Screening and biological evaluation of novel anticancer agents and lipid profiling of autophagy-related proteins

Abstract

This thesis addresses three different subjects closely related. First, a phenotypic screening measuring cytotoxicity in the gastric cancer cell line HGC-27 is reported. Next, a targeted-based assay measuring the activity of autophagy-related protein Atg4B is developed and employed to identify small-molecule inhibiting this protein. Finally, a basic research study is carried out to gain knowledge of the molecular mechanisms that control autophagy.

The first chapter of this thesis covers the study of a variety of squaramates and squaramides and their cytotoxic activity in different cancer cell lines. The squaramide 34 showed a potent and selective cytotoxicity against the human gastric cancer cell line HGC-27. Studies directed to elucidate the mechanism of induced cell death were performed. Cell cycle distribution analysis and cell death studies showed that compound 34 induces cell cycle arrest at the G0/G1 phase and caspase-dependent apoptosis implicating the intrinsic pathway and mitochondrial membrane depolarization. In conclusion, squaramide 34 can be considered a potential anticancer agent for gastric carcinoma.

Drug resistance is a major issue in oncology and a limiting factor for anticancer drug efficacy. Autophagy induction is employed by cancer cells as a survival mechanism, therefore, the employment of autophagy inhibitors as adjuvant treatment could increase the anticancer drug efficacy. The second chapter of this thesis is focused on the development of a novel AlphaScreen-based HTS assay and a Mass spectrometry- based counter screen to identify Atg4B inhibitors. A high-throughput virtual screening performed with the National Cancer Institute Open Database library and subsequent evaluation of 250 selected compounds allowed the identification of three potential inhibitors (NSC83713, NSC126353 and NSC611216). Derivatives of them were synthesised and their characterization by both techniques allowed the discovering of most active compounds 54, 55, 56 and 57. Compound 57 was chosen for additional characterization based on its high potency and good cytotoxicity profile on cell lines. Inhibition of the autophagic flux was maintained and the synergistic effect of 57 combined with oxaliplatin resulted in an enhanced cell death in the human cell line HT-29. In conclusion, the aminobenzo[cd]indol-2-[1H]-one scaffold represents a novel chemotype for the development of small molecule inhibitors of Atg4B.

The conjugation of a phosphatidylethanolamine (PE) unit at the C-terminus of LC3 is essential for the autophagy regulation. Despite the high variability described in cellular lipids, a potential role of heterogenous lipidation on protein activity has not been considered. Hence, the third chapter of this thesis is focussed on the development of a lipidomic approach for the study of the PE species conjugated to LC3/GABARAP. The method relies on the enzymatic release of the protein-bound lipids mediated by Atg4B incubation. The strategy is applied to the whole proteome and proteins isolated by immunoaffinity techniques. Preliminary results could not succeed in the analysis of the lipid bound proteins. Moreover, lipid contamination of the enzyme difficulted the establishment of the lipidomic approach.