Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei subspecies, is one of the most neglected diseases: available treatments are old, toxic, and difficult to administer; they are not efficacious against all parasite species or disease stages and drug resistance is an increasing problem. Protein kinases are well validated drug targets for a variety of human diseases with many inhibitors under development or in the clinic. The T. brucei kinome has been annotated and there is evidence of essentiality of some of the members of this family. This thesis aims at evaluating the essentiality of Glycogen Synthase Kinase 3 (TbGSK3 short; Tb927.10.13780) and chemically validating it as a potential drug target in T. brucei. TbGSK3 recombinant protein was biochemically characterised and screened against a focussed kinase library using the KinaseGlo assay method. Further repurchase and synthesis of novel compounds yielded 10 validated chemical series against TbGSK3 short. In particular two series showed anti-proliferative activity against the parasite. GSK3 07 series was further investigated by the Drug Discovery Unit with a phenotypic approach for its off-target effects, and GSK3 09 series was further validated to act “on target”. The latter series showed a good correlation between biochemical potency and cellular efficacy. Using a combination of chemical and genetic approaches TbGSK3 short was demonstrated to be specifically targeted by a GSK3 09 tool molecule in T. brucei lysates. Furthermore, the in vitro efficacy in trypanosomes could be reverted by target over-expression. Further validation of its activity “on target” was given by its ability to modulate the cell toxicity caused by TbGSK3 short over-expression. The genetic validation of TbGSK3 short by generation of conditional null mutants was not possible due to the tight regulation of the protein levels and the cell toxicity associated with protein over-expression. The validated TbGSK3 short chemical tool could be used to elucidate the functions of TbGSK3 short in T. brucei, identify its substrates and increase the chance to solve the crystal structure of this enzyme for the design of novel inhibitors with different mechanism of inhibition and/or increased selectivity.

Over the past decade, knowledge of the human genome has grown exponentially. While identifying individual genes and their protein products is crucial, understanding how these entities exist within the context of other molecules within the cell provides valuable insight into their functional significance. In particular, mapping the intricate web of interactions between proteins (or the ‘interactome’), allows for an understanding the roles of individual proteins within specific cellular processes and the potentially negative implications when these processes cannot occur. At the present time, approximately 40,000 binary, protein-protein interactions have been identified in human through low- and high-throughput, lab-based experiments; however, this number represents only a fraction of the estimated 600,000 protein-protein interactions thought to occur. With the high number of potential protein-protein pairing, experimentally testing each possible interaction is a time-consuming and near-impossible task. As a result, several computational methods have been developed to predict probable interactions for experimental verification. Previously, our group developed PIPs, a predictor of protein-protein interactions in human based on a naive Bayesian framework that has undergone two version releases (Scott et al., 2007, McDowall, 2011). In this thesis, a third version of PIPs, PIPs v. 3.0, is described. In addition to an update of the included data, PIPs v. 3.0 contains a new network analysis component, the TransMCL (Z) module, that combines the previously separate Transitive module (and associated EOCT predictor) introduced in version 1.0 and Cluster module (and associated EOCM predictor) introduced in version 2.0. This new module has allowed the two previously separate PIPs predictors to be merged into one method (the EOCZ predictor). In total, the new EOCZ predictor identifies over 500K significant interactions, made up of those predicted by the EOCT and EOCM predictors individually as well as a new set of interactions.Additionally, this thesis describes the development of PIP’NN, a new protein-protein interaction predictor built on a neural network framework with the data incorporated into PIPs. Overall, PIP’NN performs slightly better than the three PIPs predictors on multiple blind tests of varying sizes. PIP’NN identifies both interactions predicted by the three PIPs methods as well as a set of new interactions. As a result, PIP’NN is able to stand on its own as a new predictor of human protein-protein interactions or in conjunction with PIPs as a method to further narrow down the set of predicted interactions. Finally, this thesis describes the practical implementation of PIPs and PIP’NN through collaborations with two groups within the University of Dundee that have identified sets of potential interactions of interest for experimental confirmation. While these interactions have yet to be confirmed, both studies offer a proof of concept of how the predictors can be incorporated into lab-based interaction identification protocols. Additionally, the new PIPs web server will allow outside groups access to the updated PIPs prediction database.Overall, the work described in this thesis has built upon previous work both within and outside of the University of Dundee to further the identification of novel protein-protein interactions in human and increase the understanding of the human interactome.

