Profile functional E3 ligase cascades
While ubiquitination offers an exciting new avenue to approach many difficult-to-treat diseases, there are certain aspects of ubiquitination that have provided an obstacle to drug development. The first complexity is that, unlike other post-translational modifications (e.g., phosphorylation), ubiquitination relies on several interconnected steps involving multiple enzymes (i.e., E1, E2 and E3). Secondly, while E3 ligases are the most attractive targets because of their potential specificity, they are the most complex - sometimes consisting of multiple protein constituents. Furthermore, many of the substrates themselves need to be post-translationally modified before they are recognized as substrates for ubiquitination.
Eurofins overcomes the restrictions to ubiquitination drug discovery. By applying our protein production and assay expertise, we've developed a suite of assays to measure distinct ubiquitination cascades. Each assay contains the complete complement of ubiquitin enzymes (E1 activating enzyme, E2 conjugating enzyme and E3 ligase) and a biologically-relevant substrate with appropriate post-translational modifications. The incorporation of ubiquitin into the substrate is quantitatively detected by electrochemiluminescence, which allows for the identification of inhibitors and activators.
Advantages of UbiquitinProfiler™:
Figure 1.UbiquitinProfiler™ services: selectivity profiling.
Compounds were profiled at a final concentration of 100 μM against four E3 ligase cascades to assess their selectivity for individual E3 ligases. PYR41, which inhibits Ube1 (E1) activity, was included as a control.
Figure 2. UbiquitinProfilerIC50™ services: potency determinations.
Compounds identified to inhibit the ubiquitination of p27 by SCFSkp2/Cks1 E3 ligase were tested in a dose response to determine their potency. Final DMSO concentration in the assay was 2%.