Abstract 4778: Novel bioluminescence approaches for measuring fatty acid β-oxidation and production of reactive oxygen and nitrogen species

Abstract

Abstract Energy uptake and utilization in eukaryotic cells is a dynamic process regulated by a series of interacting metabolic networks. Interrogation of this complex network relies on rapid, sensitive approaches that do not require extensive sample handling and are easily adaptable to 96- and 384-well plates. Previously, using the Ultra-Glo luciferase reaction we developed a panel of bioluminescent assays that can be used to monitor numerous aspects of cellular metabolism and mitochondrial function including ATP production, glucose and amino acid metabolism, and the TCA cycle. Here, we extend the use of the luciferase reaction and report on the development of novel bioluminescence probes for studying two important metabolic cellular responses: fatty acid β-oxidation (FAO) and production of reactive oxygen and nitrogen species. For studying FAO, we developed a cell permeable probe with caged-luciferin attached to a fatty acid chain. The probe enters the cells and following FAO cycling, free luciferin is released and detected using Luciferin Detection Reagent. We validated the approach using known FAO activators and inhibitors and used it to monitor the changes in FAO during T-cell activation. For measuring reactive oxygen and nitrogen species, highly selective bioluminescence probes were developed. Upon reaction of the probes with their corresponding ROS target, they form the stable D-luciferin reporter molecule, causing luciferin to accumulate. Upon treatment with luciferase in the detection step, the generated light allows for quantification of the superoxide or nitric oxide formed. Both probes are suitable for in vitro and cell-based detection of ROS in an “add and read” format, providing for a simple workflow amenable to high throughput experimentation. Citation Format: Kim Haupt, Matt Larsen, Hui Wang, Natasha Karassina, Mike Valley, Wenhui Zhou, Jolanta Vidugiriene. Novel bioluminescence approaches for measuring fatty acid β-oxidation and production of reactive oxygen and nitrogen species. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4778.