This dissertation presents fragment screening studies against two human proteins Siah1 and SENP1, which function in post-translational modification pathways. Siah1 is an E3 ubiquitin ligase that functions as a scaffold to transfer ubiquitin bound to an E2 ubiquitin-conjugating enzyme to a substrate as part of the ubiquitination pathway. SENP1 is a cysteine protease that catalyses two essential reactions in the SUMO pathway. It processes pre-SUMO proteins to their mature form and removes SUMO from the target proteins. Siah1 interactions with other proteins involve large surface areas, while SENP1 has a small active site, making it hard to identify ligands for these proteins. The fragment-based approach has emerged as a complementary method to high-throughput screening of finding novel small molecules. The main aim of the study was to examine whether fragment screening would identify any ligands against these targets. Chapter 1 introduces post-translational modifications and presents fragment-based approach used in drug discovery. Chapter 2 describes the experimental methods used. The results from fragment screening against Siah1 using SPR and DSF are reported in chapter 3. The chapter also presents the structure of Siah1 refined to 1.95 Å that displays new parts of the structure, previously missing due to the absence of reliable electron density. Chapter 4 contains results from the fragment screens against SENP1 using DSF and NMR. The crystal structure of SENP1 was determined with a number of improvements made over earlier structures. Besides performing fragment screening, the binding between Siah1 interacting proteins reported in the literature and Siah1 was investigated. A number of Siah1 binding partners were successfully expressed and purified as described in chapter 5. One of those, SIP showed a clear interaction with Siah1, as observed by the shift on a size exclusion column of the complex relative to the individual protein species. Siah1 was reported to collaborate with PEG3 in the regulation of ß-catenin degradation. A SCAN domain, located at the N-terminus of PEG3, was tested for binding using gel filtration chromatography and NMR, but no interaction was observed. PEG3 was used in the crystallographic studies and a structure of its SCAN domain was solved using molecular replacement and refined to 1.95 Å. The structure of PEG3-SCAN domain revealed a stable homodimer with an extensive dimerization interface. The structure of a zinc-dependent cytosolic carboxypeptidase from Burkholderiacenocepacia was determined and is reported in chapter 6. This work was a side project assessing a new refinement strategy, which involved the use of the automated protocols embedded in the PDB_REDO server. The structure revealed that carboxypeptidase is a tetramer and provides details of its active site, whose spatial conformation of residues supports the notion that the protein might function as a deglutamylase.

Human African trypanosomiasis (HAT) also known as sleeping sickness is caused by a subspecies of <em>Trypanosoma brucei</em>. These parasites are transmitted by tsetse flies and endanger over 60 million people in Sub-Saharan Africa. Untreated, sleeping sickness is fatal, causing at least 48.000 deaths per year. Its treatment remains complicated since the currently available drugs show high toxicity and are too expensive to be ubiquitously distributed in the affected third world economies. Additionally, emerging drug resistance towards the most clinically relevant anti HAT drugs, drastically limits treatment options and makes it imperative to conduct research to find safer and more efficient drugs to treat this terrible disease. This thesis describes the hit identification and hit validation for two validated targets for HAT: <em>Tb</em>6PGDH and <em>Tb</em>UGP. For hit identification different techniques like in silico virtual screening, NMR lead-like fragment screening and HTS were used. For <em>Tb</em>UGP a very first drug-like, competitive inhibitor with a pIC50 of 3.53±0.04 and a Hill slope of 1.1±0.1 was discovered. Additionally this thesis describes the determination and validation of the in silico proposed binding mode using mutation studies and crystallisation techniques.

