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 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 for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z′ value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration–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 cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5–specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for β-III-spectrin ABD modulators.
|Original language||English (US)|
|Journal||Journal of Biological Chemistry|
|State||Published - Mar 2023|
Bibliographical noteFunding Information:
This work was supported by NIH grants R61NS111075, R33NS111075, and R01GM044757 to T. S. H.; R01HL139065 (formerly GM027906) and R37AG026160 to D. D. T.; and R15NS116511 to A. W. A. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2023 The Authors
- actin binding
- drug screening
- fluorescence lifetime
- spinocerebellar ataxia
- swinholide A
- time-resolved FRET
PubMed: MeSH publication types
- Journal Article