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.

Time-varying bispectral analysis of auditory evoked multi-channel scalp EEG

Time-varying bispectra, computed using a classical sliding window short-time Fourier approach, are analyzed for scalp EEG potentials evoked by an auditory stimulus and new observations are presented. A single, short duration tone is presented from the left or the right, direction unknown to the test subject. The subject responds by moving the eyes to the direction of the sound. EEG epochs sampled at 200 Hz for repeated trials are processed between -70 ms and +1200 ms with reference to the stimulus. It is observed that for an ensemble of correctly recognized cases, the best matching timevarying bispectra at (8 Hz, 8Hz) are for PZ-FZ channels and this is also largely the case for grand averages but not for power spectra at 8 Hz. Out of 11 subjects, the only exception for time-varying bispectral match was a subject with family history of Alzheimer’s disease and the difference was in bicoherence, not biphase.

Anchored deformable face ensemble alignment

At present, many approaches have been proposed for deformable face alignment with varying degrees of success. However, the common drawback to nearly all these approaches is the inaccurate landmark registrations. The registration errors which occur are predominantly heterogeneous (i.e. low error for some frames in a sequence and higher error for others). In this paper we propose an approach for simultaneously aligning an ensemble of deformable face images stemming from the same subject given noisy heterogeneous landmark estimates. We propose that these initial noisy landmark estimates can be used as an “anchor” in conjunction with known state-of-the-art objectives for unsupervised image ensemble alignment. Impressive alignment performance is obtained using well known deformable face fitting algorithms as “anchors.

Deformable face ensemble alignment with robust grouped-L1 anchors

Many methods exist at the moment for deformable face fitting. A drawback to nearly all these approaches is that they are (i) noisy in terms of landmark positions, and (ii) the noise is biased across frames (i.e. the misalignment is toward common directions across all frames). In this paper we propose a grouped $\mathcal{L}1$-norm anchored method for simultaneously aligning an ensemble of deformable face images stemming from the same subject, given noisy heterogeneous landmark estimates. Impressive alignment performance improvement and refinement is obtained using very weak initialization as "anchors".

Rank minimization across appearance and shape for AAM ensemble fitting

Active Appearance Models (AAMs) employ a paradigm of inverting a synthesis model of how an object can vary in terms of shape and appearance. As a result, the ability of AAMs to register an unseen object image is intrinsically linked to two factors. First, how well the synthesis model can reconstruct the object image. Second, the degrees of freedom in the model. Fewer degrees of freedom yield a higher likelihood of good fitting performance. In this paper we look at how these seemingly contrasting factors can complement one another for the problem of AAM fitting of an ensemble of images stemming from a constrained set (e.g. an ensemble of face images of the same person).

Sequential decision fusion of multibiometrics applied to text-dependent speaker verification for controlled errors

Reliability of the performance of biometric identity verification systems remains a significant challenge. Individual biometric samples of the same person (identity class) are not identical at each presentation and performance degradation arises from intra-class variability and inter-class similarity. These limitations lead to false accepts and false rejects that are dependent. It is therefore difficult to reduce the rate of one type of error without increasing the other. The focus of this dissertation is to investigate a method based on classifier fusion techniques to better control the trade-off between the verification errors using text-dependent speaker verification as the test platform. A sequential classifier fusion architecture that integrates multi-instance and multisample fusion schemes is proposed. This fusion method enables a controlled trade-off between false alarms and false rejects. For statistically independent classifier decisions, analytical expressions for each type of verification error are derived using base classifier performances. As this assumption may not be always valid, these expressions are modified to incorporate the correlation between statistically dependent decisions from clients and impostors. The architecture is empirically evaluated by applying the proposed architecture for text dependent speaker verification using the Hidden Markov Model based digit dependent speaker models in each stage with multiple attempts for each digit utterance. The trade-off between the verification errors is controlled using the parameters, number of decision stages (instances) and the number of attempts at each decision stage (samples), fine-tuned on evaluation/tune set. The statistical validation of the derived expressions for error estimates is evaluated on test data. The performance of the sequential method is further demonstrated to depend on the order of the combination of digits (instances) and the nature of repetitive attempts (samples). The false rejection and false acceptance rates for proposed fusion are estimated using the base classifier performances, the variance in correlation between classifier decisions and the sequence of classifiers with favourable dependence selected using the 'Sequential Error Ratio' criteria. The error rates are better estimated by incorporating user-dependent (such as speaker-dependent thresholds and speaker-specific digit combinations) and class-dependent (such as clientimpostor dependent favourable combinations and class-error based threshold estimation) information. The proposed architecture is desirable in most of the speaker verification applications such as remote authentication, telephone and internet shopping applications. The tuning of parameters - the number of instances and samples - serve both the security and user convenience requirements of speaker-specific verification. The architecture investigated here is applicable to verification using other biometric modalities such as handwriting, fingerprints and key strokes.