The alarming increase in antibacterial drug resistance indicates an urgent need to develop new drugs. This project aimed to assess and select potential antibacterial targets and carry out initial biochemical characterisation concentrating on enzymes from biowarfare agents <i>Bacillus anthracis</i>, <i>Burkholderia pseudomallei</i>, <i>Francisella tularensis</i> and <i>Yersinia pestis</i>. The overall objective is to combine genetic and chemical studies to validate, or not, targets for early stage drug discovery. In collaboration with the Dstl (Defence Science and Technology Laboratory), a series of potential targets were selected. This was carried out using essentiality prediction data from the Dstl labs, information at AEROPATH and ChEMBL databases, and the literature. Once ten different targets were selected, recombinant protein production was carried out to support structural and biochemical characterisation. Seven proteins were successfully purified and four of them prioritised for further studies. These are kynurenine formamidase (KynB), D-alanine—D-alanine ligase (Ddl), caseinolytic protease subunit P (ClpP), and the dihydrofolate synthase:folyl-poly-glutamate synthase (bifunctional protein FolC). X-ray crystallography was used to determine protein structures of KynB, Ddl and ClpP from various bacteria. Additionally, different enzymatic and binding assays were applied to assess kinetic parameters of KynB, Ddl and FolC, and compound library screenings were carried out for Ddl. In parallel, the genetic validation of these targets was being carried out by the Dstl. KynB is an important enzyme in tryptophan metabolism and predicted to be essential in Pseudomonas aeruginosa. The <i>B. anthracis</i>, <i>B. cenocepacia</i> and <i>P. aeruginosa</i> KynB structures showed an amidase fold not previously described, with a distinctive binuclear Zn²⁺ catalytic site that indicated a distinct reaction mechanism. Whilst the characterisation of the enzyme was ongoing, the Dstl lab reported the gene as non-essential and so no additional chemical validation was pursued. Ddl generates a precursor for the peptidoglycan layer and appears to be an essential protein in several Gram-negative bacteria. The structure of <i>B. pseudomallei</i> Ddl (<i>Bp</i>Ddl) in the presence of the co-factor and the reaction product D-alanyl-D-alanine (1.5 Å resolution) gives information about the substrate-binding site. Biolayer interferometry (BLI) and high-throughput (HTP) assay protocols were developed and applied. Despite testing around 22,000 compounds no inhibitors or suitable hits were found. This suggests <i>Bp</i>Ddl is a challenging target and a different approach for drug discovery might have to be considered. Fourteen ClpP subunits form a proteolytic complex which presents two different conformations; open and compressed. Structures of <i>F. turalensis</i> ClpP (<i>Ft</i>ClpP) with open and compressed conformations have been determined indicating this major conformational change is caused by a loop rearrangement at the proteasome inner canal that leads to the protease active site. FolC is an essential protein for the synthesis of folyl-poly-glutamates, a reference pathway for drug development. A new enzymatic assay, using malachite green, has been identified and used to confirm <i>Y. pestis</i> FolC activity. Such an assay could be used to determine kinetic parameters and to develop a HTP assay. The studies carried out informed about potential antibacterial target structures and biochemical properties. Protocols have been developed for protein recombinant expression, purification, crystallisation and structure determination as well as enzymatic assay development and compound library screens.

No description available.

Breast cancer remains a significant cause of morbidity and mortality. Assessment of the axillary lymph nodes is part of the staging of the disease. Advances in surgical management of breast cancer have seen a move towards intra-operative lymph node assessment that facilitates an immediate axillary clearance if it is indicated. Raman spectroscopy, a technique based on the inelastic scattering of light, has previously been shown to be capable of differentiating between normal and malignant tissue. These results, based on the biochemical composition of the tissue, potentially allow for this technique to be utilised in this clinical context. The aim of this study was to evaluate the facility of Raman spectroscopy to both assess axillary lymph node tissue within the theatre setting and to achieve results that were comparable to other intra-operative techniques within a clinically relevant time frame.

