesicles that are secreted from cells and appear to have roles in the tumour microenvironment, including in metastasis. These vesicles are therefore thought to be invaluable in both a diagnosis setting as well as therapeutic targets. Little is known about the cell surface changes in glycans and glycan-recognising proteins.  This project will explore these changes using a multidisciplinary approach that may identify potential biomarkers and therapeutic targets that could be used in diagnosis and drug discovery, respectively.

Techniques: Cell biology, Biochemistry, Glycoanalytics

Supervisors: Dr Arun Everest-Dass, Assoc Prof Chamindie Punyadeera, Prof Mark von Itzstein & Assoc Prof Daniel Kolarich

Glycomics, proteomics, cancer, cancer-biomarkers, cancer microenvironment, cancer diagnostics, multi-omics

Understanding cancer and patient-specific dynamics of protein glycosylation holds enormous yet unmined potential for cancer precision medicine. Glycosylation is a dynamic protein post translational modification in which defined sugars (so called glycans) are attached to proteins by highly individual biosynthetic pathways. Human blood groups are one example of the individuality and clinical relevance of protein glycosylation, as specific glycans form the molecular basis of the human ABO blood group system. About 2 % of human genes are dedicated to biosynthetic pathways of this glycosylation machinery. Genomics and transcriptomics can provide some information about the presence or absence of glycosylation-relevant genes. However, the biosynthetic events that regulate the glycosylation machinery are beyond direct genomic and transcriptional regulation. Glycomics and glycoproteomics approaches thus are the only technologies that can be employed to sequence the cancer glycocode.

In close collaboration with national and international clinical partners we are studying cancer glycocode to understand why cancer forms, what makes individual cancers specific and to identify the weak points that allow us to develop novel strategies to fight cancer. With a focus on cancers such as Leukaemia, Prostate cancer, Melanoma, Ovarian cancer, Head & Neck Cancer or Colon cancer we use highly sensitive and selective glycan/glycoprotein sequencing tools to study cell surface glycoconjugates and their role in pathological processes. One technology involves cutting-edge Laser capture Microdissection that allows the specific cutting of cancer cells from tissue that has revolutionised how we can read the language of cancer. As part of the Australian Centre for Cancer Glycomics (A2CG) we are now systematically applying our glycan-sequencing technologies to sequence cancer glycomes in a variety of cancers.

Be part of the cancer glyco-revolution. A number of student projects are available supporting this important endeavour that will result in a new generation of diagnostic and prognostic cancer markers. As part of this project, students will be introduced to biochemistry laboratory workflows that include (but not limited to) SDS-PAGE, Western blotting, Proteomics and Glycomics sample preparation, acquisition and data analyses and gain general knowledge in Biochemistry and Glycobiology. They will work in an interdisciplinary and multi-national team at the direct interface between the clinic and the research lab.

Techniques: mass spectrometry, glycomics, proteomics, Western blotting, Laser Capture Microdissection microscopy, basic biochemical workflows

Supervisors: Dr Larissa Dirr, Dr Alpesh Malde, Prof Joe Tiralongo, Prof Mark von Itzstein, Assoc Prof Daniel Kolarich

Glycoproteomics, biochemistry, signaling, protein structure

Receptor glycoproteins are highly important signalling molecules in controlling cell communication and interaction. Dysregulation of these signalling pathways is frequently associated with diseases such as cancer and chronic inflammatory conditions. However, the role their glycosylation plays for protein structure and interaction is still poorly understood.

Type III family of receptor tyrosine kinases such as c-KIT (also known as SCF receptor or CD117 PDGF-receptor-a and b, CSF-1 receptor and the FLT3 receptor play a vital role in the pathogenesis across different types of cancer.

As part of a larger project a variety of student projects are available that include aspects of mass spectrometry applications (proteomics, glycomics and glycoproteomics) next to protein structure, cell culture, Western Blot, electrophoresis and other standard biochemistry techniques. In combination these techniques are being employed to characterise and modulate the glycosylation of these important signalling molecules to understand how protein-specific glycosylation impacts protein function and cell signalling. This knowledge will provide opportunities for developing novel therapeutic strategies targeting these receptor proteins. As part of this project, students will be introduced to biochemistry laboratory workflows that include (but not limited to) SDS-PAGE, Western blotting, Proteomics and Glycomics sample preparation, acquisition and data analyses and gain general knowledge in Biochemistry and Glycobiology.

