Discovery of a Potent Kelch-Like ECH-Associated Protein 1‑Nuclear Factor Erythroid 2‑Related Factor 2 (Keap1–Nrf2) Protein–Protein Interaction Inhibitor with Natural Proline Structure as a Cytoprotective Agent against Acetaminophen-Induced Hepatotoxicity

The
transcription factor Nrf2 is a key regulator of cytoprotective system,
and enhancing Nrf2 activity can protect cells from various insults
and threats. Directly disrupting Keap1–Nrf2 protein–protein
interactions has been regarded as a promising way to activate Nrf2.
We reported here the first identification of amino acids as preferred
substituents to design potent Keap1–Nrf2 inhibitors. Comprehensive
structure–activity analysis identified Pro as a preferred substituent,
obtaining a potent inhibitor 35 with an IC50 of 43 nM in the competitive fluoresce polarization (FP) assay and
a Kd value of 53.7 nM for Keap1 protein
in the isothermal titration calorimetry (ITC) assay. The Pro analogue 35 exhibited tight and prolonged Keap1 binding in vitro and
in cells, and treatment with 35 activated Nrf2-regulated
cytoprotective response and antagonized acetaminophen-induced liver
injury both in cellular and in vivo models. This work not only provides
a useful tool to further explore the therapeutic potential of Keap1–Nrf2
inhibition but also enriches the diversity of chemical structures
suitable for the Keap1–Nrf2 interface.