Haselhorst Group

Supervisors: Assoc Prof Thomas Haselhorst and Prof Joe Tiralongo

Structural Biology, Computational Chemistry, Microbiology, Cell Culture, Molecular Biology, NMR spectroscopy

Project details: The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections including Invasive Aspergillosis (IA), a major cause of life-threatening fungal infections in immuno-compromised patients. An over-whelming number of reports appeared in 2020 demonstrating that COVID-19-associated pulmonary Aspergillosis (CAPA) is one of the leading factors affecting morbidity in critically ill COVID-19 patients [2] with some reports even classifying Aspergillosis as a significantly under-recognized ‘Superinfection’ in COVID-19.

Drug resistance among fungal pathogens is continuing to develop into an increasingly serious threat to public health and health-care systems worldwide. This PhD projects entails the development of novel antifungal therapies that are urgently needed using our established and unique combined in-silico/SPR drug discovery pipeline evaluating a number of new protein targets.

Techniques: Computational Chemistry including visualisation and molecular docking, in-silico screening, microbiology, protein expression, drug discovery and design.

Supervisors: Associate Professor Thomas Haselhorst and Dr Chris Day

Structural Biology, Computational Chemistry, NMR spectroscopy, Microbiology

Project details: The decade long overuse of antibiotics in poultry agriculture and consequently the transferral of antibiotic resistance to humans and the associated health problems underlines the urgent need for novel antibiotic-independent strategies, such as feed supplements (prebiotics) that augment commercial poultry performance and provide food safety. This PhD project aims to develop prebiotic treatment options to reduce the colonisation of Campylobacter jejuni in the chicken intestinal tract. Structural and biophysical investigations of glycan-glycan interactions followed by monitoring the bacterial load in chickens and potential cross-contamination into chicken will form the main part of the thesis. Expected outcomes will be the development of a potentially commercially viable non-antibiotic treatment option for poultry farmers in Australia.

Techniques: Structural investigations on Glycans in solution with NMR spectroscopy, biophysical methods and molecular modelling, developing of a virtual glycan array approach, monitoring Campylobacter bacterial strains.

Supervisors: Associate Professor Thomas Haselhorst and Dr Santosh Rudrawar

Research areas: Medicinal Chemistry, Structural Biology, NMR spectroscopy, Cell culture

Project details: Lymphoma is the most common lymphoid malignancy and is among the 10th most prevalent cancers worldwide. Non-Hodgkin’s Lymphoma (NHL) accounts for 80–85% of all lymphomas, including the common B-cell NHLs (B-NHLs). Current standard of care for relapsed/refractory NHLs are anthracyclines that are associated with cumulative cardiotoxicity with limited repeated clinical use. Rituximab-based therapy relies on complement and antibody dependent cell-mediated cytotoxicity to effect cell killing and is associated with severe side effects and in some cases form tumour lysis syndrome.

This PhD project aims to develop novel therapies with an alternative mechanism for B cell killing and improved outcome by synthesising novel carbohydrate-based ligands and conjugating ligands to toxin-loaded liposomes.

Techniques: Synthetic carbohydrate chemistry; Lipid and peptide chemistry, Medicinal chemistry, Cell biology, Cell culture, Computational Chemistry including visualisation and molecular docking, NMR spectroscopy, drug discovery and design.

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: 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: 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: 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: Dr Yaramah Zalucki, Dr Christopher Day, Assoc Prof Thomas Haselhorst & Prof Michael Jennings

Structural biology, enzyme assays, antibiotic resistance, microbiology

Signal peptidase I is an essential enzyme in bacteria that cleaves signal peptides as the final step of protein export to the periplasm. We have discovered a novel secreted protein from Bacillus subtilils, TasA, whose signal peptide can bind signal peptidase I of E. coli (LepB), but is very inefficiently cleaved. In B. subtilis, the TasA signal peptide is removed a dedicated signal peptidase I called SipW, whose protein sequence is more similar to archaeal signal peptidase I. However, it is unknown why the TasA signal peptide is inefficiently cleaved by LepB, and requires an archaeal, not bacterial signal peptidase for its efficient removal. To answer this question, the student will purify SipW, and develop an enzyme assay to measure its cleavage of signal peptides. Subsequently both SipW and LepB will be compared in enzyme assays using the same substrates (based off the TasA signal peptide sequence). The project will develop insights into how archaeal and bacterial signal peptidases differ in their ability to process signal peptides.

The techniques involved in this study include enzyme assays, protein purification and protein analysis (coomassie gels and Western blots), cloning of genes (PCR, DNA manipulation) and other general molecular biology techniques.