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<p>Targeted protein degradation/downregulation (TPD/TPDR) is a disruptive paradigm for developing therapeutics. &lt;2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit ion channels has not been demonstrated. NEDD4-2 E3 ligase regulates myriad ion channels, but its utility for TPD/TPDR is uncertain due to complex regulatory mechanisms. Here, we identify a nanobody that binds NEDD4-2 HECT domain without disrupting catalysis sites as revealed by cryo-electron microscopy and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels (Ca<sub>V</sub>2.2; KCNQ1; and epithelial Na<sup>+</sup> channel, ENaC, with a Liddle syndrome mutation) using divalent nanobodies (DiVas) strongly suppresses their surface density and function. Global proteomics indicates DiVa recruitment of endogenous NEDD4-2 to KCNQ1-YFP yields dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of NEDD4-2 recruitment for TPD/TPDR, validate ion channels as susceptible to this modality, and introduce a general method to generate ion channel inhibitors.</p>

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Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月6日

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  • Arden Darko-Boateng,
  • Emmanuel Afriyie,
  • Travis J. Morgenstern,
  • Sri Karthika Shanmugam,
  • Xinle Zou,
  • Yianni D. Laloudakis,
  • Papiya Choudhury,
  • Meera Desai,
  • Robert S. Kass,
  • Francesca Vallese,
  • Oliver B. Clarke,
  • Henry M. Colecraft

摘要

Targeted protein degradation/downregulation (TPD/TPDR) is a disruptive paradigm for developing therapeutics. <2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit ion channels has not been demonstrated. NEDD4-2 E3 ligase regulates myriad ion channels, but its utility for TPD/TPDR is uncertain due to complex regulatory mechanisms. Here, we identify a nanobody that binds NEDD4-2 HECT domain without disrupting catalysis sites as revealed by cryo-electron microscopy and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels (CaV2.2; KCNQ1; and epithelial Na+ channel, ENaC, with a Liddle syndrome mutation) using divalent nanobodies (DiVas) strongly suppresses their surface density and function. Global proteomics indicates DiVa recruitment of endogenous NEDD4-2 to KCNQ1-YFP yields dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of NEDD4-2 recruitment for TPD/TPDR, validate ion channels as susceptible to this modality, and introduce a general method to generate ion channel inhibitors.