Development of a yeast-based screening assay for NARP-related diseases and identification of CH. (A) Serial dilutions of the WT strain, five NARP mutants, and the fmc1Δ strain were spotted onto glucose and glycerol plates. The plates were incubated at the indicated temperature for 3 d (glucose) or 7 d (glycerol). Rates of ATP synthesis relative to WT are indicated in the right-hand column. (B) Candidate screen. The fmc1Δ strain was spread onto rich glycerol plates. Small sterile filters were then placed on the agar surface and DHLA or OA (chemical structures depicted at Right) were added to the filters at the indicated quantities. The plates were then incubated at 35 °C for 7 d. (C) Screening assay carried out as described in B except that DMSO was added to the upper left filter (negative control, −) and DHLA to the bottom right filter (positive control, +). At the remaining positions, compounds from the chemical libraries were added, and plates were incubated for 7 d at 35 °C. (D) The improvement of fmc1Δ growth obtained with CH is shown, and its molecular structure is depicted. (E) The dose-dependent effect of CH on three yeast mutants is shown.

Transcription profiles of fmc1Δ, fmc1Δ + CH, and WT (FMC1⁺). (A) Scatterplot of log₂-fold changes in gene expression of CH-treated (y axis) and untreated (x axis) fmc1Δ yeast relative to WT in rich galactose medium at 35 °C. CH treatment induces a genome-wide shift toward WT expression levels of ∼1/3 as evidenced by the trendline y = 0.66x. (B) Expression of genes encoding respiratory chain complex subunits. Log₂-scale normalized expression values were centered for each gene by subtracting the mean expression level from all values per gene. Subunits show a general pattern of down-regulation in fmc1Δ yeast relative to WT and partial rescue by CH treatment. (C) Overexpression of the QCR9 gene in the fmc1Δ strain (bottom three rows, compared with the fmc1Δ strain in third row and WT strain in first two rows) results in partial recovery of growth on rich glycerol medium at a nonpermissive temperature.

CH suppresses multiple phenotypes of yeast models of ATP synthase disorders. (A) Serial dilutions of the WT strain, three NARP mutants, and the fmc1Δ strain were spotted onto glycerol plates supplemented with CH (2 μM final concentration) or the equivalent quantity of DMSO (− control). The plates were incubated at 35 °C for 7 d. (B) Growth curves of the fmc1Δ strain grown at 35 °C in liquid galactose medium supplemented with 1.25 μM CH (the optimal concentration) or DMSO. (C) Mean respiration rates of the WT and fmc1Δ strains treated in vivo with 1.25 μM CH or DMSO were determined using three different substrates as indicated. Error bars represent SD. * represents significant difference compared with untreated cells (P = 0.05, one-sided Wilcoxon rank test). (D) Energization of the mitochondrial membrane was determined by rhodamine 123 fluorescence quenching with intact mitochondria from the fmc1Δ strain treated in vivo with 1.25 μM CH (Right) or DMSO (Center), and the WT isogenic strain (FMC1⁺; Left). The contents were added as follows: mitochondrial proteins (Mito), ethanol (EtOH), ADP, potassium cyanide (KCN), DCCD, and CCCP. (E) SDS-PAGE and Western blot analysis of mitochondrial proteins from the mitochondria used in D. Porin was used as a loading control. (F) BN-PAGE, ATPase activity, and Western blot analysis of extracts from isolated mitochondria. V₂ and V₁ indicate the dimeric and monomeric forms of the F₁F₀ ATP synthase complex, respectively; F₁ indicates the free F₁ particles. (G) Ultrastructure electron micrographs of fmc1Δ cells grown at 35 °C in presence of 1 μM CH (Lower) or DMSO as control (Upper). Arrowheads indicate mitochondrial cristae.

DHLA, CH, and OA are active in a human cybrid-based model of NARP syndrome. NARP and control cybrids were grown in glucose-deprived medium with DHLA, CH, and OA at the indicated final concentrations or with the equivalent quantity of DMSO as a control (0). (A) Percentage of growth after 3 d in glucose-deprived medium containing OA or CH at the indicated concentrations, compared with treatment with DHLA at 200 μM final concentration (+), a compound being tested in clinical trials for the treatment of mitochondrial encephalopathies. * represents conditions with growth significantly different from in DMSO (P < 0.05; Methods). (B) Mean percentage of external pyruvate in NARP cybrids treated with DHLA, CH, OA (left-hand side of graph) compared with control cybrids (right-hand side of graph). Error bars represent SD. * represents significant difference compared with the DMSO condition (P = 0.01). (C) Mean percentage of external lactate as in B.