Chronic pain is a debilitating condition that is poorly treated in the clinic as conventional analgesics often lack efficacy and therapeutic doses can be limited by adverse side effects. The endocannabinoid (EC) system has been implicated in the modulation of pain and the antinociceptive effects of ECs can be enhanced by inhibiting catabolic enzymes. The main aims of this thesis were to investigate the roles that EC metabolism play in nociceptive behaviour. Theses aims were addressed by measuring tissue levels of ECs in inflammatory conditions, probing the mechanisms underlying EC mediated antinociception using pharmacological approaches and developing a sensitive analytical method to facilitate the analysis of ECs in microdialysate samples. t The key pharmacological tools used in the studies were the fatty acid amide hydrolase (FAAH) inhibitor URB597, the selective COX2 inhibitor nimesulide and the putative monoacylglycerol lipase (MAG lipase) inhibitor URB602. Intraplantar injection of URB597 (25 Ng), nimesulide (50 pg) or URB602 (70 pg) was analgesic in the carrageenan model of inflammatory pain, but only COX2 inhibition was shown to be antiinflammatory. A higher dose of URB597 (100 pg), was not, however, found to be analgesic in this model. Inhibition of metabolising enzymes resulted in changes in levels of ECs in spinal and paw tissue in the presence of carrageenan induced inflammationThe analgesic effects of URB597 (25 pg) and nimesulide (50 pg) were associated with an increase in peripheral levels of the EC-like acylethanolamine palmitoylethanolamide and were found to be mediated by the peroxisome proliferator-activated receptor alpha (PPARa). The higher dose of URB597 (100 pg) was associated with an increase of the EC anandamide, which has been shown to be a ligand for the pro-nociceptive transient receptor potential channel type V1 (TRPV1). However, the disappearance of an analgesic response at the higher dose of URB597 was independent of TRPV1 activation. Treatment with URB602 (70 pg) resulted in an increase in all ECs and related compounds ipsilateral hindpaw tissue suggesting that the inhibitor blocks FAAH as well as MAG lipase activity and so is not a selective inhibitor. Two high performance liquid chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) methods, nano-HPLC-MS/MS and micro- HPLC-MS/MS, were developed to quantitatively analyse ECs and related compounds in low fmol concentrations. The micro-HPLCMS/ MS was then used to assess the time-related effects of systemic URB597 treatment on EC levels in the hippocampus of the awake, freely-moving rat and post-mortem EC levels in hippocampal tissue. Whilst intraperitoneal injection of URB597 (1 mg kg-1) appeared to reduce release of ECs in the hippocampus compared to vehicle controls, a positive correlation between dialysate and tissue EC levels was seenThe results of these studies show that EC metabolism is a key limiting factor in the analgesic action of the EC system in inflammatory pain and highlights EC metabolising enzymes as potential targets for new analgesic drugs

