Strategies for the identification of ubiquitin ligase inhibitors

Abstract

Dysregulation of the UPS (ubiquitin-proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; however, toxicity with this target remains high. E3s (Ub-protein ligases) represent an alternative attractive therapeutic target in the UPS. In this paper, we will discuss current platforms that report on E3 ligase activity and can detect E3 inhibitors, and underline the advantages and disadvantages of each approach.

Figure 1

A) Unbound Reaction Components. Biotin labeled Ub (bio-Ub) is mixed with GST-tagged E3, E1, E2 and ATP. anti-GST antibody labeled with Eu³⁺ and streptavidin-APC are then added and excited in the Eu wavelength and the fluorescence of APC is measured. B) Immobilization of Ubiquitin Ligases. GST-tagged E3 is bound to GSH coated magnetic beads. E1, E2, Ub, ATP and compounds are added to the reaction and measured by anti-Ub antibody labeled with the ORIGEN tag that gives a chemiluminescent response. C) Immobilization of Substrate. A high-binding plate is coated with the substrate, unbound substrate is washed off, and the E1, E2, FLAG-Ub, and E3 are added. After unbound proteins are removed anti-FLAG antibody conjugated to HRP is added to the well, washed and HRP substrate is added to detect a signal. D) UBA is immobilized to a high-binding plate. Unbound UBA is removed and E1, E2, E3, Ub, and ATP are added to the well. After the components are removed anti-Ub antibody is added, washed, and secondary antibody conjugated to HRP is added. HRP substrate is added after a final wash step.