Targeted therapeutics against cancer and infections Identification and characterization of human ecto-nucleotidase (CD39) and coronaviral protease (M^pro) inhibitors

Abstract

The main objectives of this thesis were the development, characterization and optimization of enzyme inhibitors (i) for the human ecto-nucleotidase CD39, and (ii) for the main protease of the human-pathogenic coronavirus SARS-CoV-2, to provide novel treatment options for tumors and infections. Inhibitors of these enzymes have the potential to abrogate the damaging proliferation of cancer cells, or viruses, respectively, with only limited or negligible harming of the human organism. The search for small molecule inhibitors was conducted by analyzing the compounds’ effects on enzyme kinetics using optical plate reader- and capillary electrophoresis-based assays. By critical evaluation of the generated data, we were able to identify and subsequently optimize potent, selective, and metabolically stable inhibitors for both target enzymes. <br /> Ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) is the most prominent enzyme hydrolyzing extracellular nucleotides, e.g. ATP and ADP, yielding AMP. Its inhibitors have the potential to increase the concentrations of extracellular adenosine triphosphate (ATP), which stimulates the cellular immune system by activation of purinergic P2X and P2Y receptors. At the beginning of the work for this thesis no potent small molecule CD39 inhibitor with suitable pharmacokinetic properties was available. Therefore, we set out to improve known inhibitors, and to identify new lead structures. For this purpose, structure-activity-relationships of anthraquinone derivatives, nucleotide derivatives and analogs, and various heterocyclic compounds were analyzed. The analog ARL67156 of the CD39 substrate ATP, and its two most potent derivatives were found to be moderately potent, competitive inhibitors of NTPDases, also inhibiting the AMP-hydrolyzing enzyme ecto-5’-nucleotidase (CD73). We discovered that these nucleotide derivatives possess only poor metabolic stability, which limits their utilization in in vivo studies. We confirmed that the unmetabolized anti-thrombotic prodrug ticlopidine acts as a non-competitive inhibitor of CD39. Furthermore, we demonstrated that closely related, but metabolically stable 2-substituted thienotetrahydropyridine derivatives have equal potencies in inhibiting CD39, but improved target selectivity. A screening approach focused on a self-assembled approved protein kinase inhibitor library was successful leading to the identification of the CD39-inhibitory activity of ceritinib. This anti-cancer drug has an excellent metabolic stability. It inhibits CD39 non-competitively with a potency in the low micromolar range, which might contribute to its potent anti-cancer activity. <br /> A project on the development of antiviral drugs against COVID-19 was initiated at the start of the pandemic in 2020. We selected the coronavirus main protease (SARS-CoV-2 M^pro) as a suitable target, since it is essential for the viral replication cycle. By collaborating within an interdisciplinary team, we quickly established an enzyme activity assay utilizing a novel fluorogenic substrate. This enabled us to screen and evaluate various inhibitor classes. The focus of this part of the thesis was on the characterization of pyridyl esters, which are covalently binding inhibitors with nanomolar potencies. Along with their time-dependent effect on protease activity, their stabilities were assessed, in order to select the most promising lead structures for future drug development.