Aasander Frostner 2017 MiP2017
Within a drug discovery program derived from a project for a pharmacological treatment of mitochondrial complex I deficiency, we have developed cell-permeable prodrugs of succinate (NV118) and malonate (NV161) suitable for research use in intact cells. Succinate is an intermediate of the Krebs’ cycle and acts as mitochondrial energy substrate by providing reducing equivalents to complex II (succinate dehydrogenase) of the mitochondrial oxidative phosphorylation pathway. As succinate is converted to malate by complex II, electrons are transferred down the pathway leading to proton pumping and ATP-synthesis. Succinate, as a dicarboxylic acid, is not cell-permeable and for exogenous succinate to enter cells the cell membrane requires permeabilization, using e.g. digitonin or perfringolysin. NV118 allows the researcher to deliver succinate to the cytoplasm without disrupting the plasma membrane. Malonate is a competitive inhibitor of complex II that binds to the active site of succinate dehydrogenase, thus preventing succinate from being metabolized. Like succinate, malonate is a dicarboxylic acid that does not readily permeate through the cell membrane. By using the same prodrug strategy as for NV118, the cell-permeable analogue of malonate, NV161, has been synthesized. NV118 and NV161 are rapidly metabolized, likely by the action of carboxyesterases, releasing succinate and malonate respectively.
Cell-permeable succinate and malonate were tested in a range of human cells and tissues, such as blood cells, fibroblasts, immortalized liver cells and human heart fibers either in the Oroboros O2k-FluoRespirometer (Oroboros Instruments, Innsbruck, Austria) or in the Seahorse Bioscience XFe96 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, USA). Dose-response curves for both prodrugs were obtained in human complex I inhibited platelets and primary fibroblasts.
NV118 and NV161 dose-dependently support and inhibit succinate-linked mitochondrial respiration in intact human platelets and fibroblasts. NV161 completely inhibits succinate-linked mitochondrial respiration at about ten times lower concentration as compared to malonate. Dimethyl succinate and dimethyl malonate have previously been reported to be cell-permeable, but did not show strong evidence of efficient cell penetration in this study.
We believe that NV118 and NV161 may prove valuable as scientific tools in mitochondrial research, enabling evaluation of complex II in intact cells and tissues. Analogues of both the succinate and malonate series optimized for in vivo use are simultaneously being developed.
Labels: MiParea: Respiration, mt-Medicine, Pharmacology;toxicology
Organism: Human Tissue;cell: Heart, Liver, Blood cells, Fibroblast Preparation: Intact cells
Regulation: Inhibitor, Substrate
Pathway: N HRR: Oxygraph-2k
- Aasander Frostner E(1,2), Ehinger JK(1,2), Piel S(1,2), Karlsson M(1,2), Chamkha I(1,2), Sjoevall F(1), Moss SJ(3), Webster LR(3), Hansson MJ(1,2), Elmer E(1,2)
- Mitochondrial Medicine, Dept Clinical Sc, Lund Univ, Sweden
- NeuroVive Pharmaceutical AB, Lund, Sweden
- Isomerase Therapeutics Ltd, Cambridge, UK. - firstname.lastname@example.org