Elesclomol restores mitochondrial function in genetic models of copper deficiency

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 115 (2018): 8161-8166, doi:10.1073/pnas.1806296115.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.This work was supported by National Institutes of Health Awards R01GM111672 (to V.M.G.), R01 DK110195 (to B.-E.K.), and DK 44464 (to J.D.G.); Welch Foundation Grant A-1810 (to V.M.G.); and Canadian Institutes of Health Research Operating Grant MOP 133562 (to S.C.L.)

Maximal interferon induction by influenza lacking NS1 is infrequent owing to requirements for replication and export.

Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent the triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection

Summary of single-cell datasets used in this study.

(A) Diagram of the NS segment in the (+) orientation showing NS1 and NEP open-reading frames. Stop codons were introduced at the indicated site to generate NS1stop. Full genome sequence in S1 File. (B) Summary of datasets presented in this paper, either generated for the express purpose of this effort, or reanalysis of a prior study from Kelly et al. [26] Analysis pipeline schematicized in S2 Fig. Genome sequence to align Kelly et al. data in S2 File (C) Fraction of cells annotated as positive for either type I or type III interferon expression were further assessed for expression of type III or type I interferon expression. Each individual single-cell experiment was analyzed seperately, labeled by cell type or donor. Type I and type III expression were associated at levels unlikely to be driven by chance (Fisher’s exact test, p values shown) across all experiments. (D) Numbers of cells meeting indicated thresholds in our NS1stop single-cell RNAseq data. Analysis used to set thresholds shown in S3, S5 and S7 Figs. Wild-type infections, for comparison, shown in S4, S6 and S8 Figs, respectively.</p

Leptomycin inhibits interferon induction by a complemented, stimulatory deletion even in the presence of NS1.

Reporter cells expressing PB1 to permit replication were infected with PB1177:385 virus at an MOI of 2. At 3 hpi, cells were treated with 10 nM LMB. This virus was previously shown to be highly stimulatory. [10, 29] RNA was harvested at 8 hpi, and indicated transcripts analyzed by qPCR against the housekeeping control, L32. Asterisks indicate significant difference, two-tailed t test, p (TIF)</p

Curated deletion junctions and number of supporting reads from single-cell sequencing for NS1_R38A A/Hamburg/4/2009 donor 2405.

Curated deletion junctions and number of supporting reads from single-cell sequencing for NS1R38A A/Hamburg/4/2009 donor 2405.</p

The impact of deletions on interferon in an NS1_stop infection is dose-dependent.

(A) Normalized genome copies to infectivity ratio in indicated populations. Genome copies were measured by a qPCR targeting the PA segment in a region retained in deletions. Infectivity was measured by tissue-culture infectious dose 50 (TCID50). The ratio of these values was set to 1 (0 on a logarithmic scale) for the average measurement across our wild-type low-defective populations, and then all values were calculated relative to that reference. High defective burden populations confirmed to have a greater genome copy to infectivity ratio. Individual points indicate technical triplicates, adjacent sets under the same heading represent biological duplicates. (B) Percentage of total cells expressing interferon when IFNL1-reporter cells are infected with the indicated populations at the indicated genome-corrected MOI, as measured by flow cytometry at 13hpi. MOI was calculated on low-defective populations, then an identical number of genome copies were added as measured in A. Asterisks denote significant differences between high- and low-defective populations, two-tailed t test with Benjamini-Hochberg multiple testing correction at an FDR of 0.05, n = 4, two replicates of each of the biological replicate populations shown in A. Detection threshold, as defined by the reporter response in uninfected cells, denoted by the dotted line. Full flow data shown in S12 Fig. (C) Mean fluorescence intensity of M2 staining in the indicated populations from B. Asterisks denote significant differences between high- and low-defective populations, two-tailed t test with Benjamini-Hochberg multiple testing correction at an FDR of 0.05, n = 4. Full distributions shown in S13 Fig. (D) Interferon positivity in MOI 5 infections from B, divided into bins of M2 expression. Asterisks indicate significant differences between high- and low-defective populations within a viral variant. Two-tailed t test with Benjamini-Hochberg multiple testing correction at an FDR of 0.05, n = 4. Statistics only calculated, and points displayed for M2 bins with greater than 100 events.</p

Full flow data for Fig 4.

Data showing full flow data and measurements of IFNL1 reporter, and M2 staining. Cells infected the indicated, particle-corrected MOI, and measurements made 13h post-infection. Individual replicates shown. Interferon-positive events colored in orange. Data subsetted to 5000 events to show equivalent numbers between conditions. (TIF)</p

Leptomycin inhibits interferon induction by A/California/4/2009 NS1_stop in undifferentiated NHBE cells.

NHBE cells were infected with NS1stop virus at an MOI of 2. At 3 hpi, cells were treated with 10 nM LMB. RNA was harvested at 13.5 hpi, and indicated transcripts analyzed by qPCR against the housekeeping control, L32. Asterisks indicate significant difference, two-tailed t test, p (TIF)</p

Curated deletion junctions and number of supporting reads from single-cell sequencing for wild-type A/Hamburg/4/2009 donor 2405.

Curated deletion junctions and number of supporting reads from single-cell sequencing for wild-type A/Hamburg/4/2009 donor 2405.</p