Pathology - Theses
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ItemDevelopment of a candidate for PET imaging of BACE1 in Alzheimer's disease( 2015)Alzheimer's disease (AD) is the most common form of dementia but there is currently no cure or disease modifying treatment. The pathological processes which lead to AD begin up to 20 years before the onset of symptoms, offering a large window for diagnosis and potential therapeutic intervention. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease involved in the pathogenesis of AD. BACE1 levels are increased in AD patients and this elevation is known to occur early in the course of the disease; however, there is currently no method for measuring cortical BACE1 levels in vivo. It is hypothesised that the development of a BACE1 positron emission tomography (PET) imaging agent could have clinical utility for AD diagnosis as well as offering other benefits to AD research. A series of analogues of the hit compound (E)-2-(5-(3,4-dimethoxyphenyl)thiazol-2-yl)-3-(4-hydroxy-3-methoxyphenyl)acrylonitrile, identified from a screen of 70 compounds against BACE1 activity, was synthesised in order to generate a structure activity relationship. Initial attempts to radiolabel the general scaffold of the compounds with the 18F isotope by direct labelling were unsuccessful due to the instability of the scaffold under radiofluorination conditions; however, a route to radiolabel the scaffold was achieved by utilising [18F]-fluorobenzaldehyde as a prosthetic labelling group. The ability of the compounds to bind to BACE1 was assessed by a surface plasmon resonance (SPR) inhibition in solution assay. While a small number of compounds displayed limited activity, IC50 values could not be generated and most compounds were inactive. In addition, most of the compounds were too insoluble to be assessed for membrane permeability using the parallel artificial membrane permeability assay (PAMPA), although a small number of more soluble compounds were shown to have high permeability. Due to the slow progress and problems encountered in developing a de novo BACE1 PET imaging candidate, it was decided to modify an existing BACE1 inhibitor to incorporate the 18F radiolabel. MK-8931 (Merck) has a high affinity for BACE1 and is the most advanced clinical candidate for BACE1 inhibition and a synthetic route was developed to introduce a fluorine atom to the molecule. Following the synthesis of MK-8931, achieved by modification of a literature procedure, a radiofluorinated analogue, (S)-2-amino-6-(4-(5-(3-[18F]-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one, was prepared; unfortunately, the amount of labelled material was low and purification was not attempted. Analysis of (S)-2-amino-6-(4-(5-(3-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one by SPR showed an IC50 of 2.05 nM compared to 5.87 nM for that of MK-8931, and the passive membrane permeability of both compounds as measured by PAMPA was very similar. These results suggest that should the radiosynthesis be optimised, (S)-2-amino-6-(4-(5-(3-[18F]-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one would be a good candidate for PET imaging of BACE1.
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ItemThe Role of metals and Aβ in excitotoxicity and Alzheimer’s disease( 2015)Background: N-methyl-d-aspartate receptors (NMDARs) are ionotropic channels gated by the excitatory amino acid, glutamate. They play an essential role in synaptic plasticity, enhancing synaptic signal strength through long-term potentiation (LTP), a process thought to underlie learning and memory. At the synapse, NMDARs mediate neuroprotective signaling pathways including the regulation of calcineurin activity and inhibition of glycogen synthase kinase (GSK3). Under pathological conditions the prolonged and enhanced exposure of NMDARs to glutamate results in an excessive flux of calcium (Ca2+) into the cell. This triggers a range of responses resulting in cell death, including increased oxidative stress, inappropriate activation of proteases such as calpain, dysregulation of Ca2+- related pathways, mitochondrial damage and an apoptotic cascade. This process, termed excitotoxicity, contributes significantly to the acute neurodegeneration in ischemia and traumatic brain injury (TBI) and is believed to underlie the chronic neurodegeneration in Huntington’s disease (HD) and more recently, Alzheimer’s disease (AD). Alzheimer’s disease (AD) is characterised by progressive cognitive impairment resulting from synaptic degeneration and neuronal loss. A proposed key event in its aetiology is the formation of oligomeric species of the beta amyloid (Aβ) peptide. Recent work has demonstrated that the soluble Aβ oligomers induce excessive calcium influx across the cell membrane resulting in neuronal death by excitotoxicity. It is believed these toxic species of Aβ oligmomerise in the synaptic cleft between neurons in the hippocampus due to high levels of zinc and copper. These metals are released upon NMDAR activity from the pre- and post-synapse, respectively and can bind Aβ, increasing its rate of oligomerisation. Subsequent excitotoxic interactions between Aβ and NMDARs are copper (Cu2+)-dependent. In contrast, Cu2+ is also neuroprotective against excitotoxicity demonstrating the crucial role of metal homeostasis in specific regions of the brain affected by neurodegenerative diseases. Objectives: This PhD project has sought to determine the contribution of metals in excitotoxicity and whether modulating their levels could provide a mechanism to protect against this form of cell death. As excitotoxicity is strongly implicated in the aetiology of Alzheimer’s disease subsequent research aimed to describe the involvement of excitotoxicity in Aβ-mediated cell death in cortical neural model and to establish whether metals played a necessary role in this process. The final goal of the research presented here was the development of a neural-based assay, which could be employed to screen various forms of Aβ to detect more toxic forms of the peptide. Results: In experiments with the metal chaperone PBT2, a therapeutic in clinical trials for chronic neurodegenerative diseases, neurons were protected against excitotoxic cell death by pretreatment with the drug. Subsequent experiments demonstrated that this was a metal-mediated effect that required zinc. Pretreatment with this drug induced preconditioning in neurons by moderate increases in intracellular levels of calcium that activated survival pathways and inhibited activation of calcineurin and GSK3 preventing cell death. In further investigations the parameters for Aβ-induced excitotoxicity in cortical neurons were determined. In the presence of non-toxic levels of glutamate, Aβ induced significant toxicity that was dependent on the presence of metals, as demonstrated by metal chelation. These findings translated to the development of a calcium flux assay, which provided a functional readout of Aβ toxicity. Finally, this assay was validated by screening species of Aβ with varied degrees of toxicity to neurons. Conclusions: This work highlights the importance of metals in neurodegenerative disease and demonstrates modulation of both Cu2+ and Zn2+ levels in hippocampal synapses provide valid targets for future therapeutic approaches by preventing the formation of toxic oligomeric species. A concurrent finding has iii been the identification of the parameters required for Aβ-induced excitotoxicity, which provides the tools to screen an array of both in vivo and in vitro Aβ species to determine their toxicity. This knowledge will enable targeted clearance of these forms of the Aβ peptide, which, along with therapies preventing oligomer formation, will show significant therapeutic affects in the treatment of Alzheimer’s Disease.