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.

Designerly analysis of a socio-physical design activity

The Making Design and Analysing Interaction track at the Participatory Innovation Conference calls for submissions from ‘Makers’ who will contribute examples of participatory innovation activities documented in video and ‘Analysts’ who will analyse those examples of participatory innovation activity. The aim of this paper is to open up for a discussion within the format of the track of the roles that designers could play in analysing the participatory innovation activities of others and to provide a starting point for this discussion through a concrete example of such ‘designerly analysis’. Designerly analysis opens new analytic frames for understanding participatory innovation and contributes to our understanding of design activities.

Participatory analysis of mobile diaries to inform the design of ridesharing systems

In many “user centred design” methods, participants are used as informants to provide data but they are not involved in further analysis of that data. This paper investigates a participatory analysis approach in order to identify the strengths and weaknesses of involving participants collaboratively in the requirements analysis process. Findings show that participants are able to use information that they themselves have provided to analyse requirements and to draw upon that analysis for design, producing insights and suggestions that might not have been available otherwise to the design team. The contribution of this paper is to demonstrate an example of a participatory analysis process.

Design of sustainable transport : opportunities

The need to find an alternative to our current transport situation is widely accepted. In most cities of the world, traffic congestion is commonplace and air pollution is normal. Road fatalities are a regular and almost accepted event. And (in most developed nations) as an indirect consequence of our transport choices, obesity is increasing at an alarming rate. The car is undeniably a major contributor to this situation. Additionally the very structure of our cities has evolved to the point that it can be creditably claimed that the city belongs to the car and not to humans. There are however alternatives. There is a plethora of experimental vehicles in all shapes and configurations. And yet, the car is still king. The question is, how do we pick a winner? What are the aspects of the car that make it so appealing? Are these aspects able to be translated into a more sustainable version? What do we need to incorporate in our designs of new vehicles to make them more appealing to the consumers? In this paper I explore these questions and propose a list of design criteria for more sustainable transport options.

Modeling player-like behaviour for game Al design

The design of artificial intelligence in computer games is an important component of a player's game play experience. As games are becoming more life-like and interactive, the need for more realistic game AI will increase. This is particularly the case with respect to AI that simulates how human players act, behave and make decisions. The purpose of this research is to establish a model of player-like behavior that may be effectively used to inform the design of artificial intelligence to more accurately mimic a player's decision making process. The research uses a qualitative analysis of player opinions and reactions while playing a first person shooter video game, with recordings of their in game actions, speech and facial characteristics. The initial studies provide player data that has been used to design a model of how a player behaves.

Reframing the design of context-aware computing

From location-aware computing to mining the social web, representations of context have promised to make better software applications. The opportunities and challenges of context-aware computing from representational, situated and interactional perspectives have been well documented, but arguments from the perspective of design are somewhat disparate. This paper draws on both theoretical perspectives and a design framing, using the problem of designing a social mobile agile ridesharing system, in order to reflect upon and call for broader design approaches for context-aware computing and human-computer Interaction research in general.

Determining the appropriate proportion of owner-provided design in design-build contracts - a content analysis approach

The Request For Proposal (RFP) with the design‐build (DB) procurement arrangement is a document in which an owner develops his requirements and conveys the project scope to DB contractors. Owners should provide an appropriate level of design in DB RFPs to adequately describe their requirements without compromising the prospects for innovation. This paper examines and compares the different levels of owner‐provided design in DB RFPs by the content analysis of 84 requests for RFPs for public DB projects advertised between 2000 and 2010 with an aggregate contract value of over $5.4 billion. A statistical analysis was also conducted in order to explore the relationship between the proportion of owner‐provided design and other project information, including project type, advertisement time, project size, contractor selection method, procurement process and contract type. The results show that the majority (64.8%) of the RFPs provide less than 10% of the owner‐provided design. The owner‐provided design proportion has a significant association with project type, project size, contractor selection method and contract type. In addition, owners are generally providing less design in recent years than hitherto. The research findings also provide owners with perspectives to determine the appropriate level of owner‐provided design in DB RFPs.

