Research Projects
Biological and Medicinal Inorganic Chemistry
- Metalloenzyme Mimics
- New Agents for the Diagnosis and Treatment of HIV
- Biosensors and Imaging Agents Based on Metal Complex Bioconjugates
- Metal Complex Bioconjugates as New Tools to Study Pathogens of Socio-economic Importance
- Bioconjugates for Application in Neuroscience
1. Metalloenzyme Mimics
Metalloenzymes utilise one or more metal ions to catalyse chemical reactions. In phospholipase C, for example, three zinc centres catalyse the cleavage of phosphate ester bonds in phospholipids. The increase in structural data on metalloenzymes is sparking interest in small molecule mimics which, apart from improving our understanding of biological function, are finding application as drugs, therapeutics, catalysts, sensors, protein purification media etc. We are developing metalloenzyme mimics which utilise derivatives of 1,4,7-triazacyclononane (tacn) in place of the metal binding residues of the enzyme in an effort to develop small molecule mimics for redox (e.g., superoxide dismutase) and hydrolytic enzymes that cleave peptides (peptidases) and phosphate esters (phosphatases). For example, our research on phosphatase models has produced some of the most active labile metal complexes reported so far and we are pursuing systems with even better activity (Matt Belousoff (PhD), Dr Bim Graham).
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F.H. Fry, L. Spiccia, et al, Inorg. Chem., 2003, 42, 5594 & 5637; Inorg. Chem., 2005, 44, 941. |
2. New Agents for the Diagnosis and Treatment of HIV
 Metal Complex Bioconjugates and HIV Treatment We are developing bioconjugates, consisting of peptide nucleic acid (PNA) – metal complex hybrids, that sense and cleave particular types of RNA and DNA (Matt Belousoff (PhD), Dr Bim Graham, Dr Gilles Gasser). PNAs are robust synthetic analogues of RNA and DNA in which a polyamide backbone replaces the sugar phosphate backbone. The fact that these mimics bind to complementary RNA and DNA sequences more strongly when compared with the analogous oligonucleotides has led to their application as antisense and antigene drugs. We are using PNAs to target particular DNA or RNA sequences (eg., HIV virus) and by attaching hydrolytically active metal complexes to the PNAs we aim to then cleave these sequences catalytically. In contrast to classical antigene and antisense approaches, based on binding affinities, our bioconjugate is released after cleavage of the target biomolecule and can carry out further reactions.
Iinhibitors of the CXCR4 co-receptor One key step in the replication cycle of HIV is the specific binding of virus particles to the CXCR4 co-receptor, a membrane protein located on the surface of T cells, prior to virus internalisation. Studies have revealed that the zinc(II) complexes of a number of ligands based on the “cyclam” macrocycle are potent inhibitors of this interaction (eg. A). These complexes bind to the CXCR4 co-receptor via the carboxylate groups of the residues Asp262, Glu288 and Asp171 (see B). We are seeking to develop new cyclam-based CXCR4 co-receptor inhibitors bearing side-chains capable of hydrogen-bonding to the carboxylate group of Asp171.
Inhibitors of the Tat-TAR interaction A further key step in the replication cycle of HIV is the formation of a complex between a messenger RNA sequence called the “trans-activation response” (TAR) element (C), and the so-called “Tat protein”. We are developing compounds that can bind with high affinity to the “bulge” region of TAR to prevent formation of the Tat-TAR complex and, thus, inhibit viral replication.
3. Biosensors and Imaging Agents Based on Metal Complex Bioconjugates
Our PNA studies include PNA-ferrocene and ruthenium(II) complex hybrids that can be applied as biosensors (Nicki Rajoo (PhD), Dr Bim Graham, Dr Gilles Gasser). In these systems, the PNA chain, which directs the conjugate to the DNA/RNA target, is covalently attached to the redox active complexes or photo-active Ru(II) complex, which detect or manipulate the DNA/RNA (redox sensors or ‘light-up probes’). For example, ferrocenyl biomolecule hybrids are currently being synthesized with the aim of sensing specific RNA/DNA sequences via electrochemical methods. With this in mind, a ferrocenyl derivative (1) has been prepared and the complexation of this assembly with Zn2+ and subsequent binding of thymine to the Zn2+ complex (Figure 1) studied by electrochemistry (G. Gasser, A. M. Bond, B. Graham, Z. Kosowski and L. Spiccia, NSTI Technical Proceedings of 2005 Nanotechnology Conference and Trade Show, Volume 1, Ch 8.2, Bio MicroSensors, 2005, p. 412 – 415.
Figure 1: Receptor 1 complexed with Zn2+ and thymine.
We are optimizing the ligand structure in order to maximize the response upon the addition of thymine. At the same time, we are investigating the detection of nucleosides such as thymidine (dT) by our redox active ferrocenyl derivative. We are also developing methods for incorporating these redox active assemblies into oligonucleotide sequences with a view to producing sequence-specific redox sensors.
