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Peptide Folding, Membrane InteractionsPeptidomimetic Drug Design and Membrane Nanotechnology Associate Professor Mibel Aguilar Email: mibel.aguilar@med.monash.edu.au
Our group focuses on peptide-based drug design and biomembrane nanotechnology. In collaboration with Assoc Prof Patrick Perlmutter (Chemistry), we are developing novel compounds that allow us to exploit the potential of peptides as drugs. We are currently applying our technology to the development of cancer vaccines (with Dr Tony Purcell, Uni Melb), and new compounds for treatments of cardiovascular disease (with Prof Ian Smith). Our membrane nanotechnology projects involve the development of new methods for membrane protein purification and analysis with application to Alzheimer’s (with Assoc Prof David Small), G protein-coupled receptor function (with Dr Wally Thomas, Baker Heart Research Institute) and new biosensor devices (with Farfield Scientific). The long-term aim of these studies is to increase our understanding of the molecular basis of peptide and protein function and allow the rational design of peptide and protein based therapeutics. 
1. Membrane-mediated biorecognitionThe interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated biochemical processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Indeed, a major target of modern biotechnology is the design of new potent pharmaceutical agents whose biological action is dependent on the binding of peptides with lipid-bilayers. 
Model of the interaction of different peptides & proteins with the plasma membrane. Antimicrobial Peptide ActionAntimicrobial peptides are being increasingly recognised as potential candidate antibacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in recent years. However, a precise understanding of the relationship between antimicrobial peptide structure and their cytolytic function in a range of organisms is still lacking. This is a result of the complex nature of the interactions of antimicrobial peptides with the cell membrane, the mechanism of which can vary considerably between different classes of antimicrobial peptides. Antimicrobial peptide action is mediated by a direct interaction with cell membranes and a common feature of these interactions is the induction of cationic amphipathic secondary structure following binding of the peptides to the membrane surface. Since selective binding to different phospholipids is central to the design of non-hemolytic antimicrobial peptides, the affinity of the peptide for the membrane surface is a critical factor in the cell-lytic process. We have developed a sensitive method based on surface plasmon resonance (SPR), which allows the real-time measurement of peptide binding to phospholipid membranes. The project area focuses on characterising the molecular basis of antimicrobial peptide action to assist the direct design of more potent and selective antimicrobial peptides as leads from new therapeutics for the treatment of bacterial infections. 
Publications:
G Protein-Coupled Receptor (GPCR) RegulationSeven transmembrane-spanning receptors that couple to heterotrimeric G proteins represent by far the largest receptor superfamily in our genome, mediating functions across the spectrum of physiology. Elucidating the mechanisms that activate/deactivate these G protein-coupled receptors (GPCRs) is fundamental to expanding our understanding of these receptors. Moreover, many therapeutics aim to target GPCRs and hence developing an accurate picture of their function is a continuing focus of the pharmaceutical industry. The participation of the plasma membrane in GPCR signaling and regulation is emerging as a key feature of GPCR structure and function and we aim to characterise the molecular details of this synergy. In collaboration with Dr Walter Thomas (Baker Heart Research Institute) we are exploring the role of membranes in the study of membrane mediated receptor activation using the G-protein coupled angiotensin (AT1) receptor as a model system. GPCRs are integral membrane proteins with a structure consisting of an extracellular amino-terminus, seven transmembrane-spanning -helices connected by alternating extracellular and intracellular loops, and a cytoplasmic carboxyl-terminus. The AT1 receptor is a 359 amino acid GPCR that mediates the important cardiovascular and homeostatic actions of the peptide hormone, angiotensin II and it has been shown that receptor expression, affinity, signaling and trafficking is crucially dependent upon the membrane interactions. 
Model of the angiotensin receptor and its interactions with the cell membrane Publications:
Role of Membrane Binding in Neurodegenerative DiseasesUnderstanding the mechanism by which accumulation of the ß-amyloid protein (Aß) in the brain contributes to the onset of dementia is one of the main unsolved problems in the field of Alzheimer’s disease (AD) research. In collaboration with Assoc Prof David Small, we have recently identified a novel mechanism by which Aß exerts its toxic effects via direct binding with the cell membrane, an effect which is mediated by the presence of cholesterol, a known risk factor in AD. In particular, our studies provide a molecular snapshot of Aß formation and aggregation and suggest that Aß binding to membranes is a potential therapeutic target for the treatment of Alzheimer’s disease. We are now applying our membrane biosensor techniques together with atomic force microscopy, in collaboration with Dr David Small, Dr Lisa Martin (School of Chemistry) and Dr Adam Mechler (School of Chemistry), to studying the role of membrane-binding in the cellular toxicity associated with amyloid formation in Alzheimer’s disease. We are also extending these studies to a number of other neurodegenerative diseases associated with protein mis-folding including transthyretin which has been demonstrated to be the predominant component of the amyloid fibrils in familial amyloidotic polyneuropathy. 