Techniques: mass spectrometry, glycomics, proteomics, cell culture, Western blotting, protein structure, basic biochemical workflows.

Supervisors: Dr Alpesh K Malde & Prof Mark von Itzstein

Molecular Modelling, Computational Medicinal Chemistry, Structural Biology

Sugars (glycans) attached to proteins play an important role in biology, especially in immunity and viral infections. Understanding glycan recognition is crucial for interpretation of the glycan’s biological roles, however current experimental approaches are limited in their ability to resolve glycans at an atomic level. This project focuses on integrating experimental glycosylation, glycan array and NMR data with 3D structure of proteins to generate ‘physiological’ glycoprotein conjugate, protein-protein and protein-glycan complex structures. Computer simulations will be used to investigate glycan interactions at an atomic level and to facilitate design of potential drugs and vaccines. The computational approach involved in the project will be based on molecular dynamics (MD) simulations, which can be used to calculate the time evolution of a system from which various structural, dynamic and thermodynamic properties of interest can be evaluated. The project involves study of receptor-binding proteins from important human pathogenic viruses including but not limited to influenza, parainfluenza, Hendra, Nipah and SARS-CoV-2 viruses.

Techniques: Computational Chemistry and Molecular Dynamics Simulations.

Supervisors: Dr Arun Everest-Dass & Prof Mark von Itzstein

Analytical Glycomics, Biochemistry

Given the universal presence of glycans on all cell surfaces, it is not surprising that several human diseases display changes in glycosylation of proteins and lipids. For example, cancer cells frequently display aberrant glycans than those observed on normal cells.  Mass spectrometry (MS) based glycomic methodologies are now regularly used for the reliable profiling of glycans from clinical samples. Although, routine mass spectrometric glycan analysis is well established and reliable, the analysis of whole tissues destroys any information relating to the spatial distribution of the analytes. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging technique that seeks to utilize the analytical advantages of mass spectrometry whilst preserving the spatial information of the biological molecule of interest inherent in the sample. The unambiguous correlation between histopathology and MALDI–MSI allows the mass measurement of glycans directly from tissue regions [1].

This project aims to develop novel MALDI MSI based imaging technology to characterise glycosphingolipid derived glycans directly from tissue sections.

Techniques: glycomics, MALDI imaging, histopathology.

[1] Everest-Dass AV et al, Mol Cell Proteomics. 2016 Sep;15(9):3003-16. doi: 10.1074/mcp.M116.059816.

Supervisors: Dr Matthew Campbell, Dr Chi-Hung Lin & Prof Mark von Itzstein

Bioinformatics and Biochemistry

Glycoenzymes are responsible for the biosynthesis of all glycans and glycoconjugates and as such provide a rich source of biocatalysts for industrial applications. Over 350,000 sequences (CAZY.com) have already been identified in genomic databases and the number is growing exponentially, but few glycoenzymes are readily available for the glycomics and biotechnology community. This project aims to develop an international glycoenzyme bank (called ZymeBank) that can be used to synthesis complex glycans for use in biological studies.

The project will focus on expressing and purifying human glycoenzymes and building a new database for storing this data along with a web-app interface. This will address a number of challenges: (i) identification of specific glycoenzyme activities for which there is a need in the community; (ii) establish screening protocols that are benchmarked against existing methods; and (iii) set standards and develop an open access databases for glycoenzyme activites and associated biological pathways.

Techniques: Bioinformatics; Databases; Protein Expression; Molecular Biology

Supervisors: Prof Mark von Itzstein, Dr Chih-Wei Chang, Dr Robin Thomson, Dr Andrea Maggioni, Dr Alpesh Malde, Dr Yun Shi & Dr Xing Yu (Hunan Normal University, China)

Molecular Modelling, Medicinal Chemistry, Biochemistry, Structural Biology

b-Glucuronidases are essential mammalian enzymes, which play a major role in the normal structuring and turnover of components of the extracellular matrix. In addition to their roles in normal human biology, over-expression of, in particular, the endo-b-glucuronidase heparanase can facilitate tumour cell growth and spread.

X-Ray structural information is now available for the important endo-b-glucuronidase human heparanase, as well as for a bacterial heparanase.  This structural information can provide new insights into the catalytic mechanism of the enzymes and offers opportunities for inhibitor development.

