Synergy of Nrf2‐Activating Electrophiles in Combination with the Nrf2‐Inhibitor KI696: A Model of Mechanistic Basis for Synergy

Abstract

Oxidative stress, which contributes to many chronic diseases such as diabetes and cancer, can be mitigated through the production of antioxidant proteins, controlled by the Nrf2 transcription factor. Combination drug treatments in disease therapy hold promise for their increased potency and therapeutic window as compared to single drug treatments. In this work, we tested combinations of three clinically relevant Nrf2 activating electrophiles (bardoxolone methyl, Tecfidera, and sulforaphane) with the Nrf2 inhibitor KI696 to see if there was a more than additive effect on ARE expression. Under basal conditions, Nrf2 is bound by Keap1 in the cytoplasm of the cell, and targeted for ubiquitination and degradation. When Keap1 C151 is modified by these electrophiles, Nrf2 escapes repression and localizes to the nucleus to bind to cognate antioxidant response element (ARE) sequences, thereby upregulating expression of antioxidant genes. KI696 binds to the Nrf2‐binding site on Keap1, allowing for accumulation of Nrf2 and subsequent ARE‐driven expression. Through an ARE‐reporter luminescence assay, we show that these three electrophiles each synergistically upregulate ARE‐driven expression with KI696 in a statistically significant manner, based on our Dose Equivalence/Zero Interaction (DE/ZI) method (Repash et al., Frontiers in Pharmacology, 2021). The combination of these clinically‐relevant electrophiles and KI696 are a model for mechanistic investigations of synergy and identification of targets other than Keap1 cysteines.