Unsupervised temporal ensemble alignment for rapid annotation

This paper presents a novel framework for the unsupervised alignment of an ensemble of temporal sequences. This approach draws inspiration from the axiom that an ensemble of temporal signals stemming from the same source/class should have lower rank when "aligned" rather than "misaligned". Our approach shares similarities with recent state of the art methods for unsupervised images ensemble alignment (e.g. RASL) which breaks the problem into a set of image alignment problems (which have well known solutions i.e. the Lucas-Kanade algorithm). Similarly, we propose a strategy for decomposing the problem of temporal ensemble alignment into a similar set of independent sequence problems which we claim can be solved reliably through Dynamic Time Warping (DTW). We demonstrate the utility of our method using the Cohn-Kanade+ dataset, to align expression onset across multiple sequences, which allows us to automate the rapid discovery of event annotations.

Injury narrative text classification : a preliminary study

Description of a patient's injuries is recorded in narrative text form by hospital emergency departments. For statistical reporting, this text data needs to be mapped to pre-defined codes. Existing research in this field uses the Naïve Bayes probabilistic method to build classifiers for mapping. In this paper, we focus on providing guidance on the selection of a classification method. We build a number of classifiers belonging to different classification families such as decision tree, probabilistic, neural networks, and instance-based, ensemble-based and kernel-based linear classifiers. An extensive pre-processing is carried out to ensure the quality of data and, in hence, the quality classification outcome. The records with a null entry in injury description are removed. The misspelling correction process is carried out by finding and replacing the misspelt word with a soundlike word. Meaningful phrases have been identified and kept, instead of removing the part of phrase as a stop word. The abbreviations appearing in many forms of entry are manually identified and only one form of abbreviations is used. Clustering is utilised to discriminate between non-frequent and frequent terms. This process reduced the number of text features dramatically from about 28,000 to 5000. The medical narrative text injury dataset, under consideration, is composed of many short documents. The data can be characterized as high-dimensional and sparse, i.e., few features are irrelevant but features are correlated with one another. Therefore, Matrix factorization techniques such as Singular Value Decomposition (SVD) and Non Negative Matrix Factorization (NNMF) have been used to map the processed feature space to a lower-dimensional feature space. Classifiers with these reduced feature space have been built. In experiments, a set of tests are conducted to reflect which classification method is best for the medical text classification. The Non Negative Matrix Factorization with Support Vector Machine method can achieve 93% precision which is higher than all the tested traditional classifiers. We also found that TF/IDF weighting which works well for long text classification is inferior to binary weighting in short document classification. Another finding is that the Top-n terms should be removed in consultation with medical experts, as it affects the classification performance.

Learning Optimal Discriminant Functions through a Cooperative Game of Automata

The problem of learning correct decision rules to minimize the probability of misclassification is a long-standing problem of supervised learning in pattern recognition. The problem of learning such optimal discriminant functions is considered for the class of problems where the statistical properties of the pattern classes are completely unknown. The problem is posed as a game with common payoff played by a team of mutually cooperating learning automata. This essentially results in a probabilistic search through the space of classifiers. The approach is inherently capable of learning discriminant functions that are nonlinear in their parameters also. A learning algorithm is presented for the team and convergence is established. It is proved that the team can obtain the optimal classifier to an arbitrary approximation. Simulation results with a few examples are presented where the team learns the optimal classifier.

On the Landau damping of sound waves in a plasma with magnetic flux tubes

The Landau damping of sound wave in a plasma consisting of an ensemble of magnetic flux tubes with reference to the work by Ryutov and Ryutova (1976) is discussed. Sound waves cannot be Landau damped in general but under certain restriction conditions on plasma parameters the possibility of absorption of these waves can exist.

Conformational analysis of D-pantothenic acid

The conformational analysis of d-pantothenic acid using classical semiempirical methods has been carried out. The pantothenic acid molecule can exist in the neutral form (I) or in the ionised form (II) with a deprotonated negatively charged carboxyl group. The neutral molecule as well as the anion is highly flexible and has an ensemble of several allowed conformations rather than one or two unique conformations. The distribution of allowed conformations indicate that the β-alanine as well as the pantoic acid part of the molecule prefers partially folded conformations. The conformation of the former is greatly affected by the ionisation state of the carboxyl group whereas that of the latter is not. Possibility of intramolecular hydrogen bonding in different allowed conformations has also been explored. A bifurcated hydrogen bond involving a carboxyl (or carboxylate) oxygen atom and a hydroxyl oxygen atom, as acceptors, and the amide nitrogen atom as the donor occurs frequently in both I and II. Amongst the two crystal structures containing pantothenic acid reported so far, the conformation of the molecule in l-lysine d-pantothenate lies in the allowed region and is stabilised by a bifurcated intramolecular hydrogen bond, whereas that in the calcium bromide salt falls in a disallowed region, presumably due to the requirement of tridentate metal coordination.

