Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Reduced variance in monozygous twins for multiple MR parameters : implications for disease studies and the genetic basis of brain structure

Twin studies offer the opportunity to determine the relative contribution of genes versus environment in traits of interest. Here, we investigate the extent to which variance in brain structure is reduced in monozygous twins with identical genetic make-up. We investigate whether using twins as compared to a control population reduces variability in a number of common magnetic resonance (MR) structural measures, and we investigate the location of areas under major genetic influences. This is fundamental to understanding the benefit of using twins in studies where structure is the phenotype of interest. Twenty-three pairs of healthy MZ twins were compared to matched control pairs. Volume, T2 and diffusion MR imaging were performed as well as spectroscopy (MRS). Images were compared using (i) global measures of standard deviation and effect size, (ii) voxel-based analysis of similarity and (iii) intra-pair correlation. Global measures indicated a consistent increase in structural similarity in twins. The voxel-based and correlation analyses indicated a widespread pattern of increased similarity in twin pairs, particularly in frontal and temporal regions. The areas of increased similarity were most widespread for the diffusion trace and least widespread for T2. MRS showed consistent reduction in metabolite variation that was significant in the temporal lobe N-acetylaspartate (NAA). This study has shown the distribution and magnitude of reduced variability in brain volume, diffusion, T2 and metabolites in twins. The data suggest that evaluation of twins discordant for disease is indeed a valid way to attribute genetic or environmental influences to observed abnormalities in patients since evidence is provided for the underlying assumption of decreased variability in twins.

Property investment, construction and economic growth : the case of Malaysia

It is generally accepted that there is a close relationship between property investment and construction activity. The construction sector plays a crucial role in economic development, especially for a developing nation such as Malaysia. However, the volume of new properties added to the property market is only a fraction of the total volume of the property market. Is the conventional assumption of the relationship between property investment and construction supported by empirical data? This paper revisits the tripartite relationships between economic growths, property investment and construction activities with official Malaysian 2000Q1-2010Q4 quarterly time series data. The Granger causality tests are used to establish the causality runs from the GDP to the value of property transactions, and the growth of construction activities to GDP growth. The result is expected to be useful for policymakers and industrial practitioners in formulating industrial policies and corporate strategies.

Assessing the real value in residential property investment

One of the major fall outs from the Global Financial Crisis has been the decline in residential property construction, home lending and residential property prices. This has lead to some extent to a reduction in the number of small investors willing to commit funds to an investment market that is not seen to perform as well as other investment assets, particularly in relation to income return.With a decreasing supply of rental accommodation in the housing markets, less public housing being constructed by both State and Commonwealth Governments, there is the potential for the residential property market to provide more substantial returns than previous years.This paper will analyse the current residential housing market in Brisbane, Australia to determine if there are sectors in this market that are outperforming the average income and total return for residential investment property and the variation in investment performance across the various housing sub-markets. The results show that property investment in residential property provides opportunities to maximize returns based on geographic location and socio-economic economic status, with lower value areas showing the highest income returns and higher value suburbs showing greater capital returns

The Profiling of Property, Plant & Equipment (PPE) Contributions in Australia and Malaysia Public Listed Construction Companies

The purpose of this paper is to study the profiling of property, plant and equipment (PPE) contributions in Australia and Malaysia construction companies. A company’s worth is usually based on the listed share price on the stock exchange. In arriving at the net profit, the contribution of PPE in the company’s assets is somehow being neglected. This paper will investigate the followings; firstly the level of PPE contribution in the construction firms by comparing the PPE contributions to the company’s asset as a whole which includes fixed (non-current) assets and current assets. This will determine the true strength of the companies, rather than relying on the share prices alone. Secondly, the paper will determine the trend of company’s asset ownership to show the company’s performance of the PPE ownership during the period of study. The data is based on the selected construction companies listed on the Australian Stock Exchange (ASX) and Malaysian Stock Exchange, known as Bursa Malaysia. The profiling will help to determine the strength of the construction firms based on the PPE holding, and the level of PPE ownerships in the two countries construction firms during the period of study.

Sale of property gifted under a will by an attorney acting under an enduring power of attorney : does the gift adeem and what is the attorney's duty?

The authors examine Moylan v Rickard and how the case illustrates the effectiveness of the Powers of Attorney Act 1998 (Qld) to provide remedies and other possible avenues of redress

Energy efficiency and sustainability in residential property

The issue of a more sustainable environment has been the aim of many governments and institutions for decades. Current research and literature has shown the continuing impact of global development and population increases on the planet as a whole. Issues such as carbon emissions, global warming, resource sustainability, industrial pollution, waste management and the decline in scarce resources, including food, are now realities and are being addressed at various levels. All levels of government, business and the public now equally share responsibility for the continued sustainable environment in general. Although these issues of global warming, climate change and the overuse of scarce resources are well documented, and constantly covered in all media forms, public attitudes to these issues vary significantly. Despite being aware of these issues many individuals consider that the problem is one for governments to tackle and that their individual efforts are not important or necessary. In many cases individuals are concerned with sustainability, but are either not in the position to take action due to economic circumstances or are not prepared to offset sustainability gains with personal interests...

