Small molecule therapeutics for Friedreich's ataxia

Scripps Research Institute, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease caused by deficiency of the nuclear-encoded mitochondrial protein frataxin. At present there is no effective cure or treatment for FRDA. The DNA abnormality found in 98 percent of FRDA patients is the unstable hyperexpansion of a GAA triplet repeat in the first intron of the frataxin gene, which adopts unusual DNA structures that interfere with gene transcription. We will explore whether synthetic DNA ligands, pyrrole-imidizole (Py-lm) polyamides, can be designed to bind GAA repeats and whether such molecules will relieve transcription inhibition by stabilizing canonical Watson-Crick B-type DNA structure in the frataxin gene. Py-lm polyamides bind predetermined DNA sequences with subnanomolar affinities, comparable to the binding affinities of natural transcriptional regulatory proteins; moreover, these molecules have been shown to access target sites in the nucleus of cultured cells. A series of Py-lm polyamides will be synthesized to target the GAA repeat sequence in the frataxin gene, and DNase I footprinting will be used to measure polyamide binding affinities and specificities for their target sequences. The effect of polyamides on GAA repeat DNA structure in plasmid DNAs will be determined. Polyamides that bind GAA repeat DNA with high affinity are expected to stabilize B-type DNA, and prevent transitions to triplex or other unusual DNA structures. The effects of polyamides on transcription of the frataxin gene will be monitored both in vitro and in cell culture experiments. Deconvolution microscopy will be used to monitor the subcellular localization and kinetics of uptake of fluorescent dye-polyamide conjugates in cultured cells. Real-time PCR will be used to determine the effects of polyamides on frataxin mRNA expression in appropriate human FRDA cell lines. The effects of polyamides on cellular frataxin protein will be determined by western blotting, and the genome-wide effects of polyamide treatment will be assessed by DNA microarray analysis.