This project offers a number of avenues for the investigation of b-glucuronidases, which can be either undertaken separately or together; computational chemistry and molecular modelling studies with enzyme X-ray structures; the chemical synthesis of probes to explore enzyme function and activity; biological evaluation of probes and known substrates or inhibitors using enzyme assay and NMR spectroscopy, and; investigation of enzyme–inhibitor complex formation by X-ray crystallography. Each of these aspects will lead to an improved understanding of this important class of enzyme.

Techniques: Computational Chemistry including visualisation and molecular docking; Synthetic Chemistry; Protein expression and purification; Enzyme assays; Advanced NMR techniques including STD-NMR; X-ray crystallography.

Supervisors: Prof Mark von Itzstein, Dr Andrea Maggioni, Dr Robin Thomson, Dr Yun Shi & Dr Xing Yu (Hunan Normal University, China)

Medicinal Chemistry, Molecular Biology, Biochemistry, Cell Biology, Structural Biology, Molecular modelling

Sialic acids are 9-carbon acidic amino-sugars, which are found predominantly at the ends of mammalian glycoproteins and glycolipids.  The terminal location of the sialic acid residues on these cell-surface sialo-glycoconjugates results in their essential involvement in processes of cell–cell, cell–microorganism, and cell–biomolecule interactions.  The amount of sialic acid expressed on a cell's surface, and sialic acid modifications such as O-acetylation, vary throughout development, and in diseases such as some cancers.  A number of microorganisms also express sialic acids on their surface, in some cases mimicking human sialo-glycoconjugate structures which can help the microbe to avoid detection by the host immune system.

We have a number of projects that examine the steps in the sialic acid biosynthetic pathway, to give natural and modified sialo-glycoconjugates.  Non-natural substrates for enzymes of this pathway, or the use of inhibitors, can be used to change the nature and/or level of sialic acid expressed at the cell surface, and so to alter subsequent biological interactions.  In the case of pathogenic bacteria which express surface sialic acids, reduction in the level of sialic acid expression may leave the bacteria more vulnerable to attack by the immune system.

These projects cross a number of disciplines. Aspects of the projects, which can be undertaken either separately or together, include; computational chemistry and molecular modelling studies with enzyme X-ray structures; the chemical synthesis of enzyme probes and inhibitors; biological evaluation of probes and inhibitors using enzyme assay and/or NMR spectroscopy; study of changes of cell surface sialic acid and modifications; and investigation of enzyme–inhibitor complex formation by X-ray crystallography. Each of these aspects will help us gain to an improved understanding of the enzymes of sialic acid metabolism.

Techniques: Computational Chemistry including visualisation and molecular docking; Synthetic Chemistry; Protein expression; Cell-based studies; NMR-based enzyme assays; Advanced NMR techniques including STD-NMR; X-ray crystallography.

Supervisors: Dr Ibraham El-Deeb & Prof Mark von Itzstein

Medicinal Chemistry

The increase in bacteria acquiring resistance to current antibiotics, and a reduction in development of new antibiotics by the pharmaceutical industry over the past years, is placing a significant burden on global health care, with the World Health Organization noting that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Our research is focussed on investigating alternative therapeutic strategies to break antibiotic resistance.  Metal-ion homeostasis is critical for bacterial survival, and elevated metal ion levels can be toxic to bacterial pathogens.  Ionophores are chemical compounds that facilitate transport of metal ions across biological membranes. Together with our collaborators, we have identified ionophores that are able to break antibiotic resistance by destabilizing bacterial metal homeostasis.¹ This project will extend our work in this area, through development and evaluation of new ionophores.

1. Bohlmann L et al., mBio 9:e02391-18 (2018).  doi: 10.1128/mBio.02391-18.

Techniques: Synthetic chemistry

Supervisors: Dr Ibraham El-Deeb, Dr Andrea Maggioni & Prof Mark von Itzstein

Medicinal Chemistry, Cell Biology

We have developed¹ a versatile synthesis of a class of potent anti-cancer agents known as the duocarmycins.  We are now using a further optimised synthesis of this class of compound to discover novel anti-cancer agents that contain added carbohydrate residues to potentially improve biological function (creating glycoconjugates of duocarmycins). Our preliminary biological evaluation of some of these compounds, in cell-based assays, provides us with optimism that such compounds may have good anti-cancer activity. This project will look at the further development of these glycoconjugates as potential anti-cancer drugs.

