Keap1-Nrf2-antioxidant response element (ARE) system regulates cellular defense mechanisms that protect cells from oxidative stress. It has been demonstrated that Nrf2 knockout mice are more sensitive to toxicological effects of carcinogens, drugs, and inflammatory stresses while Keap1 knockout mice exhibited high levels of Nrf2, high constitutive expression of cytoprotective enzymes and striking resistance to the environmental stresses. Keap1-Nrf2 interaction plays a key role in cancer chemoprevention by many chemicals like sulforaphane, curcumin, and epigallocatechin gallate derived from natural sources such as fruits, vegetables, and tea products. Modification, by these natural electrophiles, of sensitive cysteine residues found in the redox "sensor" protein Keap1 is believed to be responsible for causing the translocation of Nrf2 to the nucleus and subsequent upregulation of anti-oxidative stress cytoprotective enzymes. One approach to address concerns over the general use of purified natural thiol-reactive compounds as chemopreventive agents is to use high throughput screening assays of chemical libraries to discover and develop novel small molecules as direct inhibitors of Keap1-Nrf2 interaction at the protein-protein interface. These inhibitors will mimic the actions of electrophiles like isothiocyanates and Michael acceptors in the induction of cytoprotective enzymes but will potentially be more selective and specific against the Keap-Nrf2 axis. Two solution fluorescence-based high throughput screening assays will be developed and validated in this new R01 application using fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET). These assays will be applied to the screening of chemical libraries for the discovery of new leads in an interdisciplinary program involving medicinal chemistry, computational chemistry, X-ray crystallography, and cancer biology. These studies will provide important information about the protein-protein interaction between Keap1 and Nrf2 and facilitate the development of more potent and selective Keap1-Nrf2 inhibitors as inducers of oxidative stress response enzymes. PUBLIC HEALTH REVELANCE:HTS assays developed in this project will facilitate the discovery of novel leads for the development of new Keap1-Nrf2 inhibitors. Specific and potent Keap1-Nrf2 inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential cancer chemopreventive agents in high risk populations.