{"doi":"10.1101/2024.05.28.596281","title":"Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies","abstract":"Abstract Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins— ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis— the E2 binding site, the catalytic cysteine, and the ubiquitin exosite— as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and Ca V 2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":491052,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9539,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1336441,"name":"Arden Darko-Boateng","orcid":"0000-0002-5298-0755","position":1,"is_corresponding":false},{"id":1336442,"name":"Emmanuel Afriyie","orcid":"0009-0007-1073-2765","position":2,"is_corresponding":false},{"id":1007548,"name":"Sri Karthika Shanmugam","orcid":"0000-0002-1467-401X","position":3,"is_corresponding":false},{"id":1336776,"name":"Xinle Zhou","orcid":null,"position":4,"is_corresponding":false},{"id":899026,"name":"Papiya Choudhury","orcid":null,"position":5,"is_corresponding":false},{"id":1336777,"name":"Meera J. Desai","orcid":null,"position":6,"is_corresponding":false},{"id":352598,"name":"Robert S. Kass","orcid":"0000-0003-4377-3874","position":7,"is_corresponding":false},{"id":290817,"name":"Oliver B. Clarke","orcid":"0000-0003-1876-196X","position":8,"is_corresponding":false},{"id":395048,"name":"Henry M. Colecraft","orcid":"0000-0002-2340-8899","position":9,"is_corresponding":false},{"id":307525,"name":"Travis J. Morgenstern","orcid":"0000-0003-2634-8470","position":0,"is_corresponding":true}],"reference_count":93,"raw_metadata":null,"created_at":"2026-07-19T02:08:41.211111Z","pmid":"38854018","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":[]}