Place and Space: Community in the Internet Economy and What This Will Mean for Property

By 2020 Australia‟s National Digital Economy Strategy aims to increase household online participation and engage 12 per cent of all employees in teleworking arrangements. Achieving these goals is generally perceived as positive due to the reduced impact on the natural environment from less use of transport. However, this also will enable greater flexibility as to where people live and thus will impact upon the maintenance and formation of communities and on property use. This paper commences by clarifying what is Australia‟s internet economy before highlighting the impact of the internet on community formation and maintenance. The paper concludes by identifying what the achievement of these goals will mean for property use in the future.

Property, planning and purpose : legal concerns facing broadband rollout in Australia

The Australian government, and opposition, are committed to facilitating high-speed broadband provision. In April 2009 the (then) Labor government announced a proposal to facilitate provision by mandating “…the use of fibre optic infrastructure … in greenfield estates ….” Separately, the installation of (usually overhead) cables commenced in select brownfield areas throughout Australia. In the lead up to the 2010 federal election, the broadband policy focus of the (then) federal opposition was to enabling private investment rather than direct investment by government itself. High-speed broadband is essential for Australia’s economic future. Whether implementation is undertaken by government, government owned corporations or private investors, will impact on the processes to be followed. Who does what, also will determine the rights available to land owners. The next stage, of necessity, will involve the establishment of procedures to require the retrofitting of existing urban environments. This clearly will have major property, property rights and valuation impacts. As Horan (2000) observed “…preserving... unique characteristics … of…regions requires a compromise between economic ambitions and social, cultural, and environmental values”. The uncertainty following the federal election, and the influence of independants with individual agendas; presents unique challenges for broadband implementation. This paper seeks to identify the processes to be followed by various potential broadband investors as they work to establish a ubiquitous network. It overviews current legislative regimes and examines concerns raised by stakeholders in various government reviews. It concludes by plotting a clear way forward to the future, with particular regard to property rights and usage.

Enabling sustainable property practices by ensuring security of tenure

Sustainable property practices will be essential for Australia’s future. The various levels of government offer incentives aimed at encouraging residents to participate in sustainable practices. Many of these programmes however are only accessible by owner occupiers, or landlords and tenants with long term tenancies. Improving security of tenure for tenants, to enable longer term tenancies, would positively impact upon property practices. This article explains what security of tenure is and identifies how a lack of security of tenure adversely impacts property practices. By comparison with Genevan property practices, it concludes by making suggestions as to how security of tenure can be reinforced.

An attack on Iran: the legal basis, or lack thereof

Talk of a possible Israeli strike on Iran’s nuclear facilities has re-ignited debate over the right of self-defence under international law. Some academics, including Anthony D'Amato and Alan Dershowitz, have claimed that an attack on Iran would be a permissible act of self-defence. Others, such as Kevin Jon Heller, argue that such action would be a clear breach of international law. So, who is correct? Would military action against Iran be legal or illegal?

Its place in property education : where does property law fit into property programs?

Property law is one of the key elements in any property-based degree program. In particular, an understanding of 'property law' is one of the required knowledge fields for inclusion in property programs accredited by professional institutes such as the Royal Institution of Chartered Surveyors, the Appraisal Institute and the Australian Property Institute. Despite the importance of property law as a cornerstone element of all property programs this aspect of the program is often approached from a more generic legal perspective with teaching resources used and pedagogical approach more aligned to the study of law that property. The specificity of this type of program is rarely adequately acknowledged. The question arises as to what the study of 'property law' entails and what the composition of a 'property law' subject should be. Replicating the methodology used by Placid and Weeks (2009) in their examination of the current composition of real estate law courses in the United States, this paper examines the current composition and pedagogical approach adopted by Australian universities based on the study of three Queensland property programs. In particular the curriculum, teaching resources used, assessment and engagement strategies are considered with a view to making improvements to the way these property law courses can be more effectively tailored to property students. It is anticipated that the outcomes of this paper will be of interest to all academics who are responsible for developing and delivering property law subjects and those who manage property programs in Australia and internationally.

Reviewing the impact of the 2008/2009 global financial crisis (GFC) on international property markets and property professions

The global financial crisis that impacted on all world economies throughout 2008 and 2009. This impact has not been confined to the finance industries but has had a direct and indirect impact on the property industry worldwide from both an ownership and investment perspective. Property markets have experienced various levels of impact from this event, but universally the greatest impact has been on the traditional commercial and industrial property sectors from the investor perspective, with investment and superannuation funds reporting significant declines in the reported value of these investments. Despite the very direct impact of these declining property markets, the GFC has also had a very significant indirect impact on the various property professions and how these professions are now operating in this declining property market. Of particular interest is the comparison of the property market forecasts in late 2007 to the actual results in 2008/2009.

Submission to discussion paper : Queensland retirement villages

This submission is directed to addressing Issue 4 only – Entering a village and closure – implications for residents; and one question only – What particular items do you consider should be particularly addressed as part of the disclosure requirements for prospective retirement village residents? The recommendations below are premised on the basis that information to enable informed choice needs to be clearly presented, easy to understand and, while legal advice and assistance remains essential for any form of ‘conveyancing’ process, should enable decision making by prospective residents independent of that legal advice.