Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency

Abstract

Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.

Figure 1. Effects of mitochondrial complex II stimulation by the succinate prodrug NV189.

(a) Structures of NV118, NV189 and NV241, succinate highlighted in red. (b) Respiration in platelets (plts) with rotenone-induced mitochondrial complex I (CI) inhibition. (c) ATP-generating respiration in platelets. (d) Mitochondrial membrane potential in complex I-inhibited platelets, ratio of basal TMRM fluorescence, n=4. (e) Respiration in platelets with FCCP-induced uncoupling. (f) Respiration in digitonin-permeabilized platelets. (g) Effect on respiration in platelets with addition of the cell-permeable complex II inhibitor NV161, * indicate significant difference between NV161 and vehicle, n=4. (h) Structure of NV161, malonate highlighted in red. (i) Respiration in peripheral blood mononuclear cells (PBMCs) with rotenone-induced CI inhibition, n=4. (j) Convergent respiration in PBMCs, n=4, * indicate significant difference between pre and post dosing. (k) Respiration in human heart muscle fibres (HHMFs), n=5. (l) Lactate:pyruvate ratio in PBMCs at baseline, after rotenone CI inhibition and after treatment with NV189, n=4. * indicates significant difference using Friedmans non-parametric paired test with Dunn's multiple comparisons test of all groups against control. For three data points, pyruvate was below detection limit and the estimated lower-quantification limit was used for calculating the ratio. (m) Lactate accumulation in 2 ml buffer containing 400 × 10⁶ platelets, incubated with or without rotenone, antimycin A and NV189, n=5. (n) Lactate production in platelets, data quantification from previous panel. Mean with 95% confidence interval. All respirometric experiments in human platelets were performed with n=6 individuals donors if not otherwise stated. All data presented as mean and s.e. if not otherwise stated. In all experiments, blood cells from separate donors are used for each n. *P<0.05, **P<0.01, ***P<0.001 (two-tailed paired or unpaired Student's t-test as appropriate, difference between test compound and control if not otherwise stated).

Figure 2. Intracellular metabolism of exogenous prodrug-delivered succinate.

(a) TCA cycle intermediates in peripheral blood mononuclear cells after 20 min incubation with or without rotenone and NV189 quantified using capillary electrophoresis mass spectrometry, n=4. Data presented as mean and s.d. (b) Fraction of [¹³C] isotope labelled carbons in TCA cycle intermediates and related metabolites in human platelets incubated with [1, 2, 3, 4-¹³C₄]NV118 for 7.5, 15, 30, 120 or 240 min. Mean of n=2. 2-OG, 2-oxoglutaric acid.

Figure 3. Succinate prodrug treatment of mitochondrial complex I-deficient Leigh syndrome patient fibroblasts.

(a,b) Oxygen consumption rate (OCR) in three control fibroblast cell lines and a mitochondrial complex I-deficient cell line (recessive NDUFS2 mutation) treated with NV189 or vehicle. (c–f) Quantification of OCR in control and patient fibroblasts for each respiratory state. (g,h) Relative contribution of complex I- and complex II-linked respiration to maximum uncoupled respiration in patient cells and control cell lines. (i) Spare respiratory capacity, defined as per cent increase from endogenous baseline to maximum uncoupled respiration. Data presented as mean and s.e. of n=3 experiments from separate cell culture flasks performed with eight technical replicates each time for each cell lines. Data from the three control cell lines are pooled. *P<0.05 (two-tailed unpaired Student's t-test, difference between Leigh and control cell lines).

Figure 4. Delivery of succinate to the intracellular space via a prodrug strategy.

(a) Dysfunction in mitochondrial complex I reduces electron flow through the respiratory chain, shift metabolism towards glycolysis, induce lactate accumulation and limit ATP production. (b) Cell membrane-permeable prodrugs of succinate access the intracellular space and release succinate, enabling increased electron transport, respiration and ATP production through complex II, thus bypassing the deficiency in mitochondrial complex I.