{"doi":"10.1101/2022.06.27.497760","title":"Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput","abstract":"ABSTRACT The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor’s enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated. Abstract Figure","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":309524,"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":0.9513,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":409319,"name":"Patricia Dranchak","orcid":"0000-0002-1349-4885","position":1,"is_corresponding":false},{"id":441070,"name":"John L. Pezzullo","orcid":null,"position":2,"is_corresponding":false},{"id":805366,"name":"Rebecca Mancusi","orcid":"0000-0002-4809-5569","position":3,"is_corresponding":false},{"id":859066,"name":"Alexandra Maria Psaras","orcid":"0000-0002-4978-6003","position":4,"is_corresponding":false},{"id":268294,"name":"Ganesha Rai","orcid":"0000-0001-9763-9641","position":5,"is_corresponding":false},{"id":440306,"name":"José‐Luis Giner","orcid":"0000-0002-8092-5139","position":6,"is_corresponding":false},{"id":409320,"name":"James Inglese","orcid":"0000-0002-7332-5717","position":7,"is_corresponding":false},{"id":440307,"name":"Brian P. Callahan","orcid":"0000-0003-4826-0162","position":8,"is_corresponding":false},{"id":968279,"name":"Daniel A. Ciulla","orcid":"0000-0002-0365-2667","position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":null,"created_at":"2026-07-19T00:33:11.579763Z","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":[]}