GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf

Abstract

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated in vitro translation-mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.

Figure 1

Selection of cyclic peptides that selectively bind to State 1 of GTP-bound K-Ras(G12D) from the Random non-standard Peptide Integrated Discovery (RaPID) mRNA display library. (a) Cyclic peptide selection was performed for a total of five rounds using K-Ras(G12D/T35S)·GppNHp as the positive selection target and K-Ras(G12D/T35S)·GDP as the negative selection target. (b) Top 20 hits clustered by sequence alignment. Peptides are ranked based on the number of unique NGS reads in the output library (not shown, see Supporting Information for detailed methods). Colored shades group peptides in the same cluster; gray shades represent peptides that have higher affinity for the GDP state, which were not further studied. One peptide from each cluster (bold typeface) was chosen for further characterization. (c) Structures of three distinct peptides identified from the selection. Blue color indicates the constant regions of the cyclic peptide backbone, including sulfide bridge, the starting amino acid chloroacetyl-d-tyrosine, and the ending amino acids cysteine and glycine.

Figure 2

Cyclic peptides block the interaction of K-Ras(G12D) and effector proteins. (a) Illustration of a biochemical assay that detects Ras-Raf interaction by time-resolved fluorescence energy transfer (TR-FRET). (b) Cyclic peptides block Ras-Raf interaction with selectivity for the G12D mutant over wildtype K-Ras. (c) Illustration of a biochemical assay that monitors Sos-mediated nucleotide exchange using a fluorescent-GDP analog. (d) KD2 and KD17, but not KD1, inhibit Sos-mediated nucleotide exchange of K-Ras(G12D).

Figure 3

Crystal structure of KD2 bound to K-Ras(G12D)·GppNHp. (a) KD2 binds in the Switch II groove of K-Ras(G12D)·GppNHp. (b) 2F_o – F_c map showing the electron density of KD2, Asp12, Gln61, and relevant water molecules, contoured at 1.0σ. (c) KD2 forms an intricate hydrogen bond network intramolecularly and intermolecularly with K-Ras. (d) Comparison of K-Ras(G12D)·GppNHp·KD2 structure with unliganded K-Ras(G12D)·GppNHp (PDB: 5USJ) and K-Ras(G12D)·GDP (PDB: 4EPR). Root-mean-square deviations (RMSDs) were calculated on a residue-by-residue basis for each pairwise comparison. SI, Switch I; SII, Switch II.

Figure 4

Substitution of Thr10 in KD2 improves the potency for blocking Ras-Raf interaction. (a) Thr10 in KD2 is in proximity with Asp12 of K-Ras(G12D). (b) Structure of KD2 with Thr10 highlighted in red. (c) Thr10 mutants of KD2 are more potent inhibitors of Ras-Raf interaction. (d) Thr10 mutants of KD2 do not inhibit wildtype K-Ras-Raf interaction.

Figure 5

Bicyclic variants of KD2 exhibit improved potency for block Ras-Raf interaction. (a) Val3 and Arg9 on KD2 are solvent exposed and participate in neither interaction with K-Ras nor intramolecular interactions. (b) Structures of bicyclic variants of KD2. (c) Bicyclic variants of KD2 are more potent inhibitors of K-Ras(G12D)-Raf interaction. (d) Bicyclic variants of KD2 show improved inhibition of K-Ras(wildtype)-Raf interaction at high concentrations.