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 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 Lessene, John Silke and Rebecca Feltham ()
Additional contact information
Christoph Grohmann: The Walter and Eliza Hall Institute for Medical Research
Charlene M. Magtoto: The Walter and Eliza Hall Institute for Medical Research
Joel R. Walker: Promega Biosciences LLC
Ngee Kiat Chua: The Walter and Eliza Hall Institute for Medical Research
Anna Gabrielyan: The Walter and Eliza Hall Institute for Medical Research
Mary Hall: Promega Biosciences LLC
Simon A. Cobbold: The Walter and Eliza Hall Institute for Medical Research
Stephen Mieruszynski: The Walter and Eliza Hall Institute for Medical Research
Martin Brzozowski: The Walter and Eliza Hall Institute for Medical Research
Daniel S. Simpson: The Walter and Eliza Hall Institute for Medical Research
Hao Dong: The Walter and Eliza Hall Institute for Medical Research
Bridget Dorizzi: The Walter and Eliza Hall Institute for Medical Research
Annette V. Jacobsen: The Walter and Eliza Hall Institute for Medical Research
Emma Morrish: The Walter and Eliza Hall Institute for Medical Research
Natasha Silke: The Walter and Eliza Hall Institute for Medical Research
James M. Murphy: The Walter and Eliza Hall Institute for Medical Research
Joan K. Heath: The Walter and Eliza Hall Institute for Medical Research
Andrea Testa: Amphista Therapeutics Ltd
Chiara Maniaci: Chemistry School of Natural and Environmental Sciences, Bedson Building, Newcastle University Edwards Walk
Alessio Ciulli: School of Life Sciences, University of Dundee
Guillaume Lessene: The Walter and Eliza Hall Institute for Medical Research
John Silke: The Walter and Eliza Hall Institute for Medical Research
Rebecca Feltham: The Walter and Eliza Hall Institute for Medical Research

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: Abstract Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12F36V (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 NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4CRBN 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.

Date: 2022
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DOI: 10.1038/s41467-022-29670-1

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