Abstract 5166: In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development

Abstract

Abstract The discovery of effective anti-cancer therapies has immense consequences on global health. Vacuolar (H+)-ATPases (V-ATPases) are multi-subunit nano-motors that acidify the lumen of intracellular organelles in normal physiology. In cancer cells, the plasma membrane V-ATPases acidify the extracellular space. This is critical in tumor invasion, metastasis, and chemo-resistance. The `a2` isoform of V-ATPase (a2V) is the major pH sensing unit of this complex and is distinctly overexpressed on the surface of cancer cells making it an excellent drug target. The chemical inhibitors such as bafilomycin/concanamycin target V-ATPase subunits also critical in normal cell function. Currently, there are no selective a2V inhibitors commercially available as anti-cancer agents. Here, we employed in silico molecular modeling to analyze specific a2V binding sites in order to design targeted small molecules a2V inhibitors. We have identified several natural compounds as a2V ligands which inhibit specifically a2V in cancer. Employing human ovarian and breast epithelial cells (normal or tumor), in vitro studies measured each a2V ligand(s) effects on cancer cell proliferation. We selected five a2V ligands showing the most potent anti-proliferative activity in cancer cells and were least toxic to normal cells. We further measured each ligand(s) effects on specific ATPase activity in vitro by determining the decrease in inorganic phosphate levels. Next, the two most potent a2v ligands (KDB001, KDB002) which belonged to alkaloid and mono-terpenoid phenol groups, were selected to measure the changes in cytosolic and extracellular pH- SNARF assay and pH of cell supernatants. KDB001 induced cytosolic pH acidification in a dose-dependent manner indicating inhibition of V-ATPase activity. Based on the above experiments, the best ligand with most prominent effect on a2V activity (KDB-001) was further validated in vivo for anti-ovarian cancer efficacy in an athymic nude mouse model. KDB001 administration resulted in a significant delay in in vivo ovarian tumor growth. Studies are underway to design and synthesize more efficient analogs for KDB001 molecular modelling, toxicity assessment and chemical synthesis approach. In conclusion, the study has discovered new inhibitors of V-ATPase-a2V, positioning these promising compounds to enter the anti-cancer drug development pipeline. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Awanish Kumar, Kenneth D. Beaman. In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5166.