Fragment-growing Guided Design of Keap1-Nrf2 Protein-protein Interaction Inhibitors for Targeting Myocarditis

Small-molecule inhibitors that block the Keap1-Nrf2 protein-protein interactions are being intensely pursued as a new therapeutic strategy for oxidative stress-related diseases, such as cancer, diabetes, Alzheimer's disease, arteriosclerosis, inflammation and myocarditis. However, there are not enough studies on antioxidant treatments using small molecules in myocarditis. We herein provided a series of novel hydronaphthoquinones as the Keap1-Nrf2 interaction inhibitors targeting LPS-induced myocarditis both in vitro and in vivo. These compounds were designed through an in-silico fragment growing approach based on our previous reported compound, S47 (1). The new compounds were predicted to form additional hydrogen bonds with the S363 residue, leading to higher inhibitory activity. Among these new derivatives, compounds S01 and S05 emerged as inhibitors with significant biochemical potency, as determined by fluorescent anisotropy assay and confirmed by surface plasmon resonance (SPR) and differential scanning fluorimetry (DSF) assays. These inhibitors can dose-dependently protect the H9c2 cardiac cells against LPS-induced injury (100% at 2 μM and 4 μM) and effectively prolong survival or save the life of LPS-injured mice. Mechanistic studies showed that these inhibitors could release Nrf2 in H9c2 cells and LPS-inflammatory mouse models and translocate into the nucleus in a dose-response manner, which significantly increased the downstream genes (HO-1, NQO-1) and the pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), while ROS production dramatically decreased. Their protective effects and the mechanism of action were further confirmed by siNrf2 transfected experiment. Collectively, the novel hydronaphthoquinones can be used as promising lead compounds for the study of Keap1-Nrf2 protein-protein interactions and further anti-myocarditis drug development.