Publications:
Membrane Protein Purification & Membrane ProteomicsWith the availability of the total human genome sequence, there is now an enormous effort directed towards the development of new technologies to allow the compositional and functional analysis of the corresponding proteome. It has been estimated that 30-40% of encoded DNA codes for membrane proteins and this class of proteins therefore represents a significant proportion of the cell’s complement of protein. However, membrane protein isolation and analysis continues to be an enormously challenging task despite the significant advances made in separation sciences over the last 30 years. The lack of robust separation techniques for membrane proteins has now led to a bottleneck in both their structural elucidation (there are only ~ 50 x-ray structures of membrane proteins compared to thousands of soluble protein structures) and high-throughput functional analysis. Furthermore, it has been estimated that approximately 70% of current drugs target membrane proteins, clearly demonstrating the importance of this class of proteins to the pharmaceutical industry. We have developed new chromatographic materials, which are being applied to the isolation and analysis of membrane proteins with a particular focus on the proteomic analysis of a number of tissue sources. 
Publications:
Peptidomimetic Drug DesignThe use of peptidomimetics has emerged as a powerful means for overcoming the limitations inherent in the physical characteristics of peptides thus improving their therapeutic potential. A peptidomimetic approach that has emerged in recent years with significant potential, is the use of ß-amino acids. ß-Amino acids are similar to -amino acids in that they contain an amino terminus and a carboxyl terminus. However, in ß-amino acids two carbon atoms separate these functional termini. ß-amino acids, which results in a total of 4 possible diastereoisomers for any given side chain. The flexibility to generate a vast range of stereo- and regioisomers, together with the possibility of disubstitution, significantly expands the structural diversity of ß-amino acids thereby providing enormous scope for molecular design. The incorporation of ß-amino acids has been successful in creating peptidomimetics that not only have potent biological activity, but are also resistant to proteolysis and we are applying these techniques to a range of protein targets. Therapeutic Peptidase InhibitorsThe action of most neuropeptides is terminated by specific extracellular peptidases and these enzymes therefore play an important role in the regulation of the function of the central nervous system. The availability of inhibitors of these enzymes is important for characterising the role of these enzymes in peptide signaling in the brain and ultimately for the development of new therapeutic agents for the treatment of cardiovascular disease. In collaboration with Dr Patrick Perlmutter (School of Chemistry) and Prof Ian Smith, we are focusing on a number of enzymes. EC 3.4.24.15 (EP 24.15) is a widely-distributed enzyme involved in the regulation of blood pressure. The specific function of this enzyme is unknown, but it has been implicated in the metabolism of bradykinin. One of the major reasons that the physiological role of this enzyme is unknown is the lack of a stable enzyme inhibitor. Membrane-bound aminopeptidase P (AP-P) also participates in the degradation of bradykinin in several vascular beds. Together with angiotensin-converting enzyme, AP-P is responsible for a large proportion of the breakdown of bradykinin. Since bradykinin exhibits potent vasodilatory and cardioprotective effects, there is a therapeutic benefit to inhibiting these enzymes and increasing endogenous levels of bradykinin. ACE2 is a very recently discovered enzyme and is expressed largely in the kidney and heart suggesting important functions in cardiovascular and renal systems and currently there is an enormous interest in this enzyme as it has been proposed to be an essential regulator of heart function in vivo We are currently designing novel peptide and peptidomimetic based inhibitors of these enzymes in order to develop more effective approaches for the treatment of cardiovascular disorders. 
The proposed mechanism of action of ECE, 24.15 & 24.16. These sites are targets for new drugs. 
Publications:
Peptide-Based Vaccines - High Affinity Peptide Ligands for Class I MHC ProteinsClass I major histocompatibility complex (MHC) proteins play a key role in immune surveillance by selectively binding to intracellular peptide antigens and presenting them at the cell surface to cytotoxic T-lymphocytes (CTL). Interference of this process by analogues of peptide antigens has been shown to cause significant changes in T cell function which suggests that these analogues have significant potential as immunotherapeutic agents. However, the challenge has been to rationally design analogues of peptide antigens which cause subtle changes in antigen recognition. In collaboration with Dr Tony Purcell at the Dept of Biochemistry & Molecular Biology at the University of Melbourne, we are currently using our peptidomimetic approaches to the design of novel T-cell antagonists. 
Graphic illustration of peptide antigens presented by the MHC class I heavy chain. Alteration of such antigenic peptide structure can affect the T-cell recognition and therefore the activation of CD8+ CTL. Publications:
General Project Areas
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