{"doi":"10.1016/j.jbc.2023.102956","title":"Early-phase drug discovery of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5","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.","journal":"Journal of Biological Chemistry","year":2023,"id":363474,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9525,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":417546,"name":"Robyn T. Rebbeck","orcid":"0000-0003-3840-2286","position":1,"is_corresponding":false},{"id":431507,"name":"Sarah A. Denha","orcid":null,"position":2,"is_corresponding":false},{"id":1011231,"name":"Amanda R. Keller","orcid":null,"position":3,"is_corresponding":false},{"id":1011232,"name":"Anna L. Carter","orcid":null,"position":4,"is_corresponding":false},{"id":943990,"name":"Alexandra E. Atang","orcid":"0009-0008-9686-9742","position":5,"is_corresponding":false},{"id":430804,"name":"Bengt Svensson","orcid":"0000-0003-3932-2376","position":6,"is_corresponding":false},{"id":417547,"name":"David D. Thomas","orcid":"0000-0002-8822-2040","position":7,"is_corresponding":false},{"id":430805,"name":"Thomas S. Hays","orcid":"0000-0002-4911-6042","position":8,"is_corresponding":false},{"id":430806,"name":"Adam W. Avery","orcid":"0000-0001-8085-1822","position":9,"is_corresponding":false},{"id":431508,"name":"Piyali Guhathakurta","orcid":null,"position":0,"is_corresponding":true}],"reference_count":40,"raw_metadata":null,"created_at":"2026-07-19T01:14:28.054440Z","pmid":"36731793","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}