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Fungal infections are a serious concern to the health and wellbeing of people worldwide. Up to 1.2 billion people a year suffer from fungal infections, although the majority of these infections are of the skin such as yeast infections, thrush, or athletes' foot a considerable number are life-threatening. Between 1.5 and 2 million people a year die from invasive fungal infections. The infectious fungal species that opportunistically target those patients with a compromised immune system such as that resulting from from various cancer treatments, AIDS/HIV or organ transplant patients. The current drugs available are often ineffective due to fungal resistance or have serious side effects that can severely harm the patients. Research has shown a select number of proteins from 2 families of enzymes, Phosphatases and Kinases are essential to the growth and viability of multiple pathogenic fungi. In this study, I report an in-depth analysis, using computational tools, of some essential proteins and determine strategies to develop inhibitors that will target specific sites causing the death of the organism. I also report a complete protocol to express and purify one of the essential proteins, NimT. I demonstrate that NimT is an acid phosphatase and identify a number of inhibitors that halt the function of the protein dephosphorylation. I also take considerable steps to validate an additional site, alternative to that of the active site, as a potential drug target. In addition, we report a protocol that produces clean, soluble NimT protein that is suitable for structure determination crystallisation trials.

Protein phosphatases are regulatory enzymes with unique substrate specificity for serine/threonine/tyrosine/histidine phosphorylated proteins and phosphoinositides. They mediate critical cell processes such as cell metabolism, cell cycle, growth and differentiation. For example, phosphoinositide phosphatases act as regulatory enzymes that mediate critical cellular processes such as cell signalling, membrane trafficking, cell cycle and growth. Loss-of-function mutations in these enzymes are linked to a plethora of disease conditions. Tumour suppressor PTEN is the most frequently mutated gene in various forms of human cancer; Myotubularins are mutated in X-linked myotubular myopathy (XLMTM) and Charcot-Marie-Tooth disease type 4B (CMT4B); Synaptojanin I is mutated in early-onset progressive Parkinsonism. Engineered PTPs are therefore seen as useful tools to enhance our understanding of signalling pathways involving phosphorylation events. Here, we used a combination of structure-guided rational and computational design to alter the substrate specificity of Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP), from tyrosine-specific to PI(3,5)P2-, PI(3)P-, PI(4)P-, and PI(5)P-specific phosphatase (Low Molecular Weight Phosphoinositide Phosphatase - LMWPIP). Four LMW-PIP mutants dephosphorylate phosphoinositides with single or broad substrate specificity. We combined Rosetta Enzyme designs containing frequently introduced mutations with molecular docking studies to assess binding affinity to the ligand substrate. This approach facilitates the design selection process by reducing the number of candidate mutants to be validated experimentally. We also designed mutants to be tested for specificity towards serine/threonine phospho-peptide. Finally, we set up a system in yeast that can be used for in vivo characterisation of LMWPTPB mutants, by cloning and confirming the expression of LMWPTP in yeast using western blot and RT-PCR. Engineered LMW-PIP mutants can be a useful tool in investigating lipid-regulated cell signalling pathways, and in the long term, could serve as biotherapeutics in enzyme replacement therapy.

The process of dephosphorylation of tyrosine residues in proteins is key to a number of cellular processes and is controlled by protein tyrosine phosphatases (PTPs). Misregulation of these phosphatase enzymes is implicated in a range of human disease states, including cancer, type 2 diabetes, and a number of autoimmune conditions. Many natural substrate mimetics - including those substituting oxygen for a non-hydrolysable difluoromethylene group - lack cell permeability as a result of the ionised phosphate group at physiological pH, and many compounds are not sufficiently phosphatase selective as a result of the highly conserved active site. As a result, the development of potent, cell-permeable and selective phosphatase inhibitors has been severely limited. In chapter one, synthetic strategies for the synthesis of model phosphonodiamidate prodrugs with a difluoromethylene moiety adjacent to phosphorus are explored. Through masking the charge of the phosphonic acid, the prodrugs should be cell permeable and should be activated intracellularly. Also reported is the synthesis of a number of novel compounds displaying diversity at the carbon adjacent to phosphorus, as well as a study analysing aromatic substituent effects on the reaction pathway. In chapter two, the amination conditions developed are applied to the synthesis of a series of novel prodrugs designed to target protein tyrosine phosphatase 1B (PTP1B). Chapter three shows the report of the preliminary biochemical and in vitro analysis of these novel prodrugs and the corresponding phosphonic acids. Finally, progress toward the synthesis of precursors of novel allylic phosphonate prodrugs incorporating an internal nucleophile that can activate in the presence of catalytic amounts of palladium is explored in chapter four.

