Keap1-Kelch-targeting protein-protein interaction inhibitors, but not reversibly-binding electrophiles, increase the thermostability of Keap1 in the cellular environment

Abstract

The Kelch-like ECH-associated protein 1 / Nuclear factor erythroid 2-p45-related factor 2 (Keap1/Nrf2) partnership orchestrates the cellular defence against oxidative, inflammatory and metabolic stress. Dysregulation of Nrf2 is involved in the pathogenesis of numerous chronic diseases. Under homeostatic conditions, Keap1 continuously targets Nrf2 for ubiquitination and degradation. When Keap1 is inactivated, Nrf2 accumulates and translocates to the nucleus, where it activates transcription of genes encoding cytoprotective proteins. There are two main types of Keap1 inhibitors, electrophiles and Keap1-Nrf2 protein-protein interaction (PPI) inhibitors. Using a quantitative fluorescence-based Cellular Thermal Shift Assay (CETSA), we investigated the ability of a panel of electrophilic Nrf2 activators and PPI inhibitors to bind to Keap1 in lysates and intact cells stably expressing Keap1-mCherry or free mCherry as controls. All PPI inhibitors tested caused an increase in the thermostability of Keap1-mCherry. Surprisingly however, electrophiles that bind covalently and reversibly to thiols did not. Moreover, treatment of intact cells with the double Michael acceptors bis(benzylidene)acetone and its hydroxylated derivative bis(2-hydroxybenzylidene)acetone caused a decrease in the thermostability of Keap1. Thus, in addition to confirming target engagement of Keap1-targeting PPI inhibitors in the cellular environment, the Keap1 fluorescence-based CETSA is capable of distinguishing between the mechanism of action of the two types of Nrf2 activators in the cellular environment, and has the potential for cost-effective, high-throughput applications.