Developing novel screening assays and therapeutics for Niemann-Pick disease

Fenn, Gareth 2024. Developing novel screening assays and therapeutics for Niemann-Pick disease. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

In this thesis, screening assays, and compounds with potential therapeutic effects were developed and analysed for use in Niemann-Pick disease. Niemann-Pick disease is a rare lysosomal storage disorder characterised by lysosomal sphingolipid accumulation. In Niemann-Pick Types A&B loss of acid sphingomyelinase is the primary cause of the disease, whereas in Niemann-Pick type C, loss of NPC1 protein activity results in lysosomal lipid accumulation. We first evaluated a novel fluorescent molecule, AQ7, that we hypothesised to be NPC1 specific substrate. However, AQ7 was observed to have lysosomal entrapment, and thus led to the development of AQ7 is a novel lysosomal probe, possessing a range of improvements over other commercially available lysosomal probes. We investigated a biomarker present in Niemann-Pick disease, free zinc and its ability to be used as a marker to identify novel therapeutics. An imaging-based zinc assay was developed using TSQ as a probe, which was used to screen a 1500-compound library for modulators of intracellular free zinc in patient derived NPC1 fibroblasts. A total of 20 hit compounds that reduced lysosomal by up to 45% in 24 hours in NPC1 mutant fibroblasts were identified. Cannabidiol and copaiba, have been suggested as potential therapeutics in Niemann-Pick, and were investigated in vitro. Both cannabidiol and copaiba oil reduced lysosomal volume, sphingomyelin and cholesterol staining in patient fibroblasts. Increases in acid sphingomyelinase activity and protein expression was found upon cannabidiol and copaiba treatment, The increased acid sphingomyelinase expression, was postulated to be driven by an increase in TFE3 nuclear localisation, upon cannabidiol and copaiba treatment. We have shown that AQ7 is not a suitable substrate for NPC1, but is a novel lysosome probe, with the ability to be used for time course assays without inducing lysosomal de-acidification. We have demonstrated that screening for zinc modulators is a viable screening strategy in Niemann-Pick and has allowed us to identify 20 potential starting points for therapeutics through their ability to both modulate zinc and reducing lysosomal volume. Furthermore, we’ve identified that cannabidiol and copaiba reduce lysosomal storage in Niemann-Pick disease through activating TFE3.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	30 January 2025
Last Modified:	30 Jan 2025 14:47
URI:	https://orca.cardiff.ac.uk/id/eprint/175757

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