Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms
Christoph Grohmann, Charlene M. Magtoto, Joel R. Walker, Ngee Kiat Chua, Anna Gabrielyan, Mary P. Hall, Simon A. Cobbold, Stephen Mieruszynski, Martin Brzozowski, Daniel S. Simpson, Hao Dong, Bridget Dorizzi, Annette V. Jacobsen, Emma Morrish, Natasha Silke, James M. Murphy, Joan K. Heath, Andrea Testa, Chiara Maniaci, Alessio Ciulli, Guillaume Lessène, John Silke, Rebecca Feltham
Abstract
Abstract Modulation of protein abundance using t ag- T argeted P rotein D egrader (tTPD) systems targeting FKBP12 F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic Nano Luc-targeting PRO TACs (NanoTACs) to hijack the CRL4 CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.