Breast cancer remains the most lethal type of cancer for women. A quarter of breast cancer cases display overexpression of the HER2 protein, which is associated with poor prognosis. A major drug for treating HER2 (human epidermal growth factor receptor 2)-positive breast cancer is Herceptin, which reduces HER2 dimerization, thereby inhibiting the major cell proliferation pathways. Despite its effectiveness, resistance to Herceptin is a very significant clinical problem: only a third of patients respond to Herceptin, and two thirds of these relapse within one year due to resistance to the drug. Specific members of the Dual-specificity phosphatases (DUSPs) family act downstream of the HER2 protein in breast cancer and contribute to development of Herceptin resistance. I aimed to model the role of dual specificity phosphatases (DUSPs) and the effect of their inhibition in signalling pathways in HER2-positive breast cancer and resistance to Herceptin. This thesis focuses on the implementation of a multi-disciplinary approach to identify a specific profile of DUSPs, whose inhibition could reduce Herceptin resistance in breast cancer cells or resensitize resistant cells. A further aim was to assess druggability among these DUSPs to guide future drug discovery efforts. First, using Boolean modelling and experimental measurements of DUSP expression during initial treatment of HER2-positive breast cancer cells with Herceptin, I was able to predict regulatory mechanisms of DUSPs, where these mechanisms were partially known. Next, I used a systems biology approach together with experimental data to investigate how DUSP overexpression could favour cell proliferation in resistant HER2-positive breast cancer and to predict how this mechanism could be reversed by targeted inhibition of selected DUSPs. After showing that our Boolean models correctly accounted for resistance when overexpressed DUSPs were kept activated, I then simulated inhibition of both individual and combinations of DUSPs, and determined conditions under which the resistance could be reversed. Further on, I investigated whether inhibiting certain DUSPs resensitized Herceptin-resistant breast cancer cells to the drug. I built a series of kinetic models incorporating the key players of HER2 signalling pathways and simulating a range of inhibition intensities. I observed that when silencing certain DUSPs, resistant HER2-positive breast cancer cells became more responsive to the drug, showing a decrease in resistance, in agreement with the model predictions. Finally, by applying molecular docking and virtual screening of compound libraries together with predictions of pockets and protein-protein interactions, I could assess the druggability among DUSPs in order to guide future drug discovery efforts. Overall, this thesis highlights the value of combining a systems biology approach with wet lab experimentation and computational screening of chemical compounds, with regards to the identification of specific DUSPs playing a role in resistance to Herceptin of HER2-positive breast cancer cells.

