Identification of -III-spectrin actin binding modulators for treatment of spinocerebellar ataxia

β-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells, and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the cytoskeletal protein β-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small molecule actin-binding modulators of the L253P mutant β-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P β-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay, we screened a 2,684-compound library of FDA-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous co-sedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a SCA5-specific therapeutic. Furthermore, our validated FRET-based in vitro HTS platform is poised for screening large compound libraries for β-III-spectrin ABD modulators. ### Competing Interest Statement DDT holds equity in, and serves as an executive officer for Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study, except to provide some instrumentation, as stated in Experimental Procedures. RTR, PG, ALC, SAD, ARK, AEA, BS, TSH and AWA have no conflict of interest to disclose.