Taurine depletion and Schwann cell dysfunction in diabetic neuropathy

It is estimated that 2.6 million people in the UK suffer from diabetes, 50% of whom suffer from diabetic neuropathy. Patients with diabetes have low levels of platelet and plasma taurine and in animal models taurine supplementation ameliorates neuropathic symptoms. The mechanisms behind Taurine depletion and taurine supplementation are not well understood. Schwann cells are highly vulnerable to hyperglycaemia-induced stress which plays a key role in the pathogenesis of diabetic neuropathy, however, the mechanisms behind these effects are not well understood. In these studies I have elucidated the effect of hyperglycaemia on taurine transport in isolated human Schwann cells and the mechanisms behind the beneficial effects of taurine supplementation. I demonstrated that high glucose reduces TauT expression in a dose-dependent manner and that high glucose inhibited the pro-oxidant increase in TauT expression and taurine uptake. This high glucose response was ablated by inhibition of aldose reductase, nitric oxide synthase as well as antioxidant treatment. Taurine supplementation reduced glucose-induced increases in oxidative stress, lipid peroxidation nitrosative stress and poly(ADP-ribosyl)ation and these effects were not accompanied by changes in antioxidant defence. Taurine also restored glucose-induced increases in iNOS and nNOS expression along with phospho-p38 MAPK abundance.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Supramolecular self-associating amphiphiles (SSAs) as nanoscale enhancers of cisplatin anticancer activity

Many chemotherapeutic drugs have a narrow therapeutic window due to inefficient tumour cell permeation.Supramolecular self-associating amphiphilic salts (SSAs) are a unique class of small molecules that offer potential as nextgeneration cancer drugs and/or therapeutic enhancement agents. Herein, we demonstrate the cytotoxicity of seven SSAstowards both ovarian and glioblastoma cancer cells. We also utilize the intrinsic fluorescent properties of one of these leadSSAs to provide evidence for this class of compound to both bind to the exterior cancer cell surface and permeate the cellmembrane, to become internalized. Furthermore, we demonstrate synergistic effects of two lead SSAs on cisplatin-mediatedcytotoxicity of ovarian cancer cells and show that this correlates with increased DNA damage and apoptosis versus eitheragent alone. This work provides the first evidence that SSAs interact with and permeate cancer cell membranes and enhancethe cytotoxic activity of a chemotherapeutic drug in human cancer cells

African trypanosomiasis: Synthesis & SAR enabling novel drug discovery of ubiquinol mimics for trypanosome alternative oxidase

African trypanosomiasis is a parasitic disease affecting 5000 humans and millions of livestock animals in sub-Saharan Africa every year. Current treatments are limited, difficult to administer and often toxic causing long term injury or death in many patients. Trypanosome alternative oxidase is a parasite specific enzyme whose inhibition by the natural product ascofuranone (AF) has been shown to be curative in murine models. Until now synthetic methods to AF analogues have been limited, this has restricted both understanding of the key structural features required for binding and also how this chemotype could be developed to an effective therapeutic agent. The development of 3 amenable novel synthetic routes to ascofuranone-like compounds is described. The SAR generated around the AF chemotype is reported with correlation to the inhibition of T. b. brucei growth and corresponding selectivity in cytotoxic assessment in mammalian HepG2 cell lines. These methods allow access to greater synthetic diversification and have enabled the synthesis of compounds that have and will continue to facilitate further optimisation of the AF chemotype into a drug-like lead

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

TDP2 is a 5’-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II. TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for HTS-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanised’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1,2,3,4,5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development

Taurine depletion and Schwann cell dysfunction in diabetic neuropathy

It is estimated that 2.6 million people in the UK suffer from diabetes, 50% of whom suffer from diabetic neuropathy. Patients with diabetes have low levels of platelet and plasma taurine and in animal models taurine supplementation ameliorates neuropathic symptoms. The mechanisms behind Taurine depletion and taurine supplementation are not well understood. Schwann cells are highly vulnerable to hyperglycaemia-induced stress which plays a key role in the pathogenesis of diabetic neuropathy, however, the mechanisms behind these effects are not well understood. In these studies I have elucidated the effect of hyperglycaemia on taurine transport in isolated human Schwann cells and the mechanisms behind the beneficial effects of taurine supplementation. I demonstrated that high glucose reduces TauT expression in a dose-dependent manner and that high glucose inhibited the pro-oxidant increase in TauT expression and taurine uptake. This high glucose response was ablated by inhibition of aldose reductase, nitric oxide synthase as well as antioxidant treatment. Taurine supplementation reduced glucose-induced increases in oxidative stress, lipid peroxidation nitrosative stress and poly(ADP-ribosyl)ation and these effects were not accompanied by changes in antioxidant defence. Taurine also restored glucose-induced increases in iNOS and nNOS expression along with phospho-p38 MAPK abundance

Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes

BACKGROUND: PLCγ enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCγ enzymes. METHODS: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. FINDINGS: We define structure of a complex containing an intact, autoinhibited PLCγ1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCγ1. We define the architecture of PLCγ1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCγ1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCγ1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. INTERPRETATION: We reveal features of PLCγ enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCγ deregulation. FUND: CR UK, MRC and AstaZeneca