Developing PROTACs (Proteolysis-Targeting Chimeras) is a complex process due to the numerous factors that influence their efficiency, including linker optimization, which affects ternary complex formation, physical properties, and metabolic liabilities. Traditional methods of synthesizing and testing PROTAC iterations are time-consuming, but plate-based chemistry allows the rapid creation of multiple versions of PROTACs with minimal purification, directly used in cell assays for degradation data—a method called Direct-to-Biology (D2B).
While HiBiT is the gold standard for PROTAC screening due to the natural expression of the protein of interest (POI), this study demonstrates that similar results can be achieved using transiently expressed NanoLuc® tagged POIs. The team used a 96-well plate of crude PROTAC mixtures for initial spot test screening, followed by dilutions into a 384-well assay plate at two concentrations to minimize false negatives caused by the hook effect. Cycloheximide was added in the NanoLuc® system to inhibit protein synthesis, which is unnecessary for HiBiT.
Charnwood Discovery’s Direct-to-Biology platform accelerates the optimization of PROTACs by quickly screening partial molecules combined with novel warheads, driving rapid hit identification and lead compound selection. The data showed strong correlation between BRD4 degradation using NanoLuc® and HiBiT systems, supporting the use of both approaches for effective screening.
