Chemico-Biological Interactions 331 (2020) 109272 Available online 30 September 2020 0009-2797/© 2020 Elsevier B.V. All rights reserved. Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues Ange Mouithys-Mickalad a, * , Justine Ceusters a , Mounia Charif b , Benaïssa El Moualij b , Mathieu Schoumacher c , Simon Plyte d , Thierry Franck a, e , Lucien Bettendorff f , Bernard Pirotte a, c , Didier Serteyn a , Pascal de Tullio a, c a Center for Oxygen, Research & Development (CORD) & Center for Interdisciplinary Research on Medicines (CIRM), Institute of Chemistry, B6a, ULi` ege, All´ ee du six Août, 11, B-4000, Li` ege, Belgium b Centre de Recherche sur les Prot´ eines Prions (CRPP), ULi` ege, Quartier Hˆ opital, 15, Avenue Hippocrate, B-4000, Li` ege, Belgium c Department of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (CIRM), ULi` ege, Quartier Hˆ opital, 15, Avenue de l’Hospital, B-4000, Li` ege, Belgium d Merus, Closing in on Cancer, Immuno-Oncology, Yalelaan 62, 3584 CM, Utrecht, the Netherlands e Department of Clinical Sciences, Faculty of Veterinary Medicine, Quartier Vall´ ee 2, 5A-5D, Avenue de Cureghem, ULi` ege, B-4000, Li` ege, Belgium f Laboratory of Neurophysiology, GIGA-neurosciences, ULi` ege, Quartier Hˆ opital, 15, Avenue Hippocrate, B-4000, Li` ege, Belgium A R T I C L E INFO Keywords: Oxygen consumption rate (OCR) K ATP channel openers Cromakalim Benzopyrans ETSmax Cardiomyocytes (H9c2) ATP Mitochondrial Ca 2+ swelling ABSTRACT A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain. 1. Introduction Mitochondrion is an organelle that plays a key role in cell meta- bolism, reactive oxygen species (ROS) generation and calcium (Ca 2+ ) homeostasis [1]. Besides the crucial role of mitochondria in regulating the apoptosis of tumor cells [2], mitochondrial dysfunction has been implicated in different pathologies such as cardiovascular disease, dia- betes, metabolic syndrome, sepsis, cancer and ageing [3–6]. Recent works also report the role played by enzymatic complexes in electron transfer. The complex I (NADH-ubiquinone oxidoreductase) is considered as the primary electron entry pathway in mitochondrial electron transport chain [7]. Together with the other complexes (e.g. complex III, IV and ATP-synthase), they constitute a major proton pumping machine in the mitochondrial intermembrane which drives mitochondrial ATP production required to all cells [8]. Both complexes I and III have been reported as a major source of reactive oxygen species (ROS) involved in the cell survival and death mechanisms. Recently, complex I was described to play a crucial role in glucose-stimulated insulin secretion [9] but its precise role on β-cells in diabetic pancreas remains unknown. On the other hand, fnding a new molecule able to * Corresponding author. E-mail addresses: amouithys@uliege.be, amouithys@ulg.ac.be (A. Mouithys-Mickalad). Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint https://doi.org/10.1016/j.cbi.2020.109272 Received 18 August 2020; Received in revised form 7 September 2020; Accepted 28 September 2020