Biogenesis of the multivesicular body (MVB) organelle is an important process for regulation of signalling in the cell. Signal receptors embedded within the outer MVB membrane can be sorted into intralumenal vesicles which bud away from the cytosol to within the MVB preventing further signalling. Sorting of receptors, invagination of the membrane and release of vesicles into the MVB lumen are mediated by the endosomal sorting complexes required for transport (ESCRT) along with a range of accessory proteins including histidine domain protein tyrosine phosphatase (HD-PTP). HD-PTP is a multidomain protein which makes several interactions with ESCRT partners, including ESCRT-0, ESCRT-I and ESCRT-III. This thesis focusses specifically on the interaction between HD-PTP CC domain and Ubap1 (ESCRT-I), and the two interactions of HD-PTP Bro and PRR domains with STAM2 (ESCRT-0) SH3 and Core domains. To address the structure of HD-PTP, multiple techniques were used: X-ray crystallography, which gives high resolution structural information; small angle X-ray scattering (SAXS), which gives low resolution data for large non-crystallisable units in their solution state; and double electron-electron resonance (DEER) spectroscopy, which gives high resolution nanometre-range distance constraints between cysteines labelled with methanethiosulfonate spin label (MTSL). It was shown by X-ray crystallography that HD-PTP has an elongated CC domain, in stark contrast to its homologues ALIX and Bro1 which both have V-shaped CC domains. The CC domain showed limited flexibility both by SAXS and DEER. Further investigation showed that there was no significant conformational change upon binding its ESCRT-I partner Ubap1. The multidomain structure of HD-PTP Bro1-CC-PRR was described by SAXS, showing that these domains form an extended arrangement in solution. In addition, SAXS was also used to analyse the structure of these domains in complex with STAM2 (ESCRT-0), which showed that STAM2 is simultaneously tethered by the Bro1 domain and PRR. The Bro-CC-PRR portion of HD-PTP, has 9 cysteines, so with the aim of measuring local structural information in the CC domain alone, alternative spin labelling methods were investigated. Use of a bromoacrylaldehyde spin label (BASL), instead of MTSL, allowed more selective labelling of surface exposed cysteines, and avoided labelling most of the cysteines in the Bro1 domain. This novel method allowed the shape of the CC domain to be monitored during STAM2 binding and showed that there is no induced conformational change.

Mutations in LPS-Induced TNF Activating Factor/Small Integral Membrane Protein of the Late Endosome (LITAF/SIMPLE) cause Charcot-Marie-Tooth (CMT) type 1C disease, an autosomal-dominant demyelinating disorder of the peripheral nervous system. The pathogenesis has not been characterized due to little knowledge about the cellular roles of LITAF. N-terminus of LITAF contains PPXY and P(T/S)AP motifs that interact with E3 ubiquitin ligase Itch/Nedd4 and ESCRT-I subunit TSG101, respectively. C-terminus of LITAF consists of a hydrophobic region bracketed by two proposed "CXXC" zinc knuckles, termed the LITAF-like domain or the SIMPLE-like domain (SLD). Mammalian cells also express an uncharacterized Cell Death Inducing P53 target 1 (CDIP1), which has conserved PPXY and P(T/S)AP motifs as well as the LITAF-like domain. Here, we show that LITAF/CDIP1 belong to a novel class of monotopic integral membrane proteins and localise to multiple endocytic compartments. LITAF/CDIP1 are associated with EGFR-containing endosomes and also interact with HD-PTP. Knockdown of LITAF and CDIP1, affect the morphogenesis of Multivesicular Bodies (MVBs). We also show that LITAF (SLD) localises to the Tubular recycling endosome (TRE). LITAF/CDIP1 depletion cause expanded TRE which is involved in the recycling of Clathrin-independent cargoes. Introducing disease-linked mutations into the LITAF (SLD) abolished its TRE association. Hence, our study proposes that the CMT1C disease is caused due to the malformation of TRE and MVBs.