Developing a transition pedagogy : intentional curriculum design in first and final year law programs

This chapter will first consider the rationale for a transition pedagogy for first and final year law students. It then discusses the elements of a transition pedagogy for both years, noting the synergies and differences between programs designed to assist transition into and out of a law degree. In doing so, the authors attempt to explore the extent to which the first year curriculum principles identified by Sally Kift under an Australian Learning and Teaching Council (ALTC) Senior Fellowship may also be applied to the final year university experience. During the course of the discussion, examples are drawn from universities and Law Schools in Australia and internationally which seek to address these imperatives...

Influences of green star rating tool on HVAC system design

Buildings are one of the most significant infrastructures in modern societies. The construction and operation of modern buildings consume a considerable amount of energy and materials, therefore contribute significantly to the climate change process. In order to reduce the environmental impact of buildings, various green building rating tools have been developed. In this paper, energy uses of the building sector in Australia and over the world are first reviewed. This is then followed by discussions on the development and scopes of various green building rating tools, with a particular focus on the Green Star rating scheme developed in Australia. It is shown that Green Star has significant implications on almost every aspect of the design of HVAC systems, including the selection of air handling and distribution systems, fluid handling systems, refrigeration systems, heat rejection systems and building control systems.

Design and implementation of a virtual world training simulation of ICU first hour handover processes

Nursing training for an Intensive Care Unit (ICU) is a resource intensive process. High demands are made on staff, students and physical resources. Interactive, 3D computer simulations, known as virtual worlds, are increasingly being used to supplement training regimes in the health sciences; especially in areas such as complex hospital ward processes. Such worlds have been found to be very useful in maximising the utilisation of training resources. Our aim is to design and develop a novel virtual world application for teaching and training Intensive Care nurses in the approach and method for shift handover, to provide an independent, but rigorous approach to teaching these important skills. In this paper we present a virtual world simulator for students to practice key steps in handing over the 24/7 care requirements of intensive care patients during the commencing first hour of a shift. We describe the modelling process to provide a convincing interactive simulation of the handover steps involved. The virtual world provides a practice tool for students to test their analytical skills with scenarios previously provided by simple physical simulations, and live on the job training. Additional educational benefits include facilitation of remote learning, high flexibility in study hours and the automatic recording of a reviewable log from the session. To the best of our knowledge, we believe this is a novel and original application of virtual worlds to an ICU handover process. The major outcome of the work was a virtual world environment for training nurses in the shift handover process, designed and developed for use by postgraduate nurses in training.

Analysis of selection criteria for design-builders through the analysis of Request for Proposals (RFPs)

Selecting an appropriate design-builder is critical to the success of DB projects. The objective of this study is to identify selection criteria for design-builders and compare their relative importance by means of a robust content analysis of 94 Request For Proposals (RFPs) for public DB projects. These DB projects had an aggregate contract value of over US$3.5 billion and were advertised between 2000 and 2010. This study summarized twenty-six selection criteria and classified into ten categories, i.e.: price, experience, technical approach, management approach, qualification, schedule, past performance, financial capability, responsiveness to the RFP, and legal status in descending order of their relative importance. The results showed that even though price still remains as the most important selection category, its relative importance declines significantly in the last decade. The categories of qualification, experience, past performance, by contrast, have been becoming more important to DB owners for selecting design-builders. Finally, it is found that the importance weighting of price in large projects is significantly higher than that in small projects. This study provides a useful reference for owners in selecting their preferred design-builders.

