E3 Ligase Screening and PROTAC Profiling Services
Ubiquitin E3 ligases are the largest family of proteins in human genome (about 800 members). The excitement in E3 ligase drugs is not only to target a ligase and discover selective drugs such as SCF ligases where substrate recognizing subunit cereblon binds to Tholidamide derivatives and highjacks it to ubiquitylate selective proteins in the cell but also due to the discovery of PROTACs (proteolysis-targeting chimeras). PROTACs are designed to cross-link E3 ligase and the target protein to induce polyubiquitylation and proteasomal degradation of the target protein within cells.
Despite heroic efforts to screens for ligase drugs, industry has failed to discover selective molecules that regulate E3 ligase function. There are number of reasons for this failure. Lack of sensitive and selective catalytic, biochemical and biophysical assays has hampered discovery of credible small molecules that target ligases. Discovery of a selective molecule requires number of orthogonal assays.
LifeSensors has developed platform of technologies to characterize and profile compounds that modulate E3 ligase activity. LifeSensors has established variety of catalytic, biochemical, biophysical assays for number of ligases. The vast library of ubiquitin ligase proteins allows LifeSensors to adapt and customize unique assays that fits your need. Selective ligase inhibitors will not be discovered by testing compounds in one assay platform but will require that the compound be evaluated in number of assays and evaluated for selectivity by screening against the same family of ligases (HECT or Ring Finger).
The LifeSensors proprietary E3 ligase activity assay involves Terbium-labeled Tandem Ubiquitin Binding Entities (TUBEs) that bind to polyubiquitn chains synthesized by E3 ligases. Terbium and fluorescein are a FRET pair, so polyubiquitin chains containing fluorescein-labeled ubiquitin will yield a FRET signal when bound by terbium-TUBE. This signal can be monitored over time in a homogenous, high-throughput format, making it ideal for small-molecule screening.
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.