title: Primary and secondary drug screening assays for Friedreich ataxia.
abstract: Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: M Grazia
authors.initials: MG
authors.lastname: Cotticelli
authors.forename: Lynn
authors.initials: L
authors.lastname: Rasmussen
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Kushner
authors.forename: Sara
authors.initials: S
authors.lastname: McKellip
authors.forename: Melinda Ingrum
authors.initials: MI
authors.lastname: Sosa
authors.forename: Anna
authors.initials: A
authors.lastname: Manouvakhova
authors.forename: Shuang
authors.initials: S
authors.lastname: Feng
authors.forename: E Lucile
authors.initials: EL
authors.lastname: White
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Maddry
authors.forename: Jill
authors.initials: J
authors.lastname: Heemskerk
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Oldt
authors.forename: Lea F
authors.initials: LF
authors.lastname: Surrey
authors.forename: Rachel
authors.initials: R
authors.lastname: Ochs
authors.forename: Robert B
authors.initials: RB
authors.lastname: Wilson
mesh_terms: D000818:Animals; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005621:Friedreich Ataxia / Q000188:drug therapy / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D033862:Iron-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013778:Tetrazolium Salts / Q000737:chemistry / Q000378:metabolism; D000096923:Frataxin
chemical_list: D033862:Iron-Binding Proteins; D007506:Iron-Sulfur Proteins; D013778:Tetrazolium Salts; D000096923:Frataxin; C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium