{"doi":"10.17615/g5ha-5183","title":"A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction","abstract":"The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high-throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z′-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z′-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.","journal":"UNC Libraries","year":2020,"id":138241,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9601,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":437994,"name":"David P. Siderovski","orcid":"0000-0002-0688-8210","position":1,"is_corresponding":false},{"id":228423,"name":"Anton Simeonov","orcid":"0000-0002-4523-9977","position":2,"is_corresponding":false},{"id":262007,"name":"Ajit Jadhav","orcid":"0000-0001-7955-1451","position":3,"is_corresponding":false},{"id":599694,"name":"Mark Hughes","orcid":"0000-0003-3278-130X","position":4,"is_corresponding":false},{"id":105527,"name":"Adam J. Kimple","orcid":"0000-0003-1670-8401","position":5,"is_corresponding":false},{"id":225306,"name":"Christopher P. Austin","orcid":"0000-0001-7770-0708","position":6,"is_corresponding":false},{"id":409320,"name":"James Inglese","orcid":"0000-0002-7332-5717","position":7,"is_corresponding":false},{"id":599695,"name":"Gordon C. Ibeanu","orcid":"0000-0002-7673-2529","position":8,"is_corresponding":false},{"id":226892,"name":"Francis S. Willard","orcid":"0000-0002-4260-2451","position":9,"is_corresponding":false},{"id":593126,"name":"Robin E. Muller","orcid":null,"position":10,"is_corresponding":false},{"id":233839,"name":"Adam Yasgar","orcid":"0000-0001-7350-1402","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:17:02.849477Z","pmid":null,"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":[]}