Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.
J Biol Chem
; 299(3): 102956, 2023 03.
Article
en En
| MEDLINE
| ID: mdl-36731793
ABSTRACT
ß-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 ß-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 ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-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 ß-III-spectrin ABD modulators.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Actinas
/
Espectrina
/
Ataxias Espinocerebelosas
Límite:
Humans
Idioma:
En
Revista:
J Biol Chem
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos