Abstract 2872: Methods of assessment of polysialyltransferase inhibitors for treatment of tumor cell dissemination
Authors: Xiaoxiao Guo, Jodie R. Malcolm, Anjana Patel, Marrwa M. Ali, Goreti Ribeiro Morais, Steven D. Shnyder, Paul M. Loadman, Laurence H. Patterson, Robert A. Falconer
Journal: Cancer Research (2021)

Abstract Polysialic acid (polySia) decorates the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumors, notably neuroblastoma and small cell lung cancer, and is strongly associated with poor prognosis and aggressive disease in patients in the clinic [1]. PolySia modulates tumor cell-cell and cell-matrix adhesion, migration, invasion and metastasis. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumors, abrogates tumor cell migration and invasion. PolyST is a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1]. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimized for identification of novel polyST inhibitors. We describe the development of cell-free and cell-based assays that enable assessment of polysialyltransferase inhibition. Development of the HPLC-fluorescence-based enzyme assay includes a comprehensive optimization of assay conditions, including evaluation of metal ion composition, enzyme concentration, substrate and acceptor concentration, 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 optimized conditions. Under these optimized 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. We additionally report optimized HPLC-based and ELISA-based methodologies for assessment of polyST inhibition in neuroblastoma cells, using endoneuraminidase N as control, and assessment of ICT3176 (a polysialylation inhibitor) as a test agent. In conclusion, in vitro cell-free and cell-based assays for accurate measurement of polysialyltransferase (polyST) inhibition are described, specifically designed for routine identification of potential polyST inhibitors, generation of kinetics data and assessment of mode of inhibition, and assessment of effects on cellular polysialylation. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, these are vital tools to enable preclinical identification and evaluation of novel polyST inhibitors for neuroblastoma therapy. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939. Citation Format: Xiaoxiao Guo, Jodie R. Malcolm, Anjana Patel, Marrwa M. Ali, Goreti Ribeiro Morais, Steven D. Shnyder, Paul M. Loadman, Laurence H. Patterson, Robert A. Falconer. Methods of assessment of polysialyltransferase inhibitors for treatment of tumor cell dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2872.