1. El-Deeb IM et al, Org Biomol Chem. 12(24):4260-4 (2014). doi: 10.1039/c4ob00842a.

Techniques: Synthetic carbohydrate chemistry, Cell biology

Supervisors: Dr Darren Grice & Prof Mark von Itzstein

Medicinal Chemistry

From previous studies^1,2, it is clear that the use of Proteolysis-targeting chimera (PROTAC) molecules can result in the effective degradation of target-proteins. PROTAC techniques involve the exploitation of normal protein degradation essential for cellular maintenance and hijacking the system to specifically target proteins of interest (POI) for degradation.

To achieve an effective PROTAC design the molecule must provide high affinity binding to both the protein of interest and a suitable ubiquitin ligase and maintain these interactions whilst not inhibiting the overall ubiquitination (or tagging for destruction) process.

Work is underway within the Institute for Glycomics to synthesise novel PROTAC molecules to achieve the successful proteolysis of a cancer-associated protein, which is known to be intimately involved in cancer progression. This research will be further progressed in this ongoing project.

Techniques: The project will involve synthetic organic/carbohydrate chemistry, along with NMR spectroscopy, mass spectrometry and other associated techniques for structural characterisation of the synthesised PROTACs followed by assessment of biological activity.

1. Winter, et al. Science. (2015) Jun 19;348(6241):1376–81.

2. Gu, Cui, Chen, Xiong, Zhao. (2018). Bioessays Apr 40(4), e1700247.

Supervisors: Dr Chih-Wei Chang & Prof Mark von Itzstein

Medicinal Chemistry

Glycosaminoglycans (GAGs), large negatively-charged polysaccharides, exist universally on the cell surface and have various functions that include sustaining the integrity of the extracellular matrix (ECM), and acting as ligands for recognition and binding of biological molecules. In particular, their role as biological ligands has received significant attention in the fields of glycobiology and biomaterials. Variation in length, sequence, sulfation degree, and conformational flexibility of the GAG polysaccharide chains give rise to a large number of complex GAG sequences. Detailed study of GAG functions requires homogenous GAG fragments, however, due to this structural complexity, syntheses can be long and challenging, and reliable production of GAGs for in vitro and subsequent in vivo experiments can be a limiting factor.

In this project our aim is to look for alternative GAG-mimetic scaffolds that can replace native GAG sequences.  We will explore these new scaffolds in interactions with specific GAG-recognising proteins and look at their effect(s) on GAG biological functions.

Techniques: Synthetic carbohydrate chemistry

Supervisors: Dr Chih-Wei Chang & Prof Mark von Itzstein

Medicinal Chemistry

Glycosaminoglycans (GAGs), found either on cell membranes or in the extracellular matrix, are classes of large linear polysaccharides carrying negatively charged groups, that are involved in a wide range of physiological processes. The GAG family includes heparan sulfate (HS) and chondroitin sulfate, among others. Most of the roles of GAGs in interacting with proteins, and modulating the host of diverse biological activities, are still poorly understood. Better understanding of the biological interactions between GAGs and GAG-binding proteins requires the use of pure GAG fragments.

The synthesis of complex GAGs in a pure form is not trivial. In this project, we aim to develop new synthetic strategies to access a discrete heparan sulfate (HS) fragment library. The homogeneous HS fragments, that will incorporate 2-O-, 6-O- and N-sulfate groups in a defined manner, will be used to elucidate the interactions between the specific GAG sequences and proteins associated with a variety of diseases including cancer, virus infection and diabetes.

Techniques: Synthetic carbohydrate chemistry

Supervisors: Dr Chi-Wei Chang, Dr Benjamin Bailly, Dr Crystall Swarbrick, Dr Mehfuz Zaman, Dr Robin Thomson & Prof Mark von Itzstein

Medicinal Chemistry, Virology, Structural Biology

The picornavirus Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease in children less than 5 years old worldwide. While the disease usually presents with mild symptoms, it can sometimes spread to the central nervous system and cause severe neurological complications such as flaccid-like paralysis or encephalitis. There are currently no treatments or vaccines against EV71 infection.

EV71 is thought to infect cells by binding to various cellular receptors including glycosaminoglycans (GAGs) and sialylated glycans. While most efforts in anti-EV71 drug discovery are focussed on inhibiting the various viral proteases, we take advantage of the scaffold of naturally occurring glycan receptors to investigate the potential of functionalised glycans and GAG-mimetics to inhibit the virus binding to cells.¹ This project therefore involves medicinal carbohydrate chemistry for the design and synthesis of glycans and their mimetics, virology techniques for cell-based screening and evaluation of compounds, and X-ray crystallography and STD-NMR technologies for the characterisation of virus/glycan binding events.

