Our long-term goal is to develop drugs that target the aberrant interaction of ß-III-spectrin with actin, for treatment of the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). This condition is marked by cerebellar hypoplasia, the loss of Purkinje cells in the cerebellar cortex, and loss of Purkinje cell dendritic branching. Recently, we resolved the structural mechanism for a SCA5 mutation (L253P), located in the actin-binding domain (ABD) of ß-III-spectrin, which contributes to pathology via dramatically enhanced actin-binding affinity. Our unpublished results suggest that other SCA5 mutations outside of the ABD also cause increased actin-binding affinity, potentially through an allosteric effect on the ABD. In the R61 phase, we will refine and validate for high-throughput screening (HTS), a cell-based assay to identify small molecules that disrupt the binding of the mutant L253P ß-III-spectrin ABD to actin. In the R33 phase, we will use this assay to perform HTS, followed by in vitro binding assays to further characterize the specificity of hits. Using fluorescent fusion proteins and fluorescence resonance energy transfer (FRET), we have performed initial development of an assay that senses the binding of the L253P mutant ß-III-spectrin ABD to actin filaments in cells. Novel fluorescence lifetime (FLT) technology enables the translation of this FRET tool into an ultrasensitive HTS assay to monitor the interaction of ß-III-spectrin with actin. Successful completion of this IGNITE project is assured by four critical assets: (1) initial development of a FRET biosensor and detection system that senses binding of mutant ß-III-spectrin to actin in cells, (2) demonstrated application of this FRET system to HTS, (3) a unique high-precision FLT plate reader (FLT-PR) exclusively available at the University of Minnesota, and (4) the established record of our collaborators in previous applications of this technology for drug discovery on other targets. In a pilot screen of a small collection of known bioactive compounds, we have shown that ß-III-spectrin-targeted HTS is feasible. During the R61 phase of this project, we will refine and validate a HTS-compatible FRET assay that reports on the binding of the L253P ß-III-spectrin ABD to actin in living cells. This involves FRET assay optimization to enhance specificity, robustness and sensitivity (Aim 1), which will be closely followed by screens to identify assay positive controls (Aim 2) and screening of a small compound library (1k compounds) to gauge assay metrics for HTS compatibility (Aim3). During the R33 phase, we will use this FRET assay to perform a 50K-compound screen followed by dose-response analysis to identify FRET hits (Aim1), followed by secondary in vitro assays to validate hits and characterize their specificities and biophysical mechanisms (Aim2), and medicinal chemistry to analyze and refine the hits (Aim 3). Following completion of this project, including the achievement of well-defined milestones, and HTS, we will have identified several lead compounds that will further be developed, under BPN or private sector funding, for preclinical testing.
