Elsevier

Tetrahedron

Volume 70, Issue 42, 21 October 2014, Pages 7714-7720
Tetrahedron

Direct Ras inhibitors identified from a structurally rigidified bicyclic peptide library

https://doi.org/10.1016/j.tet.2014.05.113Get rights and content

Abstract

A one-bead-two-compound (OBTC) library of structurally rigidified bicyclic peptides was chemically synthesized on TentaGel microbeads (90 μm), with each bead displaying a unique bicyclic peptide on its surface and a linear encoding peptide of the same sequence in its interior. Screening of the library against oncogenic K-Ras G12V mutant identified two classes of Ras ligands. The class I ligands apparently bind to the effector-binding site and inhibit the Ras–Raf interaction, whereas the class II ligand appears to bind to a yet unidentified site different from the effector-binding site. These Ras ligands provide useful research tools and may be further developed into therapeutic agents.

Introduction

The Ras family proteins are small GTP-binding proteins that play critical roles in many signaling pathways and regulate cell proliferation, differentiation, and survival. The three main family members, K-Ras, H-Ras, and N-Ras, are highly homologous in their N-terminal catalytic domains and differ mainly in the C-terminal membrane anchoring sequences.1 While all three Ras proteins have been shown to drive cancer formation and progression, K-Ras is the most frequently mutated isoform, occurring in ∼30% of human cancers. Wild-type K-Ras oscillates between an active, GTP-bound form and an inactive GDP form.2, 3 The GTP-bound form interacts with multiple effector proteins, such as Raf, PI3K, and Ral-GDS, via its Switch I and Switch II regions. Single point mutations in K-Ras (e.g., G12V) abolish GTPase-activating protein (GAP)-mediated hydrolysis of bound GTP through steric hindrance, rendering the mutant K-Ras constitutively active and causing sustained activation of downstream effector pathways. Ample experimental data suggest that inhibition of oncogenic K-Ras, especially its interaction with effector proteins, should have therapeutic benefits in cancer patients.4, 5 Unfortunately, K-Ras has been a very challenging target for small-molecule drug discovery, because its binding sites for effector proteins involve flat surfaces without any obvious pockets. As a result, most of the efforts have been focused on inhibition of signaling molecules immediately upstream and downstream of K-Ras or the posttranslational processing/membrane anchoring of K-Ras.6, 7 Several small-molecule inhibitors have recently been reported to inhibit the nucleotide exchange activity of K-Ras, but they are generally weak inhibitors, with IC50 values in the high μM to low mM range.8, 9, 10, 11, 12 Covalent inhibitors have recently been developed to selectively target the G12C mutant Ras.13, 14 Weak peptide ligands against Ras have also been reported.15, 16, 17 However, despite the intense efforts during the past three decades, no effective treatment for Ras mutant tumors is yet available. In particular, compounds that bind directly to Ras and inhibit the Ras–effector interaction are lacking.

We recently discovered a cyclic peptide inhibitor against K-Ras (KD of 0.83 μM), which blocks the interaction between K-Ras and its effector proteins Raf, PI3K, and Ral-GDS, demonstrating the feasibility of developing macrocyclic compounds as direct Ras inhibitors.18 Further development of the cyclic peptide was problematic, however, because the compound was synthetically cumbersome and its lactone moiety was susceptible to hydrolytic degradation. Meanwhile, we devised a general methodology for synthesizing and screening bicyclic peptide libraries displayed on rigid small-molecule scaffolds.19 Screening of a bicyclic peptide library against tumor necrosis factor-alpha (TNFα), a protein considered as ‘undruggable’ by the small-molecule approach, identified a potent low-molecular weight TNFα antagonist. This suggests that structurally rigidified bicyclic peptides are effective for binding flat protein surfaces, such as the interfaces of protein–protein interactions (PPIs). In this work, we screened the bicyclic peptide library against the K-Ras G12V mutant to identify direct Ras inhibitors as well as assess the generality of bicyclic peptides as PPI inhibitors.

Section snippets

Results and discussion

The bicyclic peptide library consisted of a random peptide sequence of 6–10 residues ‘wrapped’ around a trimesoyl group (Fig. 1a).19 Peptide cyclization was mediated by the formation of three amide bonds between trimesic acid and the N-terminal amine, the side chain of a C-terminal l-2,3-diaminopropionic acid (Dap), and the side chain of a fixed lysine within the random region. The resulting bicyclic peptides contained 3–5 random residues in each ring and 24 different amino acids at each random

Conclusion

We discovered several relatively potent ligands against K-Ras, a previously ‘undruggable’ protein target, by screening a naïve bicyclic peptide library. Together with our earlier success against TNFα,18 our study demonstrates that structurally rigidified bicyclic peptides are effective (and perhaps even privileged) for recognizing flat protein surfaces, such as the interfaces of protein–protein interactions. Moreover, out of the five bicyclic peptides with confirmed binding to their target

Materials

Reagents for peptide synthesis were purchased from Peptides International (Louisville, KY), NovaBiochem (La Jolla, CA), Anaspec (San Jose, CA), Chem-Impex International Inc. (Wood Dale, IL), or Aapptec (Lousiville, KY). N-Hydroxysuccinimidyl biotin was purchased from Chem-Impex International (Wood Dale, IL) and N-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) was from Advanced ChemTech. Phenyl isothiocyanate (PITC) was purchased in 1-mL sealed ampoules from Sigma–Aldrich, and a freshly

Acknowledgements

This work was supported by the National Institutes of Health (GM062820 and CA132855).

References and notes (36)

  • K. Rajalingam et al.

    Biochim. Biophys. Acta, Mol. Cell Res.

    (2007)
  • K. Walker et al.

    Curr. Opin. Genet. Dev.

    (2005)
  • A. Singh et al.

    Cancer Cell

    (2009)
  • W. Wang et al.

    Bioorg. Med. Chem. Lett.

    (2012)
  • A.G. Taveras et al.

    Bioorg. Med. Chem.

    (1997)
  • D. Barnard et al.

    Biochem. Biophys. Res. Commun.

    (1998)
  • M. Hintersteiner et al.

    Chem. Biol.

    (2009)
  • J.P. Leyris et al.

    Anal. Biochem.

    (2011)
  • T.J. Mosmann

    Immunol. Methods

    (1983)
  • K. Giehl

    Biol. Chem.

    (2005)
  • S. Gysin et al.

    Genes Cancer

    (2011)
  • Y. Wang et al.

    J. Med. Chem.

    (2013)
  • T. Maurer et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (2012)
  • Q. Sun et al.

    Angew. Chem., Int. Ed.

    (2012)
  • H. Waldmann et al.

    Angew. Chem., Int. Ed.

    (2004)
  • F. Shima et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (2013)
  • J.M. Ostrem et al.

    Nature

    (2013)
  • S.M. Lim et al.

    Angew. Chem., Int. Ed.

    (2014)
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