Ve Group

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

Supervisors: Dr Thomas Ve, Dr Yun Shi & Assoc Prof Daniel Kolarich

Structural Biology, Biochemistry, Microbiology, Innate Immunity

In both animals and plants TIR domain enzymes have important immune functions. While bacterial TIR proteins have long been recognised, their biochemistry and function remain poorly understood. Some TIR domain containing proteins with NAD+ cleavage activity have been reported to be involved in (i) subversion of host innate immunity (4) and (ii) in antiphage defence systems, but the mechanism of how these proteins utilise NAD+ and its metabolites to modulate the immune system, or provide resistance against phage infection has not yet been explored. As the bacterial TIR domain family is widespread and highly sequence diverse the characterised NAD+ cleaving bacterial TIR domains is likely to only comprise a small fraction of this family’s enzyme diversity and a kingdom wide analysis of them will allow systematic identification of new bacterial signalling nucleotides as well as potential agonists/antagonists of the innate immune system in animals and plants. Mechanistic understanding of bacterial defence systems has previously led to the development of revolutionary biotechnological tools such as restriction enzymes and CRISPR-Cas. Understanding the mechanism of new defence systems such as the ones containing TIR domains may facilitate strategies for developing new useful molecular tools.

This multidisciplinary project can include work on one, or several, topic, including: (i) Characterise the structural basis of TIR domain NADase activity; (ii) Explore the diversity of nucleotide signals produced by bacterial TIR domain containing proteins (iii) Identify the mechanisms that regulate TIR domain NADase activity; and (iv) Define the interactome of TIR domain produced nucleotide signals.

Techniques: X-ray Crystallography, Cryo-EM, NMR, Enzyme assays, HPLC, mass spectrometry.

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 Thomas Ve & Assoc Prof Haselhorst

Innate Immunity, Biochemistry, Structural Biology, Molecular Modelling

Toll-like receptors (TLRs) detect pathogens and endogenous danger, initiating innate immune responses that lead to the production of pro-inflammatory cytokines. At the same time, TLR- mediated inflammation is associated with a number of pathological states, including infectious, autoimmune, inflammatory, cardiovascular and cancer-related disorders. This dual role of the pathways in protecting against infection and contributing to pathological conditions has attracted widespread interest from pharmaceutical and biotechnology industries.

Cytoplasmic signaling by TLRs starts by their TIR (Toll/interleukin-1 receptor) domain interacting with TIR-containing adaptor proteins MyD88 (myeloid differentiation primary response gene 88), MAL (MyD88 adaptor-like/TIRAP), TRIF (TIR-containing adaptor inducing interferon-β/IFNβ), and TRAM (TRIF-related adaptor molecule) Combinatorial recruitment of these adaptors via TIR:TIR interactions orchestrates downstream signaling pathways, leading to induction of the pro-inflammatory genes. Although TLR pathways have been well characterized, molecular information on the signaling proteins is limited, impeding the development of therapeutic strategies and the understanding of the effects of polymorphic variants on human disease

This project aim to identify new inhibitors of TLR4 signalling, which will involve screening of small molecule compound libraries using an in vitro TIR domain assembly assay established for the TLR4 adaptor proteins MAL and MyD88. Compounds shown to inhibit assembly formation will be characterised in more detail for protein interaction using Saturation Transfer Difference (STD) NMR, surface plasmon/isothermal titration calorimetry, and X-ray Crystallography. This information will then be used with molecular modelling and structure analysis to generate more effective small-molecule inhibitors as potential leads for drugs.

Techniques:Production and purification of the TIR domains from the TLR adaptor proteins MAL and MyD88 using established protocols, screening of small molecule libraries using an established biochemical TIR domain assembly assay, STD-NMR, surface plasmon resonance or isothermal titration calorimetry, X-ray crystallography, in silico structure analysis and molecular modelling.

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.