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. 2020 Feb 4;6(3):368–381. doi: 10.1021/acscentsci.9b00927

Figure 4.

Figure 4

Identification of macrocyclic peptide inhibitors of Keap1/Nrf2 interaction. (A) Crystal structure of Keap1 Kelch domain (KKD) in complex with the Nrf2 regulatory domain (pdb 2FLU).83 (B) Libraries of semirandomized O2beY-linked peptide macrocycles (X = NNK codon) displayed on phages and hit sequences identified by deep sequencing (relative abundance = n/56 000 sequences) after library panning against immobilized KKD. KD values correspond to the FLAG-tagged CBD-fused peptide macrocycles. (C) Binding curves for selected macrocyclic peptides as determined using a direct binding assay with plate-immobilized KKD and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. (D) Competition assay in which binding of FLAG-KKD-m1 to immobilized KKD is inhibited by a Nrf2-derived peptide (IC50 = 2.8 ± 0.1 nM).