Features of Idebenone and Related Short-Chain Quinones that Rescue ATP Levels under Conditions of Impaired Mitochondrial Complex I

Short-chain quinones have been investigated as therapeutic molecules due to their ability to modulate cellular redox reactions, mitochondrial electron transfer and oxidative stress, which are pathologically altered in many mitochondrial and neuromuscular disorders. Recently, we and others described that certain short-chain quinones are able to bypass a deficiency in complex I by shuttling electrons directly from the cytoplasm to complex III of the mitochondrial respiratory chain to produce ATP. Although this energy rescue activity is highly interesting for the therapy of disorders associated with complex I dysfunction, no structure-activity-relationship has been reported for short-chain quinones so far. Using a panel of 70 quinones, we observed that the capacity for this cellular energy rescue as well as their effect on lipid peroxidation was influenced more by the physicochemical properties (in particular logD) of the whole molecule than the quinone moiety itself. Thus, the observed correlations allow us to explain the differential biological activities and therapeutic potential of short-chain quinones for the therapy of disorders associated with mitochondrial complex I dysfunction and/or oxidative stress

NQO1-Dependent Redox Cycling of Idebenone: Effects on Cellular Redox Potential and Energy Levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders

Effect of quinones on basal lipid peroxidation is dependent on their logD value.

<p>(A) Cellular levels of basal lipid peroxidation were measured using BODIPY-C₁₁ dye and correlated with the respective logD value. Response to DMSO was set to 100%. Compounds #69 and 70 represent two mitochondrially targeted idebenone molecules. The values are means ± SD, n = 3 replicate wells. (B) Lipid peroxidation values were correlated to ATP rescue efficiency of each quinone. Data represent values for L6 cells. Similar results were obtained for human immortalized hepatic cells (HepG2) and human myoblasts (9Te) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s003" target="_blank">Figure S3</a>). For reasons of clarity, the highly prooxidative (>200%) compounds (# 36,44,52,63 64) and error bars were omitted from graphs but standard deviation values can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a>, which lists all results.</p

Efficacy of ATP rescue in the presence of rotenone by different quinones.

<p>(A) ATP rescue by 4 different quinones was determined in rotenone-treated rat myoblasts (L6). ATP rescue is defined as the percentage of quinone-induced increase in ATP levels in the presence of rotenone, relative to the ATP reduction by rotenone alone. Bars represent the mean of 3 wells, error bars represent standard deviation. (B) ATP rescue by different quinones is not cell type- or species-dependent. ATP rescue by 70 quinones was determined in primary human myoblasts (9Te), rat myoblasts (L6) and human immortalized hepatic cells (HepG2). Correlation of values derived from rat myoblasts (L6) and human myoblasts (9Te), are shown (R² = 0.8434). Similar results were obtained for rat myoblasts (L6) vs. human immortalized hepatic cells (HepG2) as well as human myoblasts (9Te) vs. human immortalized hepatic cells (HepG2) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s001" target="_blank">Figure S1</a>). For reasons of clarity, error bars were omitted from (B) but standard deviation (SD) values can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a> which lists all results.</p

Reduction of quinones by NQO1 in a cell free assay system is dependent on their logD values.

<p><i>In vitro</i> reduction of 70 quinone derivatives by recombinant NQO1 was correlated to their calculated logD values. Each data point represents the mean from 2 independent experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s004" target="_blank">Table S1</a> for details about the quinones used). For reasons of clarity, error bars were omitted from graphs but standard deviation values can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a> which lists all results.</p

Cellular ATP rescue by different quinones in the presence of rotenone is dependent on their logD value.

<p>(A) Cellular ATP rescue capacity was correlated to the rate of reduction by recombinant NQO1 enzyme in a cell-free assay. Clear outliers (boxed compounds, red circles) are either mitochondrially targeted (#69, 70) or have logD values <2. (Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone-0036153-g003" target="_blank">Figure 3B</a>). Similarly, some compounds demonstrated good ATP rescue activity while being poorly reduced by NQO1 in vitro (boxed compounds, blue circles). Of all compounds tested, about 14% demonstrated very good (>80%) ATP rescue activity (green circles). (B) ATP rescue ability was correlated to the logD value of each compound. Data represent values for rat myoblasts (L6). Similar results were obtained for human immortalized hepatic cells (HepG2) and human myoblasts (9Te) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s002" target="_blank">Figure S2</a>). The data represent one typical experiment out of three experiments, which yielded similar results. The values are means ± SD, n = 3 replicate wells. Color coding of results is analogous to (A). For reasons of clarity, error bars were omitted from graphs but standard deviation values can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a>, which lists all results.</p

Schematic representation of the requirements for cytoplasmic-mitochondrial electron transfer and ATP rescue.

<p>Only quinones with a logD<7 are reduced by cytoplasmic NQO1 or other reductases. Once reduced, and depending on permissive lipophilicity characteristics (27), they can shuttle electrons into the mitochondrial respiratory chain by reducing complex III or integrate into lipid membranes to prevent lipid peroxidation. However, these effects manifest only, if the compounds upon oxidation can return to the cytoplasm to be reduced again, which allows them to act in a catalytical manner. (N: no, Y: yes).</p