Pharmacological evaluation of novel polysialyltransferase inhibitors as anti-metastatic agents and development of analytical methods for assessment of polysialylation inhibition. In vitro assessment of the effects of novel polysialyltransferase inhibitors on tumour cell function and development of quantitative HPLC-based methods for evaluation of novel polysialyltransferase inhibitors
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PhD Thesis (9.942Mb)
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Publication date
2016Supervisor
Falconer, Robert A.Loadman, Paul M.
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The University of Bradford theses are licenced under a Creative Commons Licence.
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University of BradfordDepartment
Institute of Cancer TherapeuticsAwarded
2016Metadata
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Polysialic acid (polySia) is a carbohydrate polymer highly expressed during embryonic development but rarely expressed during postnatal development. Two polysialyltransferase (polyST) enzymes are responsible for the synthesis of polySia: ST8SiaII and ST8SiaIV. During oncogenesis polySia is re-expressed and it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in neuroblastoma and many other tumours. PolyST inhibition thus presents a novel, selective and largely unexplored therapeutic opportunity to reduce tumour dissemination. Progress towards development of polyST inhibitors has been limited by lack of an efficient technique for quantitative assessment of enzyme activity. We have validated a highly sensitive cell-based and cell-free high throughput HPLC-based inhibition assays. Using isogenic cell lines (C6-STX: polySia+/ST8SiaII+ and C6-WT: polySia-/ST8SiaII-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y), a set of ST8SiaII inhibitors designed and synthesised in house were evaluated for their ability to reduce polySia expression and to modulate cell migration in vitro. We have identified CMP-sialic acid precursors, including ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These effects were only found in cell lines expressing ST8SiaII and polySia. Furthermore, we have investigated the possible additive anti-migratory effect of combining polyST inhibition with the inhibition of certain signalling pathways that have been previously suggested to be modulated by polySia expression. Out of these combinations it was found that combining ST8SiaII and C-MET/ALK inhibition had a synergistic effect on inhibiting cancer cell migration. Additionally, the effect of polySia expression on cancer cell behaviour under hypoxic conditions was examined, where it was found that polySia expression enhanced cell migration and survival and inhibits cell adhesion. In summary, polyST inhibitors which dramatically decrease cell migration in vitro through modulation of polySia assembly were identified, using optimised cell-free and cell-based assays. Initial investigations into the role of polySia in hypoxia were also accomplished. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.Type
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Synthesis of inhibitors of polysialyltransferases PST and STX. Development of routes to synthesis, preparation and purification of carbohydrate and carbacycle-based potential inhibitors of the polysialyltransferase enzymes PST and STXPolySialic acid (polySia) is a linear carbohydrate homopolymer of α- 2,8-linked sialic acids and a posttranslational modification of NCAM (neural cell adhesion molecule), biosynthesized by combined action of two polysialyltransferase enzymes, ST8SiaIV(PST) and ST8SiaII(STX). PolySia alters NCAM-dependent cell adhesion that is crucial for the CNS development. In adulthood, polySia expression is largely absent persisting only in areas of the brain associated with neuronal plasticity. Significantly, a number of malignant tumours re-express polySia and there is considerable evidence that its presence is related to higher malignancy, invasion and metastasis. The hypothesis underpinning this project is that inhibition of polySia biosynthesis will prevent (or reduce) tumour cell migration and invasion, thereby reducing the incidence of metastasis, which will lead to higher patient survival. The work reported in this thesis describes efforts towards the synthesis polysialyltransferase inhibitors that are structural analogues of CMP-Neu5Ac, the natural substrate. Specifically, development of methodology to synthesise building blocks suitable for conjugation as inhibitors is described. Quinic acid-based substrate analogues were explored, with a focus on development of chemistry to achieve substitution of C1-OH. Several protected quinic acid-based compounds were synthesized, and deoxygenation of the C1-OH through the use of a Barton-McCombie reaction was accomplished successfully, allowing an attempt to introduce different aliphatic groups at C1 position using the Mukayiama reaction. Synthesis of a cytidine building block, suitable for conjugation to either quinic acid or sialic acid is also reported. In parallel, studies towards the development of sialoside disulfide analogues are described, with novel conditions identified for their synthesis.
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Investigation of inhibitors of polysialyltransferase as novel therapeutics for neuroblastoma. Development of in vitro assays to assess the functionality and selectivity of novel small-molecule inhibitors of polysialyltransferases for use in neuroblastoma therapyPolysialic acid is aunique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acidis an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma,strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST(ST8SiaIV)and STX(ST8SiaII),withSTX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferasecould lead to control of tumour dissemination and metastasis.The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferaseinhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells(SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acidsurface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acidsurface expression and invasion. Of the potential polysialyltransferase inhibitorsevaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p<0.05)reduction in cell-surface polysialic acidre-expression and invasion in polysialic acid expressing cells.Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over othermembers of the wider sialyltransferase family (α-2,3-and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitrocell based assays to discovery more potent derivatives.
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Pharmacological evaluation of the inhibition of polysialyltransferases as a therapeutic strategy in cancer. Characterisation of models for evaluating polysialic acid as a potential therapeutic target and pharmacological assessment of novel polysialyltransferase inhibitorsNeuroblastoma is a highly metastatic and invasive tumour with poor prognosis. Despite recent advances in the treatment of neuroblastoma, mortality is still high due to uncontrolled metastatic disease, and novel therapeutic approaches for the treatment of neuroblastoma are therefore desperately needed. A potential novel approach for therapy of neuroblastoma relates to the polysialic acid decoration of the neural cell adhesion molecule (PSANCAM). PSA-NCAM is selectively re-expressed in a number of tumours including neuroblastoma, where it is thought to modulate tumour dissemination. Expression is strongly associated with poor clinical prognosis and an aggressive tumour phenotype. Inhibition of the enzymes responsible for synthesis of PSA, the polysialyltransferases (polySTs) presents a novel and selective therapeutic opportunity. The aims of the studies described in this thesis are to evaluate PSANCAM expression and function in neuroblastoma, and to develop and utilise cell-based models to pharmacologically investigate novel polyST inhibitors. PSA-NCAM was seen to be highly expressed in neuroblastoma clinical specimens and associated with phenotypes of tumour aggressiveness. A screening panel consisting of cell lines with a range of PSA-NCAM expression types was established and utilised to develop assays for pharmacologically assessing novel polyST inhibitors. Using cytidine monophosphate (CMP), a naturally-occurring inhibitor of polySTs, the robustness of the assays was confirmed before progression to evaluate novel molecules. From 16 compounds identified in an in vitro screen of polyST inhibition, three promising polyST inhibitors were identified. These promising polyST inhibitors modulated PSA-NCAM expression on the tumour cell surface and led to a significant reduction in cell migration. Therefore the work presented in this thesis suggests that targeting polySTs is a promising novel therapeutic strategy for neuroblastoma and further research in this area is warranted.
