Title: Development of next-generation voltage-gated calcium channel inhibitors using engineered nanobodies
Description: High-voltage activated calcium channels underlie many critical functions in excitable cells and their dysfunction has been implicated in a myriad of cardiovascular and neurological diseases. These channels are multimeric protein complexes composed of α1, β, and α2δ subunits; currently, all calcium channel blockers target either the pore-forming α1 or extracellular-facing α2δ auxiliary subunit. These pharmacological agents have been invaluable in delineating the individual function of each subunit within excitable cells that express multiple calcium channels. Yet, no current tool allows similar pharmacological dissection of individual cytosolic β subunits, preventing our understanding of how distinct β subunits affect the function of calcium channel complexes. Further, small-molecule calcium channel blockers are highly-valued therapeutics for certain conditions, yet their propensity for off-target effects precludes their use in other diseases. In certain applications, genetically-encoded calcium channel blockers may enable channel inhibition with greater tissue-precision and versatility than is achievable with small molecules.

Previous work that found the family of RGK proteins powerfully inhibits high-voltage activated calcium channels in part via an association with the β subunit. However, the myriad functions of RGK proteins limit the utility of this approach. In this work, we circumvent this issue by isolating single-domain antibodies (nanobodies) that target the auxiliary CaVβ subunit. We then paired these nanobodies with the powerful enzymatic activity of the HECT domain E3 ubiquitin ligase Nedd4L, to selectively target the calcium channel for ubiquitination. We found this strategy effectively eliminated functional calcium channels from the surface of HEK293 cells, myocytes, and DRG neurons. This modular design permitted us to characterize a pan-β inhibitor (CaV-aβlator) in chapter 2 while refining the approach with a β1-selective channel inhibitor in chapter 3. In chapter 4 I demonstrate that it is possible to hijack the endogenous ubiquitin machinery of the cell by creating Divas: divalent nanobodies that are capable of recruiting endogenous Nedd4L to regulate the calcium channel.

Finally, we demonstrate the potential for these genetically-encoded calcium inhibitors to be employed as therapeutic agents by targeting CaV-aβlator to sensory neurons in order to reduce the onset of neuropathic pain. Altogether, this work lays the foundation for nanobody-based genetically-encoded calcium channel inhibitors that have the potential to achieve superior precision in regards to molecular and tissue specificity.