Development of a homogeneous time-resolved fluorescence energy transfer (TR-FRET) assay for the inhibition of Keap1-Nrf2 protein-protein interaction

The transcription factor Nrf2 plays a major role in regulating the antioxidant defense system through the Keap1-Nrf2-ARE pathway. Small molecule inhibitors targeting Keap1-Nrf2 protein-protein interaction (PPI) decrease the rate of Nrf2 degradation by the 26S proteasome and thus increase the intracellular level of Nrf2, which translocates into the nucleus leading to upregulated expression of cytoprotective and antioxidant enzymes. Such inhibitors can be developed into potential preventive and therapeutic agents of diseases caused by oxidative damage. In order to more effectively identify promising Nrf2 activators through the inhibition of Keap1-Nrf2 PPI, a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed in this work by indirectly labeling the Keap1 Kelch domain protein with Tb-anti-His antibody as the donor and using, as the acceptor, FITC-labeled 9mer Nrf2 peptide amide, the same fluorescent probe that has been used in the earlier fluorescence polarization (FP) assay. Assay conditions including concentrations of the various components, buffer type and incubation time were optimized in the TR-FRET competition assay with known small molecule inhibitors of Keap1-Nrf2 PPI. Under the optimized conditions, the Keap1-Nrf2 TR-FRET assay exhibited great sensitivity with a high dynamic range and considerable stability up to 5 h. The Z' factor was determined to be 0.82, suggesting that the assay is suitable for high-throughput screening and lead optimization of inhibitors of Keap1-Nrf2 PPI. Furthermore, the TR-FRET assay is capable of differentiating potent inhibitors of Keap1-Nrf2 PPI down to subnanomolar K i .