Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools Monitoring Targeted Protein Degradation

Target protein degradation (TPD) has emerged as a revolutionary approach in drug discovery, leveraging the cell's intrinsic machinery to selectively degrade disease-associated proteins. Proteolysis-Targeting Chimeras (PROTACs) exemplify this strategy, exploiting heterobifunctional molecules to induce ubiquitination and subsequent degradation of target proteins. The clinical advancement of PROTACs underscores their potential in therapeutic intervention, with numerous projects progressing through clinical stages. However, monitoring subtle changes in protein abundance induced by TPD molecules demands highly sensitive assays. Nano-luciferase (nLuc) fusion proteins, or the NanoBiT technology derived from it, offer a robust screening platform due to their high sensitivity and stability. Despite these advantages, concerns have arisen regarding potential degradation artifacts introduced by tagging systems due to the presence of lysine residues on them, prompting the development of alternative tools. In this study, we introduce HiBiT-RR and nLuc