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

Piyali Guhathakurta, Robyn T. Rebbeck, Sarah A. Denha, Amanda R. Keller, Anna L. Carter, Alexandra E. Atang, Bengt Svensson, David D. Thomas, Thomas S. Hays, Adam W. Avery

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

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 languageEnglish (US)
Article number102956
JournalJournal of Biological Chemistry
Volume299
Issue number3
DOIs
StatePublished - Mar 2023

Bibliographical note

Funding 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.

Publisher Copyright:
© 2023 The Authors

Keywords

  • actin binding
  • drug screening
  • fluorescence lifetime
  • SCA5
  • spinocerebellar ataxia
  • swinholide A
  • time-resolved FRET
  • β-III-spectrin

PubMed: MeSH publication types

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

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