1. Earley D; Bailly B et al, ACS Infect. Dis. 5: 1708−17 (2019).  doi: 10.1021/acsinfecdis.9b00070.

Techniques: Synthetic carbohydrate chemistry; Virology; Cell biology; Crystallography; NMR techniques including STD-NMR.

Supervisors: Prof Mark von Itzstein, Dr Robin Thomson & Assoc Prof Thomas Haselhorst

Molecular modelling, Medicinal Chemistry, Structural Biology

Rotaviruses are double stranded RNA viruses that are the leading cause of infantile gastroenteritis globally. The resulting dehydrating diarrhoea following infection is responsible for 33% of all hospitalisation of infants.

The triple layered Rotavirus virion must be delivered across host cell membranes into the cytoplasm in order to initiate viral gene expression. Cell-attachment and entry mechanisms are promising targets for therapeutic and preventative interventions against rotavirus diarrhoea.

Rotavirus outer capsids comprise a coat glycoprotein and a spike protein that mediate infection. VP8* is the 18 kDa protein fragment forming the spike tip and binds a cell-surface carbohydrate (sialic acid) during virus attachment to cells. The overall aim of this project is design of carbohydrate based compounds that could bind and block the active site of VP8* thus preventing the virus particle from attaching to the host cell and causing infection. X-Ray crystal structures of VP8* proteins alone and in complex with natural sialic acids are available for use in structure-based design of synthetic ligands, and potential inhibitors of VP8* interactions.

This project offers the potential to combine computational structure-based modelling and design, with chemical synthesis of carbohydrate-based compounds as potential VP8* inhibitors, and evaluation of the VP8*–compound interactions using the technique of STD NMR spectroscopy.

Techniques: Computational Chemistry including visualisation and molecular docking; Synthetic carbohydrate chemistry; Advanced NMR techniques including STD-NMR.

Sialic acid dependence in rotavirus host cell invasion. T. Haselhorst, F.E. Fleming, J.C. Dyason, R.D. Hartnell, X.Yu, G. Holloway, K. Santegoets, M.J. Kiefel, H. Blanchard, B.S. Coulson, M. von Itzstein.(2009)  Nat. Chem. Biol. Feb;5(2):91-93

Supervisors: Prof Mark von Itzstein, Dr Chih-Wei Chang & Dr Robin Thomson

Medicinal Chemistry

Interactions between cells, and between cells and microorganisms, are often based on multiple, simultaneous or sequential, interactions between protein receptors and their carbohydrate ligands. Mimicking these interactions by the use of multivalent arrays of receptor ligands – for example dendritic structures terminated with biologically relevant molecules or displays of molecules conjugated to nanoparticles or liposomes – has been successful for a number of carbohydrate-recognising proteins. This project involves the design and synthesis of multivalent structures, carrying functionalised carbohydrates, that will then be examined as probes and potentially inhibitors in a range of biological systems, for example in cell-binding studies of human pathogenic viruses.

Techniques: Synthetic carbohydrate chemistry.

Supervisors: Prof Mark von Itzstein, Dr Chi-Hung Lin & Dr Robin Thomson

Medicinal Chemistry

Human cell-surface carbohydrates (glycans) on glycoproteins and glycolipids are involved in important cell–cell and cell–biomolecule interactions. They also often form the initial attachment point for invading pathogenic microorganisms. Studies in glycobiology often require the use of a natural, or specifically modified, glycan to characterise and increase understanding of a specific biological interaction. However, not all natural glycans are commercially or readily available.  While methods of chemical glycan synthesis are advancing, there are significant advantages in the use of enzymes to construct both complex monosaccharides, and the linkages between monosaccharide units to form a glycan. Combining chemical manipulation of monosaccharide residues, or of a final glycan structure, with enzymatic linkage formation, it is possible to produce both natural and specifically modified complex glycan structures.  This project will incorporate the use of both traditional carbohydrate chemistry techniques and the use of carbohydrate biosynthetic enzymes, to prepare glycans for use in a range of biological studies.

Techniques: Synthetic chemical and enzymatic carbohydrate chemistry.

Supervisors: Prof Mark von Itzstein & Dr Robin Thomson

Medicinal Chemistry

Bacterial resistance to antibiotics is a growing problem and is driving the search for novel antibacterial therapies. Importantly, bacterial cell membrane components often contain carbohydrate units and structural linkages that are not found in mammalian systems.  The biosynthetic pathways to these structures are therefore attractive targets for the development of antimicrobial agents that affect the growth and integrity of, specifically, bacterial cell membranes. This project, as part of a continuing antimicrobial drug discovery programme, will involve the preparation of carbohydrate-based compounds for the investigation of bacterial cell wall biosynthetic enzymes, and their evaluation as inhibitors of bacterial growth.

