Ehinger 2015 Abstract MiPschool London 2015

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Cell-permeable mitochondrial complex II substrates – a new compound class for treatment of complex I-linked mitochondrial disease.

Link:

Ehinger JK, Morota S, Piel S, Karlsson M, Sjoevall F, Ford R, Fliri H, Hansson Magnus J, Elmer E (2015)

Event: MiPschool London 2015

Pediatric mitochondrial disorders display great diversity in severity and clinical presentation but certain pathophysiological mechanisms are shared between several syndromes. Partial or isolated complex I (CI) dysfunction of the mitochondrial electron transfer-pathway is part of the pathophysiology in a majority of cases. The endogenous mitochondrial complex II (CII) substrate succinate cannot support respiration when administered to intact cells due to inadequate cell membrane permeability. We introduce a group of new cell permeable chemical entities able to bypass a defect CI and restore mitochondrial respiration and cellular ATP-production by providing substrate to CII, in an attempt to address the unmet medical need for these conditions.

Effects of succinate and the newly designed prodrugs of succinate on mitochondrial respiration were assayed using high-resolution respirometry of intact human peripheral blood cells. Several protocols were used – with and without rotenone inhibition of CI in the screening cascade. The three prodrugs NV118, NV189 and NV241 met the predefined efficacy criteria of cell permeability and positive effect on mitochondrial respiration.

Oxygen utilization by intact human blood cells with rotenone-induced CI inhibition was instantly and substantially increased upon addition of either prodrug. In intact cells with normal CI function with endogenous substrates, the respiration was also increased, demonstrating the synergy of convergent substrate supply through both CI and CII with the addition of the cell-permeable CII substrates. Uncoupling or auto-oxidation as cause of the increase in oxygen consumption was ruled out using specific respirometry protocols and flow cytometry (assessing mitochondrial membrane potential using the probe TMRM). In metabolomic assays of human platelets and white blood cells treated with the compounds, increases in succinate and other Kreb’s cycle intermediates were seen, further demonstrating that mitochondrial substrates are delivered to the intracellular space and the mitochondria. The general applicability of these findings was confirmed by repeating key experiments in cultured human fibroblasts and human heart muscle fibers. To further validate that cell-permeable succinate prodrugs can support aerobic respiration, we determined lactate production of rotenone-inhibited platelets over several hours. The succinate prodrugs, but not succinate or monomethyl succinate, attenuated the rotenone-induced lactate production of intact human platelets. Also lactate production induced by the anti-diabetic drug Metformin (a drug known to cause lactic acidosis as a rare but severe side effect) was counteracted by coincubation with succinate prodrugs.

We demonstrate for the first time that ATP-coupled mitochondrial respiration can be increased in intact human cells using cell-permeable prodrugs of succinate. This novel class of pharmaceutical agents offers a potential treatment specifically directed towards supporting cells suffering from energy decompensation due to primary or secondary CI dysfunction.


O2k-Network Lab: JP Tokyo Uchino H, SE Lund Elmer E


Labels: MiParea: Instruments;methods 


Organism: Human  Tissue;cell: Heart, Blood cells, Fibroblast  Preparation: Intact cells, Permeabilized tissue  Enzyme: Complex I 


Pathway: N, S  HRR: Oxygraph-2k 


Affiliations

1-Mitochondrial Medicine, Dept Clinical Sciences, Lund Univ, Sweden. - johannes.ehinger@med.lu.se

2-NeuroVive Pharmaceutical AB, Medicon Village, Lund, Sweden

3-Div Cancer Biol, Group Research Mol Functions Targets, Nat Cancer Center, Tokyo, Japan

4-Mitopharm Ltd, c/o Selcia Ltd, UK