Early-phase drug discovery of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5

J Biol Chem. 2023 Mar;299(3):102956. doi: 10.1016/j.jbc.2023.102956. Epub 2023 Jan 31.


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.

Keywords: SCA5; actin binding; drug screening; fluorescence lifetime; spinocerebellar ataxia; swinholide A; time-resolved FRET; β-III-spectrin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actins* / genetics
  • Actins* / metabolism
  • Drug Discovery
  • Humans
  • Neurons / metabolism
  • Spectrin* / metabolism
  • Spinocerebellar Ataxias* / drug therapy
  • Spinocerebellar Ataxias* / genetics
  • Spinocerebellar Ataxias* / metabolism


  • Actins
  • Spectrin
  • SPTBN2 protein, human