Evaluation of design-builder qualifications through the analysis of Requests for Qualifications (RFQs)

The two-phrase best-value process has been widely used by public agencies for Design and Build (DB) procurement, with a key issue in the first phase of pre-qualification being the determination of evaluation criteria. This study identified a set of general qualification criteria for design-builders and compares their relative importance by a thorough content analysis of 97 Requests for Qualification (RFQ) for public DB projects advertised between 2000 and 2011 in various regions of the USA. The thirty-nine qualification criteria found are summarized and classified into eight categories comprising: experience; project understanding and approach; organizational structure and capacity; past performance record; professional qualifications; responsiveness to RFQs, office location and familiarity with local environment; and legal status in descending order of their relative importance. A comparative analysis of different types of projects shows that the relative weightings of the qualification criteria vary according to different characteristics of the DB projects involved.

Identification of selection criteria for operational variations of the design-build system : a delphi study in China

The design-build (DB) system has been demonstrated as an effective delivery method and has gained popularity worldwide. However it is observed that a number of operational variations of DB system have emerged since the last decade to cater for different client’s requirements. After the client decides to procure his project through the DB system, he still has to choose an appropriate configuration to deliver their projects optimally. However, there is little research on the selection of DB operational variations. One of the main reasons for this is the lack of evaluation criteria for determining the appropriateness of each operational variation. To obtain such criteria, a three-round Delphi survey has been conducted with 20 construction experts in the People’s Republic of China (PRC). Seven top selection criteria were identified. These are: (1) availability of competent design-builders; (2) client’s capabilities; (3) project complexity; (4) client’s control of project; (5) early commencement & short duration; (6) reduced responsibility or involvement; and (7) clearly defined end user’s requirements. These selection criteria were found to have a statistically significant agreement. These findings may furnish various stakeholders, DB clients in particular, with better insight to understand and compare the different operational variations of the DB system.

Developing a fuzzy multi-criteria decision-making model for selecting design-build operational variations

Many academic researchers have conducted studies on the selection of design-build (DB) delivery method; however, there are few studies on the selection of DB operational variations, which poses challenges to many clients. The selection of DB operational variation is a multi-criteria decision making process that requires clients to objectively evaluate the performance of each DB operational variation with reference to the selection criteria. This evaluation process is often characterized by subjectivity and uncertainty. In order to resolve this deficiency, the current investigation aimed to establish a fuzzy multicriteria decision-making (FMCDM) model for selecting the most suitable DB operational variation. A three-round Delphi questionnaire survey was conducted to identify the selection criteria and their relative importance. A fuzzy set theory approach, namely the modified horizontal approach with the bisector error method, was applied to establish the fuzzy membership functions, which enables clients to perform quantitative calculations on the performance of each DB operational variation. The FMCDM was developed using the weighted mean method to aggregate the overall performance of DB operational variations with regard to the selection criteria. The proposed FMCDM model enables clients to perform quantitative calculations in a fuzzy decision-making environment and provides a useful tool to cope with different project attributes.

Key competences of design-build clients in China

Purpose-- DB clients play a vital role in the delivery of DB system and the clients’ competences are critical to the success of DB projects. Most of DB clients, however, remain inexperienced with the DB system. This study, therefore, aims to identify the key competences that DB clients should possess to ensure the success of DB projects in the construction market of China. Design/Methodology/Approach -- Five semi-structured face-to-face interviews and two rounds Delphi questionnaire survey were conducted in the construction market of China to identify the key competences of DB clients. Rankings have been assigned to these key competences on the basis of their relative importance. Findings-- Six ranked key competences of DB clients have been identified, which are, namely, (1) the ability to clearly define project scope and objectives; (2) financial capacity for the projects; (3) capacity in contract management; (4) adequate staff or consulting team; (5) effective coordination with DB contractors and (6) experience with similar design-build projects. Calculation of Kendall’s Coefficient of Concordance (W) indicates a statistically significant consensus of panel experts on these top six key competences. Practical implications—Clients should clearly understand the competence requirements in DB projects and should assess their DB capability before going for the DB option. Originality/Value-- The examination of DB client’s key competences will help the client deepen the understanding of the DB system. DB clients can also make use of the research findings as guidelines to improve their DB competence.