Medicinal Chemistry and Pharmacology of Ectonucleotidases: Assay Development and Characterisation of Inhibitors

Abstract

The tumour microenvironment components work together for a common goal, i.e. to avoid defence mechanisms. The immunosuppressive environment helps tumours evade immune surveillance. This work discusses primary cancers where an overexpression of ectonucleotidases is reported. Newly developed high-throughput screening assays investigated some representative tumour cell lines, including U87, triple negative breast cancers and astrocytoma. The assays were optimised for monitoring ATPases, AMPases, NADases, ADPR, and Ap₄A-hydrolases. These newly developed assays were highly sensitive, measuring relative fluorescence units. A comprehensive review was performed for major ectonucleotidases (NPP1, NPP3, NPP4, CD38, CD39, CD73) regarding their substrate specificity, tissue expression, pathological outcome, monitoring assays, and small-molecule inhibitors with both nucleotidic and non-nucleotidic scaffolds. Our search for small-molecule inhibitors for these enzymes yielded positive results, including the most potent competitive inhibitors of NPP3 described so far that are active under physiological conditions (neutral pH). We also discovered competitive inhibitors of NPP1 with comparable Ki to the previously reported most active scaffold. Multitarget inhibitors of CD38, CD39, and NPP1 are reported. And finally, CD39-selective nucleotide inhibitors were discussed. The results of this thesis provide a basis for future research in the field of ectonucleotidases, and they may contribute to the development of novel anti-cancer drugs.