Techniques: Synthetic carbohydrate chemistry; Bacterial cell growth assays

Supervisors: Prof Nicolle Packer, Prof Riccardo Dolcetti, Prof Mark von Itzstein & Assoc Prof Daniel Kolarich

Glycoproteomics, cancer biology, Understanding cancer immunotherapy

Cancer therapies have experienced a tremendous revolution with the introduction of therapies that use monoclonal antibodies that specifically target cancer cell surface targets and immune-checkpoint receptors. More than 95% of the protein receptors targeted by these immunotherapy agents are in fact glycoproteins, but to date the impact of receptor glycosylation in precision medicine is still not understood.

In this close collaboration with colleagues from the Peter MacCallum Cancer Centre students will be introduced to biochemistry and immunology laboratory workflows that include (but not limited to) SDS-PAGE, Western blotting, Proteomics and Glycomics sample preparation, acquisition and data analyses and gain general knowledge in Biochemistry and Glycobiology. Cancers that are being targeted in this project include Leukaemia, prostate cancer, melanoma, Head & Neck Cancer, Hepatocellular carcinoma or Colon cancer are investigated.

Techniques: mass spectrometry, glycomics, proteomics, cell culture, Western blotting, basic biochemical workflows

Supervisors: Assoc Prof Thomas Haselhorst, Dr Yun Shi & Prof Mark von Itzstein

Molecular Modelling, Structural Biology

Many carbohydrate-recognising proteins, eg Siglec 2 and trans-sialidases, have been implicated in clinically significant diseases such as non-Hodgkin’s lymphoma and trypanosomiasis, respectively. Recently, the method STD-NMR was developed to screen compound libraries against various protein targets. This method is suitable for determining an epitope map of a ligand within the protein’s binding site as only regions of the ligand that are in contact with the protein’s binding site are observed in the NMR spectrum.

Techniques: Computational Chemistry including visualisation and molecular docking; Advanced NMR techniques including STD-NMR; Protein purification

Supervisors: Assoc Prof Thomas Haselhorst, Dr Andrea Maggioni & Prof Mark von Itzstein

Structural Biology, Biochemistry

Abbott laboratories have published a new NMR spectroscopic method called “SAR by NMR” to identify binding ligands and simultaneously to detect amino acids within the protein binding sites which play a key role in the binding event. This project will involve the expression and purification of ¹⁵N labelled rotavirus VP8* protein in minimal media and the analysis of the purified labelled protein by means of high-resolution NMR spectroscopy. ¹⁵N/¹H-HSQC experiments of the apo protein and complexed with potential binding ligands are acquired. For amino acids involved in the binding event a change in their chemical shifts is likely. This valuable information can then result in lead structures for the design of new anti-viral drugs.

Techniques: Chemical Characterisation including Proton and Carbon-13 NMR; Advanced NMR techniques including STD-NMR; Protein purification.

Supervisors: Dr Thomas Ve, Dr Yun Shi & Prof Mark von Itzstein

Structural Biology, Biochemistry, Medicinal Chemistry

Axon loss is a common theme in some of the most prevalent neurological diseases, including peripheral neuropathies, traumatic brain injury, Parkinson's disease and glaucoma, but no treatments currently exist that effectively target axonal breakdown. The protein SARM1 is a central player in axon loss. In healthy nerve cells, SARM1 (sterile alpha and TIR motif 1) is present but inactive. Disease and injury activate SARM1, which results in rapid breakdown of the essential “helper molecule” nicotinamide adenine dinucleotide (NAD+) and ultimately destruction of the axon. We have demonstrated that it is SARM1 itself that cleaves NAD+ upon activation through self-association and we hypothesise that detailed structural knowledge of the SARM1 catalytic mechanism and defining the molecular mechanisms upstream and downstream of SARM1 enzyme activity can yield inhibitors as leads to anti-neurodegenerative disease therapeutics. This project can include work in one, or several, areas, including Cryo-EM, X-ray crystallography, NMR and inhibitor design.

Techniques: Cryo-EM, X-ray Crystallography, NMR, Enzyme assays, Computational Chemistry including visualisation and molecular docking; Synthetic Chemistry