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bioRxiv · 10.1101/2024.05.28.596281

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

Morgenstern, T. J.·Darko-Boateng, A.·Afriyie, E.·Shanmugam, S. K.·Zou, X.·Choudhury, P.·Desai, M.·Kass, R. S.·Clarke, O. B.·Colecraft, H. M.
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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 [A] cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) 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.

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Morgenstern, T. J., Darko-Boateng, A., Afriyie, E., Shanmugam, S. K., Zou, X., Choudhury, P., Desai, M., Kass, R. S., Clarke, O. B., Colecraft, H. M.. 2024-05-31. Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies. https://doi.org/10.1101/2024.05.28.596281

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