A bioenergetic functional screening assay workflow to identify, validate, and characterize anti-cancer compounds

Abstract As bioenergetic metabolism is recognized as a critical activity supporting cell proliferation, differentiation, and even death, many proposed targets for cancer therapy are functionally linked to cellular bioenergetic metabolism. Here, we introduce an assay workflow for screening and analyzing potential anticancer drugs affecting cellular metabolism using extracellular flux analysis. The Agilent Seahorse XF Real-Time ATP rate assay can be used as a starting point to identify therapeutic compounds that induce a perturbation in metabolic phenotype. It provides a quantitative comparison of basal mitochondrial and glycolytic activities using the universal unit of ATP production rate. Through this initial comparative screening, the effect of different compounds on basal energetic state and pathway-specific metabolic perturbations can be identified providing information that cannot be assessed by intracellular ATP level measurements or cytotoxicity assays. A more comprehensive functional analysis can then be applied to validate the target mode of action through pathway-specific analysis tools such as the XF Cell Mito Stress Test and the XF Glycolytic Rate Assay. In this study, we tested the proposed workflow in three different NSCLC cell lines with different genetic backgrounds using a mock panel of known metabolic modulators including thiosemicarbazones, EGFR inhibitors, KRAS inhibitors, a glutaminase inhibitor, a Glut1 inhibitor, a chemotherapy drug, and a radiotherapy sensitizer candidate. The ATP production rate data from lung cancer cell lines exposed for 1 hour or 24 hours to the compounds, successfully identified the effects of metabolic pathway-specific modulation as well as metabolic phenotypic switching. In addition, dose-response studies using cell lines with different genetic backgrounds demonstrated differential susceptibility to the tested compounds. The workflow was then applied using a broader panel of 72 potential mitochondrial toxicants compounds. The assay could identify potent mitochondrial and glycolytic suppressor compounds effective on A549, a lung cancer cell line. A more comprehensive XF analysis then validated the mode of action for the identified metabolic suppressors. These results demonstrate the benefit of using the XF Real-Time ATP rate assay as an initial assay to uncover novel metabolic targets in early drug discovery research, guiding further research toward functional validation with the appropriate comprehensive XF assays. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. A bioenergetic functional screening assay workflow to identify, validate, and characterize anti-cancer compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2323.