Pharmaceutical industries aim for continuous improvement in the manufacturing process of producing medicines. Demands on the pharmaceutical industries are to produce quality products in a quick and cost effective way. Designing a robust crystallization process so as to produce quality crystals with the desired polymorphic form, morphology, size and size distribution, will contribute towards meeting these demands. The Food and Drug Administration regulating body encourages the development of quality by design (QbD) approaches, involving the use of process analytical technology (PAT) for the design of the crystallization process. This method enables the design of the crystallization process to be more flexible in terms of variation in operating conditions and process parameters so long as the quality of the product is maintained. The aim of this thesis work is to use QbD approaches involving the use of PAT tools and solid state analytical (SSA) techniques to increase process knowledge and understanding, which is required for the robust design of crystallization processes. Discovery of all possible polymorphic forms of an active pharmaceutical ingredient (API) is important for the design of a robust crystallization process in which product quality is consistent during scale up and to prevent late stage failures. This thesis work shows the importance of using PAT tools and SSA techniques for monitoring polymorphic transformations and for the discovery of new polymorphic forms that have not yet been reported in the literature. A range of PAT tools including the FBRM, turbidity probe and ATR-UV/Vis spectrometer detected polymorphic transformations during both cooling and antisolvent crystallization experiments using the model system sodium benzoate in water and a propan-2-ol (IPA)/water mixture. Information obtained from a range of SSA techniques provided supporting evidence for the discovery of a new channel hydrate, channel solvate and an anhydrous form of sodium benzoate. The problem of crusting (solid depositing on vessel walls) occurring within crystallization vessels has been investigated with the use of a combination of PAT tools and SSA techniques. The FBRM and turbidity probe detected a change occurring during the cooling crystallization process of para-amino benzoic acid (ABA) in ethyl acetate. Repeats of the experiments using the ATR-UV/Vis confirmed that the change was due to crusting forming on vessel walls and not a polymorphic transformation. PAT tools also detected changes occurring during a pH controlled polymorphic cooling crystallization experiment (Chapter 9), which was subsequently confirmed by SSA methods to be due to the formation of a mixture of products and not a polymorphic transformation. This research work shows the importance of using a combination of PAT tools and SSA techniques for gaining a deeper understanding of the crystallization process and to prevent misinterpretation of results, saving both time and money. Also this research work highlights the need for improvement within industrial scale vessel design, such as vessels with variable jacket height, to prevent the problems of crusting. Robust MSZW measurements are obtained at laboratory scale using the model crystallization systems para and meta-ABA in water. PAT tools used include the FBRM and turbidity probe. The robust MSZW takes into consideration variation in process parameters including ramp rate, vessel size (1 mL and 1 L), agitator speed and type. This robust MSZW can be used for the design of scale up experiments (pilot plant and industrial scale), increasing the likelihood of producing a quality product. Nucleation orders used within crystallization models were determined from the MSZW measurements. Results showed that the nucleation order varied within different crystallization set-ups (vessel size and mixing conditions) using the model system meta-ABA in water. Therefore model-based design and scale-up of crystallization processes must be used carefully and more detailed mechanistic models, which take into consideration the effect of mixing need to be designed to improve the generality and applicability of crystallization models. pH controlled polymorphic crystallization experiments were performed using the model systems meta and para-ABA in ethanol and water. A combination of 5 PAT tools were used in a single vessel to monitor the cooling crystallization process. PAT tools used include FBRM, ATR-UV/Vis, PVM, pH and a temperature probe. Various parameters including mixing conditions, solvent, pH of solution, strength and type of acid were varied to investigate the best conditions to produce salts. Results showed that careful selection of design parameters and salt selection is important for producing quality crystals of the desired morphology so as to prevent problems in downstream processing.

The purpose of the work carried out in this thesis was to examine hormonal effects on central nervous system serotonergic (5-hydroxtryptamine; 5-HT) receptor function. Experiments were carried out using established rodent (mouse and rat) models of central 5-HT receptor function. Corticosteroids were administered, both acutely and chronically, and the effects on these models assessed. Corticosteroids were found to have relatively selective effects: in the mouse and rat certain models of 5-HT<SUB>1A</SUB> receptor function were attenuated and in the mouse corticosteroids enhanced behavioural models of 5-HT release. No effects were found on models of other central 5-HT receptor functions. These effects were found to be both time and dose dependent and to reverse after the administration of corticosteroids ceased. Removal of endogenous corticosteroids, by adrenalectomy, was found to cause an enhancement of 5-HT<SUB>1A</SUB> receptor function. A sex difference, in rodents, in 5-HT<SUB>1A</SUB> receptor function was found which was abolished by ovariectomy. The action of corticosterone in producing these effects could not be replicated by selective corticosteroid receptor antagonists. Further experiments were carried out in healthy human male volunteers. Buspirone was shown to induce a hypothermic response significantly greater than placebo. The effects of hydrocortisone administration (20mg, orally, twice daily) on the sensitivity of brain 5-HT<SUB>1A</SUB> receptors in healthy volunteers were then studied using a buspirone challenge paradigm. These experiments show that the effects of corticosteroids are relatively selective to 5-HT<SUB>1A</SUB> receptors. The effect of corticosteroids on 5-HT<SUB>1A</SUB> receptors may be the mechanism whereby exogenous corticosteroids affect mood. Furthermore, the effects of endogenous corticosteroids on 5-HT receptor function may be important in the pathophysiology of mood disorders.