Yes / The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors. / Yorkshire Cancer Research, Wellcome Trust

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.

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.

PolySialic 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.

Yes / Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated posttranslational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (Ki = 10 μM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers. / Yorkshire Cancer Research; EPSRC; Association for International Cancer Research; Jordanian Government PhD scholarship

Yes / α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Polysialic acid (PSA) is a developmentally regulated glycan made of repeating sialic acid monomers with α2-8 linkages. PSA has very limited expression in adults, and modifies only a few cell-surface proteins. However, PSA is overexpressed in several human cancers and is associated with metastasis and poor prognosis. We have described a derivative of PSA containing a mixture of de-N-acetyl and N-acetyl neuraminic acid residues (dPSA) found intracellularly in many normal human tissues but expressed at much higher levels on the cell surface of many human cancer cell lines. The proteins modified with dPSA and dPSA function in normal and abnormal human biology are unknown. The purpose of this study was to identify protein(s) modified with PSA and possible dPSA-dependent functions in cancer cell lines that express dPSA antigens. Using co-immunoprecipitation with the anti-dPSA monoclonal antibody SEAM 2 and mass spectroscopy, we identified membrane-associated nucleolin that is either directly modified or associated with dPSA. In addition, knocking down expression of the polysialyltransferase ST8SiaII (STX) in SK-MEL-28 human melanoma cells nearly eliminated dPSA and nucleolin from membranes but had no effect on the levels of nuclear nucleolin, and resulted in aberrant cell morphology, cell adhesion, and motility. The data suggest that cell-surface nucleolin depends on modification with dPSA, and that dPSA-modified nucleolin has an important role in cell adhesion and migration.

The full text will be available at the end of the embargo period: 5th March 2027

Polysialic acid is a unique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acid is 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), with STX 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 polysialyltransferase could lead to control of tumour dissemination and metastasis. The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferase inhibitors. 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 acid surface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acid surface expression and invasion. Of the potential polysialyltransferase inhibitors evaluated, 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 acid re-expression and invasion in polysialic acid expressing cells. Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over other members 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 vitro cell based assays to discovery more potent derivatives.

Polysialic 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.