Elesclomol restores mitochondrial function in genetic models of copper deficiency

Abstract

Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

Keywords: copper; cytochrome c oxidase; elesclomol; mitochondria.

Fig. 1.

ES supplementation rescues CcO assembly defects by restoring mitochondrial copper levels of coa6Δ cells. (A) Serially diluted WT and coa6Δ cells were seeded on the indicated plates and incubated at 30 °C and 37 °C for 2 d (YPD) or 4 d (YPGE) before imaging. (B–F) WT, coa6Δ and coa6Δ cells supplemented with 20 nM ES were cultured in YP galactose medium until early stationary growth phase followed by (B) oxygen consumption rate (OCR) measurement, (C) BN-PAGE/Western analysis of mitochondrial respiratory chain supercomplexes containing Complex IV (CIV; also called CcO), (D) quantification of supercomplexes, (E) in-gel activity staining for Complex IV, and (F) quantification of CcO activity, (G) mitochondrial copper levels, and (H) total cellular copper content. Error bars represent mean ± SD (n = 3, two-tailed unpaired Student’s t test, *P < 0.05 and **P < 0.005). Data shown in A, C, and E are representative of at least three independent experiments.

Fig. 2.

ES can rescue the respiratory growth deficiency of yeast mutants of copper metabolism. (A) List of yeast genes and their human orthologs implicated in cellular and mitochondrial copper metabolism and the clinical phenotypes associated with mutations in the human or murine genes. (B) Serially diluted WT cells and the indicated mutants were spotted on YPD, YPGE, and YPGE supplemented with 10 nM of ES. The plates were incubated at 37 °C and allowed to grow for 2 d (YPD) or 2 d and 4 d (YPGE) before imaging.

Fig. 3.

ES supplementation rescues the steady-state levels of copper-containing subunits of CcO in mammalian cell lines with genetic defects in copper metabolism. (A) Immunoblot analysis of CTR1 in Ctr1^+/+ and Ctr1^−/− H9c2 cells. The arrowheads labeled “g” and “t” indicate the full-length glycosylated and truncated forms of CTR1, respectively. (B) Total copper levels measured by ICP-MS in H9c2 cells. Data are presented as mean ± SD (n = 4, two-tailed unpaired Student’s t test, **P < 0.001). (C) Immunoblot analysis of CCS, COX4, and GAPDH protein levels in Ctr1^+/+ and Ctr1^−/− H9c2 cells. GAPDH serves as a loading control. (D) The Ctr1^+/+ and Ctr1^−/− H9c2 rat cardiomyocytes and (E) MEFs were cultured for 3 d with the indicated doses of ES followed by Western analysis of COX1 protein levels. ATP5A is used as loading control. (F) Control (MCH46) and SCO2 patient cell lines were cultured for 3 or 6 d in the presence of the indicated concentrations of ES or a copper–histidinate complex (Cu-His) in DMEM with 10% FBS. The cellular COX2 levels were detected by SDS/PAGE/Western blot analysis. β-Actin (ACTB) was used as a loading control.

Fig. 4.

ES supplementation rescues pigmentation and the Cox1 deficiency of ctr1-KO zebrafish. (A–C) Clutches of embryos from a WT zebrafish and from a cross of a pair of ctr1^+/− heterozygous zebrafish were imaged at 48 hpf following treatment with and without 10 nM ES. Arrows in B indicate homozygous ctr1^−/− embryos with a pigmentation defect. (D) Mitochondria were isolated from 10-dpf WT zebrafish and homozygous ctr1^−/− zebrafish treated with and without 10 nM ES. Mitochondrial samples were subjected to SDS/PAGE and immunoblotted using the Complex IV-specific antibody anti-Cox1. The mitochondrial protein Atp5a was used as a loading control.