Uveal melanoma (UM) is a sight and life-threatening malignancy of the human eye. The potential for progress in translational UM research is, however, hampered by the short supply of clinical samples due to its rarity and also the lack of an informative animal model which would allow experimental intervention to dissect the molecular machinery governing tumor development. Towards this end, we aimed to generate a genetically relevant model of human UM in zebrafish that can be used to study the roles of key genetic determinants in tumor initiation and progression in vivo and also establish a valuable resource for future preclinical studies. Given the pervasive role of Gαq proteins in driving UM pathogenesis, we engineered transgenic zebrafish to express oncogenic GNAQQ209P in the melanocyte lineage. This resulted in hyperplasia of uveal (choroidal) melanocytes, but with no evidence of malignant progression nor perturbation of RPE or skin melanocytes. However, combining expression of oncogenic GNAQQ209P with tp53 inactivation resulted in an earlier onset and even more extensive hyperplasia of choroidal melanocytes that then progressed to UM. While NRASQ61L and BRAFV600E potently stimulate ERK1/2-MAPK signalling pathway, immunohistochemical analysis revealed only sporadic immunoreactivity to phosphorylated ERK1/2 in hyperplastic choroidal lesions and also uveal tumors driven by oncogenic GNAQQ209P, in contrast to an abundant immunoreactivity in oncogenic HRASG12V-driven cutaneous tumors. Rather, ubiquitous positive staining for nuclear YAP was observed in GNAQQ209P-driven uveal tumor specimens. In keeping with a lesser role of GNAQ in regulating ERK1/2-MAPK signalling in UM, we showed that downregulation of oncogenic GNAQQ209P/L or inhibition of PLC-β in the majority of human UM cells expressing oncogenic GNAQQ209P/L barely affected ERK phosphorylation. In summary, this study demonstrates the insufficiency of oncogenic GNAQQ209P alone in driving UM development which only became evident with a second genetic hit involving tp53 inactivation. Our findings also demonstrate a weak correlation between oncogenic GNAQ mutations and sustained ERK1/2-MAPK activation, implying that ERK1/2 signalling is unlikely to be instrumental in the maintenance of GNAQQ209P-driven uveal tumors.

Trypanosomiases and leishmaniases are amongst the world’s most neglected infectious diseases. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are the primary human pathogens of the trypanosomatidae family and the causative agents of African sleeping sickness, Chagas’ disease and cutaneous leishmaniasis, respectively. The molecular mechanism controlling each parasite’s life cycle and virulence is poorly understood however protein phosphatases are expected to play a critical role. This study presents the biochemical characterisation data of two groups of phosphatases from the trypanosomatids: the LRR-DSP and LM phosphatases. The LRR-DSPs are a group of twelve proteins, characterised by a unique domain architecture: a leucine-rich repeat (LRR) domain together with a dual- specificity phosphatase (DSP) catalytic domain. In this study, the recombinant expression of a representative LRR-DSP orthologue from T. brucei (TbLRR-DSP), proved to be highly problematic in E. coli. Most full-length and catalytic domain constructs were expressed at very low levels or as insoluble proteins. Soluble protein was obtained by denaturation, treatment with detergents, non-denaturing extraction from inclusion bodies and fusion to solubility-enhancing proteins. However, no method yielded protein with catalytic activity or detectable secondary structure. Soluble expression of TbLRR-DSP was achieved using baculovirus-infected insect cells, but the protein co-purified with endogenous chaperones and exhibited no catalytic activity thus implying a lack of correct folding. In the second part of this study, two phosphatases specific to Leishmania major, LM1 and LM2, were characterised and structural studies were initiated. LM2 was shown to readily hydrolyse phospho-tyrosine substrates in vitro, but not phosphoinositides like its homologue, LM1. Both proteins therefore have a differentiated catalytic profile and are likely to have different functions in vivo. Purification protocols for both proteins were established and crystallisation screenings set up. Preliminary hits were obtained for LM2 and a mutagenesis strategy was developed to improve chances of obtaining diffraction quality crystals. Recombinant LM1 samples exhibited heterogeneity and therefore will require additional engineering to improve chances of crystallization. Promising pilot NMR data was also obtained for both phosphatases. In conclusion, this study demonstrates that the recombinant expression of multi- domain trypanosomatid proteins (like the LRR-DSPs) can be highly problematic and may pose a challenge for their biochemical characterisation and functional elucidation. Future work into trypanosomatid phosphatases, however challenging, will improve our understanding of their cell biology and potentially identify therapeutic targets.