Limitations in a frataxin knockdown cell model for Friedreich ataxia in a high-throughput drug screen.
BACKGROUND
Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.


METHODS
Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.


RESULTS
The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.


CONCLUSIONS
Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.
Calmels
Nadège
N
IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), CNRS/INSERM/Université Louis Pasteur, Illkirch cedex, France. nadege.calmels@chru-strasbourg.fr
Seznec
Hervé
H
Villa
Pascal
P
Reutenauer
Laurence
L
Hibert
Marcel
M
Haiech
Jacques
J
Rustin
Pierre
P
Koenig
Michel
M
Puccio
Hélène
H
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line / Q000166:cytology; D049109:Cell Proliferation; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D005621:Friedreich Ataxia / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D033862:Iron-Binding Proteins / Q000235:genetics*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000096923:Frataxin
D033862:Iron-Binding Proteins; D007506:Iron-Sulfur Proteins; D024101:Mitochondrial Proteins; D000096923:Frataxin
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