Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency

Introduction

Paediatric mitochondrial disease due to complex I (CI) deficiency is a heterogeneous group of disorders, and can be due to alterations in either the nuclear or mitochondrial genome. It is the most prevalent defect in the respiratory chain in paediatric patients and often leads to serious or fatal neurological presentations, such as Leigh syndrome1. There are currently very limited evidence-based treatment options directed towards mitochondrial respiratory chain dysfunction2,3. Succinate is a mitochondrial substrate metabolized through complex II (CII). It is not cell membrane-permeable and exogenously given succinate has limited uptake into cells.

Here we describe that cell membrane-permeable prodrugs of succinate provide increased ATP-linked mitochondrial oxygen consumption in CI-deficient human cells and tissues, which offers a potential future intervention for patients with metabolic decompensation due to mitochondrial CI dysfunction.

Results

Drug development and screening

In a drug discovery program, >50 different prodrugs of succinate4 were designed, synthesized and evaluated for cell permeability and ability to support respiration independent of CI in human peripheral blood cells from healthy donors (platelets and mononuclear cells (PBMCs)) using an Oroboros O2k respirometer. Three compounds were selected for further evaluation: NV101-118 (NV118, diacetoxymethyl succinate), NV101-189 (NV189, bis-(1-acetoxy-ethyl) succinate) and NV101-241 (NV241, 1-acetoxyethyl acetoxymethyl succinate) (Fig. 1a). This article focuses on NV189, but qualitatively the results for all three prodrugs were similar and data on the other compounds are presented asSupplementary Figs.

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).