Fig 1: Prediction and design of amino acid substitutions in each amino acid position of KRpep-2d. The complex of ribbon diagram of K-Ras(G12D)GDP and stick model of KRpep-2d (PDB ID: 5XCO) are shown in the center. Purple amino acids are Cys5 and Cys15. Orange amino acids indicate pharmacophore sequence (Pro6–Val14). Cyan amino acids present Arg1–4,16–19 residues. Chemical structures of representative amino acid examples that were predicted to improve biochemical functions of KRpep-2d in Leu7, Ile9, and Tyr11 are listed. In the upper left side box, Arg residues at both termini of the KRpep-2d were excluded for clarity. The figure was made using PyMOL.
Fig 2: Biochemical characters of KS-58. (A) Binding activity of biotinylated peptides to recombinant K-Ras(G12D) protein in ELISA (n = 4, ± SEM). (B) Binding activity of different concentrations (from the highest 3 µM and subsequent threefold dilution) of Biotin-KS-58 to recombinant Ras proteins as measured by ELISA (n = 4, ± SEM). (C) Inhibition activity of peptides to intracellular Ras–effector proteins interactions. Intracellular PPI in the presence or absence of peptides are detected as luminescence, and the PPI is showed as % value (30 µM, n = 4, ± SEM). (D) Proposed K-Ras(G12D)-inhibition mechanism of KS-58.
Fig 3: Screening of KRpep-2d derivatives. (A) K-Ras(G12D)-binding activity of Biotin-KRpep-2d in ELISA (n = 4, ± SEM). (B) Competitive inhibition activities of KRpep-2d and KS-36 against the interaction of plate-coated K-Ras(G12D) and Biotin-KRpep-2d (100 nM) (n = 4, ± SEM). (C) Amino acid structures introduced into KRpep-2d derivatives and their K-Ras(G12D)-binding activities. Positions of the substituted amino acids in KRpep-2d and introduced amino acids are listed. K-Ras(G12D)-binding activities are listed as fold-value compared to the parental KRpep-2d set as 1.0 (n = 4, ± SEM). 2d-amide is an amide bond (Dap5–Asp15) cyclized KRpep-2d, and 2d-nc is main chain cyclized KRpep-2d.
Fig 4: MD simulations of KS-58, KS-58(monocyclic), and KS-58(Ser6Dap). (A) Structural comparison of K-Ras(G12D)GDP-binding mode and K-Ras(G12D)GTP-binding mode of KS-58 (stable snap shots) with K-Ras(G12D)GDP (PDB ID: 4EPR), K-Ras(G12D)GDP/KRpep-2d (PDB ID: 5XCO), K-Ras(G12D)GppNHp (PDB ID: 5USJ), and K-Ras(G12D)GppNHp/KD2 (PDB ID: 6WGN). The magnesium ions are presented as large spheres. Dash lines indicate hydrogen bonds. Arg residues at both termini of the KRpep-2d were excluded for clarity. (B) Lipid membrane accessibility of peptides in MD simulations. Line graph shows moving positions of underlined atoms of Asp8 (Ca–CH2–COOH, red) and Nle3/Anon5 (Ca–CH2–CH2–CH2–CH3, Ca–CH2–CH2–CH2–CH2–CH2–CH2–CH3, green), respectively. KS-58: left panel, KS-58(monocyclic): center panel, KS-58(Ser6Dap): right panel.
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