We are also interested in PNA–metal complex hybrids that incorporate gadolinium, a metal used widely as a paramagnetic contrast agent in Magnetic Resonance Imaging (MRI). PNAs will allow contrast enhancement of intracellular compartments. In collaboration with Prof. Holger Stephan (Institute for Bioinorganic and Radiopharmaceutical Chemistry, Dresden, Germany), peptide bioconjugates incorporating ligands that bind the Cu-64 radionuclide strongly are being explored as imaging and anticancer agents.
The vascular structure of cancer is porous, and unlike normal cells contains pores which are ~ 400nm in size. This means that cancer therapy or diagnosis can make use of nanoparticles that are able to enter cancer cells but not normal cells. We are interested in combining various metal oxide nanoparticles with cancer diagnostic and therapeutic agents (radionuclides, MRI agents and hydrolytic cleavage agents, etc.).
4. Metal Complex Bioconjugates as New Tools to Study Pathogens of Socio-economic Importance
(Collaboration with Prof Robin Gasser, Dept of Veterinary Science, The University of Melbourne)
 Australia derives substantial earnings from the export of quality certified animals and animal products. A key component to maintaining its competitive position in international trade flows from an advantageous animal health status and “clean-green image” globally. Parasites of animals (see Figs.on RHS) are of major relevance since the current financial losses caused by parasites to agriculture have a major impact on farm profitability.Parasites are mainly controlled through the use of chemotherapeutic agents (anthelmintics). This type of control is expensive and, in most cases, only partially effective. Also, the excessive and uncontrolled use of such agents has resulted in serious resistance problems and the use of such drugs poses risks of residue problems in meat, milk and the environment, as well as potential risks of resistance in pathogens.
Given the increasingly stringent demands placed on maximum residue levels, the ongoing development of novel and improved control strategies are crucial. The possibilities include the rational development of diagnostic tests and/or the selective eradication of the parasite responsible for the disease. We are exploring the exciting prospect of using metal complex bioconjugates in the detection and eradication of common parasites.
5. Bioconjugates for Application in Neuroscience
Collaboration with Prof David Reutens, Faculty of Medicine, Monash University
Alzheimer’s Disease
As the Australian population ages, the prevalence of Alzheimer’s disease is set to reach epidemic proportions. It is predicted that by 2040, 500,000 Australians will have Alzheimer’s Disease, a disorder characterized by the progressive loss of memory and other faculties. Already, dementia costs $6.6 billion per year in Australia. This project aims to develop new biomarkers for Alzheimer’s disease. This will confer a number of benefits, allowing patients to be diagnosed and staged, and allowing pre-symptomatic identification of patients and monitoring of treatment effects. The characteristic pathologic feature of Alzheimer’s disease is the neuritic plaque comprising Aβ amyloid and neurofibrillary tangles made of paired helical fragment (PHF)-tau aggregates. The aim of this project is to develop biomarkers which can be labeled with relatively long half-life radioisotopes.
The current view is that the amyloid precursor protein (APP) contains an Aβ region which, if metabolized incorrectly by enzymes, generates a β–amyloid peptide (Aβ) consisting of 39-42 amino acids that can aggregate into fibrils. The Aβ peptide contains a histidine rich region which can bind strongly to metal ions, such as copper(II) and zinc(II), and this process may promote aggregation. The fact that copper(II) binds strongly to the Aβ peptide will be used to advantage in this project, which aims to develop new organic molecules that: (i) bind rapidly to copper(II) ions so that radiolabelling with Cu-64 is achieved quickly; (ii) are able to cross the blood brain barrier and to reach the region of interest; and (iii) are able to bind to the Aβ peptide or fibrils.
Imaging Gene Expression
The revolution in molecular genetics now allows us to study the effects of mutant genes causing inherited human diseases. This is an important step in discovering potential cures for these disorders and in understanding the mechanisms of disease causation in similar non-inherited disorders.
One key methodology is the use of the transgeneic mouse, in which the mutant gene is inserted into an experimental animal. Molecular genetic methods allow the expression of the mutant gene to be turned on and off during development. Until recently, it has been difficult to study gene-expression non-invasively and to determine the localization and extent of gene expression (protein formation). This is an important capability if we are to understand the delayed effects of expression of the mutant gene. This project aims to develop new reporter molecules to indicate gene expression in a transgenic model of generalized epilepsy. The molecules of interest will be bioconjugates consisting of, for example, PNAs attached to reporter groups which can sense and measure forms of DNA/RNA responsible for protein expression by a particular gene. The approach will be to 'knock-in' a reporter gene downstream from the gene of interest, which is transcribed when the gene of interest is transcribed and which will interact with the tracer or biomarker, e.g., the Na-I symporter that leads to iodine uptake or thymidine kinase activity. |
