Ternary Complex Formation
Formation of the E3 ternary complex, which contains the target, degradation compound and E3 ligase, is a crucial step in targeted protein degradation. This step represents a critical parameter in the development and optimization of effective degrader compounds. Learn about cell-based NanoBRET® Assays and biochemical Lumit™ Assays for measuring ternary complex formation.
View this poster about antibody-based approaches to monitor ternary complex formation with Lumit® technology.
Does My PROTAC Form a Ternary Complex?
Cell-Based Approaches
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.
Multiplexing PROTAC-induced BRD4 VHL/CRBN ternary complex formation and BRD4 protein levels using live-cell endpoint detection. Cells (BRD4/VHL assay) transfected with a 1:100 ratio of NanoLuc®-BRD4 donor plasmid to HaloTag®-VHL or HaloTag®-CRBN acceptor plasmid, pretreated with MG132 (or DMSO control), subsequently treated with 1µM MZ1 (VHL-based PROTAC) or DMSO. The NanoBRET® ratio indicates ternary complex formation (Panel A) and NanoLuc® luminescence indicates target protein levels (Panel B) caused by PROTAC treatment.
Kinetically monitoring BRD4/VHL ternary complex formation following PROTAC treatment. HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line using CRISPR/Cas9 gene editing. A stable clone was transfected with HaloTag®-VHL acceptor plasmid. Cells were pretreated with MG132 prior to PROTAC treatment. Signal was measured using the NanoBRET® Nano-Glo® Kinetic Detection System to monitor ternary complex formation in real time.
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Biochemical Approaches
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.
To learn more about antibody-based approaches to monitoring ternary complex formation with Lumit™ technology, download our poster or contact us.
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