Coenzyme Q4 is a functional substitute for coenzyme Q10 and can be targeted to the mitochondria

Abstract

Coenzyme Q10 (CoQ₁₀) is an important cofactor and antioxidant for numerous cellular processes, and its deficiency has been linked to human disorders including mitochondrial disease, heart failure, Parkinson's disease, and hypertension. Unfortunately, treatment with exogenous CoQ₁₀ is often ineffective, likely due to its extreme hydrophobicity and high molecular weight. Here, we show that less hydrophobic CoQ species with shorter isoprenoid tails can serve as viable substitutes for CoQ₁₀ in human cells. We demonstrate that CoQ₄ can perform multiple functions of CoQ₁₀ in CoQ-deficient cells at markedly lower treatment concentrations, motivating further investigation of CoQ₄ as a supplement for CoQ₁₀ deficiencies. In addition, we describe the synthesis and evaluation of an initial set of compounds designed to target CoQ₄ selectively to mitochondria using triphenylphosphonium. Our results indicate that select versions of these compounds can successfully be delivered to mitochondria in a cell model and be cleaved to produce CoQ₄, laying the groundwork for further development.

Keywords: antioxidant; bioenergetics; coenzyme Q10 (CoQ10); ferroptosis; membrane lipid; mitochondrial respiratory chain complex; mitochondrial therapeutics; pyrimidine biosynthesis; ubiquinone.

Figure 1

Short-chain CoQ analogs support OxPhos.A, percent live wild-type or COQ2^−/− HepG2 cells after 72 h of incubation in galactose media with 20 μM indicated CoQ additive. Cells were pretreated with CoQ additives for 48 h to ensure sufficient uptake. Live cells defined as not staining for AAD-7 or annexin-V. n = three independent technical replicates, and error bars indicate standard deviation. Significance calculated using a one-way ANOVA. B, survival of COQ2^−/− HepG2 cells in galactose as in (A) over a titration of CoQ₄ and CoQ₁₀ concentrations. n = two independent technical replicates, and error bars indicate standard deviation. C and D, oxygen consumption rates (OCR) indicating (C) basal respiration and (D) maximal respiration after 3 μM FCCP treatment in COQ2^−/− and wild-type HepG2 cells after 24 h incubation with CoQ additives. n = four independent technical replicates, and error bars indicate standard deviation. Significance calculated using a one-way ANOVA. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. For exact p values, see Table S1. CoQ, coenzyme Q; OxPhos, oxidative phosphorylation.

Figure 2

CoQ₄can participate in ferroptosis suppression and pyrimidine biosynthesis.A, cell death of wild-type and COQ2^−/− HepG2 cells after 24 h of treatment with GPX4-inhibitor RSL3. Cell death determined by high levels of Cytotox Red labeling. n = three independent technical replicates, and error bars indicate standard deviation. Significance calculated using a two-way ANOVA. B, cell death of COQ2^−/− HepG2 cells after 24 h of treatment with RSL3 following 24 h of preincubation with 20 μM CoQ₄ or CoQ₁₀. n = three independent technical replicates, error bars indicate standard deviation. Significance is calculated using a two-way ANOVA. C, cell death of COQ2^−/− HepG2 cells after 24 h of treatment with RSL3 or iFSP1 following 24 h of preincubation with indicated concentrations of CoQ₄ or CoQ₁₀. n = three independent technical replicates, and error bars indicate standard deviation. Significance calculated using a two-way ANOVA. D, total ion counts of dihydroorotate normalized to cell count in HAP1 wild-type and COQ2^−/− cells after 24 h of treatment with 20 μM CoQ₄ or CoQ₁₀. n = three independent technical replicates, and error bars indicate standard deviation. ns, not significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. For exact p values see Table S1. CoQ, coenzyme Q; DHO,dihydroorotate.

Figure 3

CoQ₄can ameliorate statin-induced loss of cell viability. Cell viability of C2C12 myocytes after 48 h of treatment with 50 μM simvastatin, 50 μM linolenic acid (C18:3), or a combination. Cells were pretreated with 20 μM CoQ₄ or CoQ₉ for 24 h prior to addition of statin and polyunsaturated fatty acids to ensure adequate uptake. Cell viability was assessed with crystal violet, with normalization to vehicle-treated cells with no CoQ pretreatment. n = nine independent technical replicates, error bars indicate standard deviation. Significance calculated using a two-way ANOVA. ns, not significant, ∗∗∗∗p < 0.0001. For exact p values, see Table S1. CoQ, coenzyme Q.

Figure 4

Schematic of CoQ₄-TPP hydrolysis and synthetic scheme.A, schematic of CoQ₄-TPP hydrolysis by mitochondrial esterases to produce CoQ₄ and a carboxy-TPP side product (TPP-COOH). B, chemical synthesis of CoQ₄-TPP. Reaction conditions: (1) NaBH₄ in methanol/isopropanol (2) 4-carboxybutyl triphenylphosphonium bromide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, 4-dimethylaminopyridine in dichloromethane. C, structures of CoQ₄-TPP compounds synthesized for this study. CoQ, coenzyme Q; TPP, triphenylphosphonium.

Figure 5

CoQ₄-TPP can be hydrolyzed to produce CoQ₄.A and B, CoQ production from CoQ-TPP species after 24 h of incubation with porcine liver esterase, normalized to equivalent CoQ₄ (A) or CoQ₁₀ (B) area indicating 100% turnover. CoQ levels measured by HPLC-ECD. n = three independent technical replicates, and error bars indicate standard deviation. C and D, levels of CoQ₄ after 24 h of incubation of wild-type HepG2 cells with unmodified CoQ₄ (C) or CoQ₄-TPP (D) measured by HPLC-ECD after cellular lipid extraction. n = three independent technical replicates, and error bars indicate standard deviation. E and F, CoQ₄ levels in different subcellular compartments after 3 h of incubation of HepG2 cells with 10 μM CoQ₄ (E) or CoQ₄-TPP4 (F). G, CoQ₄-TPP levels in different subcellular compartments after 3 h of incubation of HepG2 cells with 10 μM CoQ₄-TPP4. n = three independent technical replicates, and error bars indicate standard deviation. Significance calculated with a two-tailed, unpaired t test. ns, not significant, ∗p < 0.05. For exact p values see Table S1. CoQ, coenzyme Q; TPP, triphenylphosphonium.