New light on the optical equivalence theorem and a new type of discrete diagonal coherent state representation

In many instances we find it advantageous to display a quantum optical density matrix as a generalized statistical ensemble of coherent wave fields. The weight functions involved in these constructions turn out to belong to a family of distributions, not always smooth functions. In this paper we investigate this question anew and show how it is related to the problem of expanding an arbitrary state in terms of an overcomplete subfamily of the overcomplete set of coherent states. This provides a relatively transparent derivation of the optical equivalence theorem. An interesting by-product is the discovery of a new class of discrete diagonal representations.

A semi-analytical particle filter for identification of nonlinear oscillators

Particle filters find important applications in the problems of state and parameter estimations of dynamical systems of engineering interest. Since a typical filtering algorithm involves Monte Carlo simulations of the process equations, sample variance of the estimator is inversely proportional to the number of particles. The sample variance may be reduced if one uses a Rao-Blackwell marginalization of states and performs analytical computations as much as possible. In this work, we propose a semi-analytical particle filter, requiring no Rao-Blackwell marginalization, for state and parameter estimations of nonlinear dynamical systems with additively Gaussian process/observation noises. Through local linearizations of the nonlinear drift fields in the process/observation equations via explicit Ito-Taylor expansions, the given nonlinear system is transformed into an ensemble of locally linearized systems. Using the most recent observation, conditionally Gaussian posterior density functions of the linearized systems are analytically obtained through the Kalman filter. This information is further exploited within the particle filter algorithm for obtaining samples from the optimal posterior density of the states. The potential of the method in state/parameter estimations is demonstrated through numerical illustrations for a few nonlinear oscillators. The proposed filter is found to yield estimates with reduced sample variance and improved accuracy vis-a-vis results from a form of sequential importance sampling filter.

Reservoir performance under uncertainty in hydrologic impacts of climate change

Relatively few studies have addressed water management and adaptation measures in the face of changing water balances due to climate change. The current work studies climate change impact on a multipurpose reservoir performance and derives adaptive policies for possible futurescenarios. The method developed in this work is illustrated with a case study of Hirakud reservoir on the Mahanadi river in Orissa, India,which is a multipurpose reservoir serving flood control, irrigation and power generation. Climate change effects on annual hydropower generation and four performance indices (reliability with respect to three reservoir functions, viz. hydropower, irrigation and flood control, resiliency, vulnerability and deficit ratio with respect to hydropower) are studied. Outputs from three general circulation models (GCMs) for three scenarios each are downscaled to monsoon streamflow in the Mahanadi river for two future time slices, 2045-65 and 2075-95. Increased irrigation demands, rule curves dictated by increased need for flood storage and downscaled projections of streamflow from the ensemble of GCMs and scenarios are used for projecting future hydrologic scenarios. It is seen that hydropower generation and reliability with respect to hydropower and irrigation are likely to show a decrease in future in most scenarios, whereas the deficit ratio and vulnerability are likely to increase as a result of climate change if the standard operating policy (SOP) using current rule curves for flood protection is employed. An optimal monthly operating policy is then derived using stochastic dynamic programming (SDP) as an adaptive policy for mitigating impacts of climate change on reservoir operation. The objective of this policy is to maximize reliabilities with respect to multiple reservoir functions of hydropower, irrigation and flood control. In variations to this adaptive policy, increasingly more weightage is given to the purpose of maximizing reliability with respect to hydropower for two extreme scenarios. It is seen that by marginally sacrificing reliability with respect to irrigation and flood control, hydropower reliability and generation can be increased for future scenarios. This suggests that reservoir rules for flood control may have to be revised in basins where climate change projects an increasing probability of droughts. However, it is also seen that power generation is unable to be restored to current levels, due in part to the large projected increases in irrigation demand. This suggests that future water balance deficits may limit the success of adaptive policy options. (C) 2010 Elsevier Ltd. All rights reserved.

Development and transient performance results of a single stage activated carbon - HFC 134a closed cycle adsorption cooling system

A laboratory model of a thermally driven adsorption refrigeration system with activated carbon as the adsorbent and 1,1,1,2-tetrafluoroethane (HFC 134a) as the refrigerant was developed. The single stage compression system has an ensemble of four adsorbers packed with Maxsorb II specimen of activated carbon that provide a near continuous flow which caters to a cooling load of up to 5W in the 5-18 degrees C region. The objective was to utilise the low grade thermal energy to drive a refrigeration system that can be used to cool some critical electronic components. The laboratory model was tested for it performance at various cooling loads with the heat source temperature from 73 to 93 degrees C. The pressure transients during heating and cooling phases were traced. The cyclic steady state and transient performance data are presented. (C) 2010 Elsevier Ltd. All rights reserved.