Identification and characterization of novel Nrf2 inducers designed to target the intervening region of Keap1.

Transcription factor Nrf2 regulates a battery of genes encoding detoxifying enzymes. Under basal conditions, Nrf2 is sequestered in the cytoplasm by a protein known as Keap1. In response to oxidative stress, Keap1-mediated ubiquitination of Nrf2 is decreased significantly and the Nrf2 pathway is turned on. Residues C273 and C288 at the intervening region (IVR) domain of Keap1 are necessary for Keap1 to repress Nrf2, indicating a critical role of the IVR domain in the functional interaction of Keap1 with Nrf2. To identify chemical modulator targeting the IVR domain of Keap1, we built a 3D structural model of the Keap1 IVR domain and demonstrated this structural model is effective in retrieving novel Nrf2 inducers from chemical databases, BM10, 31, and 40 increase concentration of nuclear Nrf2, with a potency comparable to that of sulforaphane. We showed C297S mutation partially abolished the Nrf2-inducing effect of BM31, suggesting BM31 may target C297 in the IVR domain of Keap1. Further, BM31 and BM40 potently induce expression of ARE-regulated enzyme gamma-glutamylcysteine synthetase. We demonstrated that BM31 provides protections for the MCF-7 cells from cytotoxic damage of carcinogen benzo[a]pyrene.