A novel Keap1 inhibitor iKeap1 activates Nrf2 signaling and ameliorates hydrogen peroxide-induced oxidative injury and apoptosis in osteoblasts

An ultra-large structure-based virtual screening has discovered iKeap1 as a direct Keap1 inhibitor that can efficiently activate Nrf2 signaling. We here tested its potential effect against hydrogen peroxide (H 2 O 2 )-induced oxidative injury in osteoblasts. In primary murine and human osteoblasts, iKeap1 robustly activated Nrf2 signaling at micromole concentrations. iKeap1 disrupted Keap1-Nrf2 association, causing Nrf2 protein stabilization, cytosol accumulation and nuclear translocation in murine and human osteoblasts. The anti-oxidant response elements (ARE) activity and transcription of Nrf2-ARE-dependent genes (including HO1 , NQO1 and GCLC ) were increased as well. Significantly, iKeap1 pretreatment largely ameliorated H 2 O 2 -induced reactive oxygen species production, lipid peroxidation and DNA damage as well as cell apoptosis and programmed necrosis in osteoblasts. Moreover, dexamethasone- and nicotine-induced oxidative injury and apoptosis were alleviated by iKeap1. Importantly, Nrf2 shRNA or CRISPR/Cas9-induced Nrf2 knockout completely abolished iKeap1-induced osteoblast cytoprotection against H 2 O 2 . Conversely, CRISPR/Cas9-induced Keap1 knockout induced Nrf2 cascade activation and mimicked iKeap1-induced cytoprotective actions in murine osteoblasts. iKeap1 was ineffective against H 2 O 2 in the Keap1-knockout murine osteoblasts. Collectively, iKeap1 activated Nrf2 signaling cascade to inhibit H 2 O 2 -induced oxidative injury and death of osteoblasts.