Does My PROTAC Form a Ternary Complex?
NanoBRET™ technology is ideally suited to study ternary complex formation in a live-cell format, allowing for both endpoint or kinetic analysis.
In this assay, target protein serves as the energy donor (bioluminescence), expressed in the cell as an exogenous transient NanoLuc® fusion or an endogenously tagged HiBiT fusion in a LgBiT-expressing cell. HaloTag® fusions with von Hippel-Lindau (VHL) or cereblon (CRBN) E3 ligase components are expressed exogenously and labeled with fluorescent ligand to serve as the energy acceptor.
A schematic depiction of NanoBRET™ ternary complex formation.
We also offer biochemical methods to monitor ternary complex formation across different E3 ligases, target proteins and PROTACs. The example shown here detects PROTAC-mediated ternary complex formation with cereblon and VHL complexes.
Schematic overview of a biochemical approach to monitor ternary complex formation. With BRD3(BD2) as the target protein, PROTAC-mediated ternary complex formation can be monitored using anti-FLAG-LgBiT and anti-GST-SmBiT Lumit™ reagents.
Lumit™ PROTAC assays can determine relative potencies of different PROTACs. Panel A. Ternary complex formation with cereblon complex + BRD3(BD2) and dBET1 or dBET6. Panel B. Ternary complex formation with VHL complex + BRD3(BD2) and ARV-771 or MZ1. These assays demonstrate dBET6 is more potent than dBET1 and show similar potencies between ARV-771 and MZ1. In both systems, the characteristic hook effect is observed at high PROTAC concentrations.