Identifying novel therapeutics for early onset dystonia

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Early onset torsion dystonia (DYT1) is a dominantly inherited disorder causing severe motor impairment thought to be due, at least in part, to abnormal dopaminergic neurotransmission in the basal ganglia without neurodegeneration. Most cases are caused by a specific mutation in the gene encoding torsinA, an AAA+ protein residing in the endoplasmic reticulum (ER) and nuclear envelope. Recent studies support a role for torsinA as an ER chaperone protein involved in processing proteins through the secretory pathway with mutant torsinA decreasing secretory capacity in cultured cells. The goal of our research is to identify compounds which may attenuate the functional effects of mutant torsinA expression, thereby serving as potential therapeutic strategies for DYT1 dystonia. Towards this end, we have developed a novel screening method to identify small molecules which bind directly to wild-type torsinA and/or the dystonia- related mutant form. Using this approach, we recently identified a set of candidate molecules, one of which appears to significantly normalize secretion of a reporter protein, Gaussia luciferase (Gluc), from cultured fibroblasts obtained from DYT1 patients. The proposed studies are designed to further characterize new drug candidates for dystonia in three stages. In stage one, we will use the same screening method to survey microarrays consisting of 20,000 additional compounds from diverse classes to identify new molecules which bind wild-type and/or mutant torsinA. Screening positives will then be tested for their ability to improve secretion of the Gluc reporter in primary fibroblasts from DYT1 patients and control subjects. In stage two, we will define structure-activity relationships (SAR) for our top candidates by synthesizing new structural analogs which will be tested in the Gluc secretion assay. In stage three, we will examine which proteins, in addition to torsinA, are cellular targets of our top compounds. Biotinylated analogs of our top molecules will be synthesized and used in pull-down assays to precipitate target proteins from cultured cells, followed by mass spectrometry for protein identification. By funneling compounds through the proposed screening pipeline, we hope to establish a set of well characterized drug candidates which may be suitable for future evaluation in DYT1 animal models.