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E3 Ligase Screening and Profiling Services

With 10 years of experience with E3 ubiquitin ligases, you can be confident in our capable hands.  We have the tools and proteins to establish the relevant E2 for your E3, and have expertise in choosing substrates for in vitro ubiquitylation assays.  Using this knowledge, we can establish customized assays to screen for inhibitors or activators of E3 ligases.  Similarly, we can determine the selectivity of your small molecule inhibitors or activators against our catalog of E3s.

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E2 Identification

LifeSensors has developed quantitative plate-based assays to determine the most optimal E2 for your E3.  The E2 can determine the ubiquitin chain-linkage type produced, and the ability of your E3 to ubiquitylate substrates.  Thus, E2 selection is closely intertwined with the physiological consequences of E3 ligase activity.

Ub Chain Identification

Different types of ubiquitination linkage patterns can have different functional consequences for the substrate. Knowing the type of chain and branching can give you insights into the signaling mechanism and role of your E3 or substrate of interest. We use a variety of technologies for this work, including but not limited to the following:

  • Mutant Ub (K6, K11, K27, K29, K33, K48, K63, and Met1) gel/WB
  • Ubiquitin linkage K-GG LC-MS/MS

E3 Screening and Compound Profiling

Targeting E3 ligases can be difficult because of the many components in the ubiquitin cascade, but LifeSensors can help you identify potent and selective compounds. Using our in-house library or your own library, we can identify and develop inhibitors and activators of your E3 of interest. In fact, with our E3 TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay you can simultaneously screen multiple E3s for activation. Development includes establishing selectivity, specificity, and binding. Following the in vitro work, we can help you determine in vivo target engagement and biomarkers with our TUBE technology. Contact us now!

Inhibition Frequency Distribution: Typical frequency distribution of compound inhibition levels from a screening campaign and subsequent primary confirmation. As expected, the library screen produces a normal distribution of inhibition percentages, while compounds selected for follow-up exhibit significant, reproducible inhibition of E3 ligase activity.

E3 hit selection by single point inhibition. Selectivity profile of E3 ligase inhibitors for E3 ligases X and Y at a single concentration, measured by TR-FRET assay. Compounds with high percent inhibition for E3 Y only are considered to be selective, while those with percent inhibitions greater than 30% for E3 X are considered nonselective. 

E3 ligase selectivity profiling: Selectivity profile of a potent E3 ligase inhibitor (Compound Y) measured by TR-FRET assay. As demonstrated by the IC50 curves, the compound is several orders of magnitude more potent against E3s A and B than E3s C and D. 

Contact us to learn more about this service.