Eurofins Discovery has developed a panel of E3 ligase binding assays, known as E3scan™ to enable discovery of novel E3 ligands for the design of targeted protein degraders. E3 ligases are a large family of proteins that play key roles in protein degradation. Targeting E3 ligases with potent and selective small-molecule ligands can reprogram the E3 ligase substrate specificity to degrade a disease-causing protein target. Assays for commonly known E3 ligases like CRBN, VHL_Elongin B/C, xIAP as well as novel E3 ligases like ITCH, KEAP1 and KLHL2 are available.
There are major advantages in using the E3scan platform for Targeted Protein Degradation programs:
- Accurate, precise and reproducible data
- High sensitivity and broad dynamic range in KD measurements for competitor compounds
- Internal reference compounds that have been characterized for binding affinity
- All assays are run in parallel on a single platform, allowing for direct comparison of data between targets
- The largest assay panel available on a single technology platform for E3 ligases, all without having issues of compound interference in the assay readout
- Assays are performed in a high-throughput, 384-well format allowing for library profiling
- Custom assay development is offered on a case-by-case basis for the E3 ligase of interest
Overview and Assay Principle
This first-in-class E3 ligase ligand binding assay platform uses proprietary KINOMEscan™ technology. KINOMEscan technology utilizes DNA-tagged proteins and an ultra-sensitive qPCR readout to measure binding affinity (KD) between a protein and a compound.
Figure 1: E3scan ligand binding assay principle. Three main components to the assay: an E3 ligase tagged with DNA, a known E3 ligase ligand immobilized on a solid support, and a test compound or solvent control. The DNA tagged E3 ligase of interest is incubated with a known ligand immobilized to the solid support (Panel A). Compounds that bind the active site and directly (sterically) or indirectly (allosterically) prevent E3 ligase binding to the immobilized ligand, will reduce the amount of DNA tagged E3 ligase captured on the solid support (Panel B). Conversely, test molecules that do not bind have no effect on the amount of E3 ligase captured on the solid support (Panel C). Screening “hits” are identified by measuring the amount of E3 ligase captured in test versus control samples by using a quantitative, precise and ultra-sensitive qPCR method that detects the associated DNA label (Panel D). In a similar manner, dissociation constants (KD) for test compound E3 ligase interactions are calculated by measuring the amount of E3 ligase captured on the solid support as a function of the test compound concentration.
The E3scan platform is available in two screening formats, scanELECT and KdELECT. The scanELECT format enables test compounds to be screened at a single concentration and compound activity is reported as a percent of an internal control. The KdELECT format offers compound screening at 11 concentrations in duplicate and the data report includes dose response curves with KD binding affinities
