1788-P: AMPK Activation by ATX-304 Is Not Secondary to Changes in ATP Levels

Introduction and Objective: ATX-304 is a novel, clinical-stage, small molecule that increases AMP-activated protein kinase (AMPK) activity and increases mitochondrial respiration via increased proton leak. Compounds that increase mitochondrial proton leak may increase AMPK activity secondary to decreased mitochondrial membrane potential (MMP) and decreased ATP levels. ATX-304 effects on MMP, ATP levels and AMPK activation were investigated up to a solubility limit of 20 µM. Methods: Isolated mitochondria from mouse skeletal muscle were preincubated for 30 mins with 5 µM ATX-304 before oxygen consumption rate (OCR), reactive oxygen species (ROS, via Amplex Red assay of H2O2 emission), and MMP (via TMRM) were assayed. MMP (via TMRE) and ROS (via DCFDA) were assayed in intact cultured mouse embryonic fibroblasts (MEF) following 1 hour incubation with 2-20 µM ATX-304. AMPK phosphorylation at α-T172 (reflecting AMPK activation) was assayed in wild-type (WT) HEK-293T cells and HEK-293T cells expressing AMP-insensitive AMPK mutant (γ1-R299G; RG) following 2 hour incubation with 20 µM ATX-304. ATP levels compared to control were assayed (via CellTiter-Glo® Assay) in human hepatocytes following 48 hours incubation with 0.2-20 µM ATX-304. Results: ATX-304 increased OCR and decreased MMP and ROS emission in isolated mitochondria from mouse skeletal muscle, consistent with an increase in proton leak. In contrast, ATX-304 decreased ROS but did not decrease MMP in intact MEF cells, unlike the reduction in both ROS and MMP seen with the positive controls, TBHP and FCCP, respectively. ATX-304 increased α-T172 phosphorylation of WT and RG mutant AMPK in HEK-293T cells to a similar extent, unlike the reduced activation of RG mutant AMPK from a 2-deoxyglucose control. ATX-304 did not reduce ATP compared to control in human hepatocytes. Conclusion: MMP and ATP levels are preserved upon exposure to ATX-304 up to 20 µM in intact cells. AMPK activation by ATX-304 up to 20 µM is not secondary to a change in ATP levels. Disclosure E.J. Schneider: Employee; Current; Cambrian BioPharma, Amplifier Therapeutics AB. R.I. Thieroff-Ekerdt: Employee; Current; Cambrian Biopharma. J. Peyer: Board Member; Current; Cambrian Biopharma. A. Gudiksen: None. H. Pilegaard: None. N.X. Ling: None. J.S. Oakhill: None. J. Hall: None.