Advertisement

Random Mutagenesis of Peptide Aptamers as an Optimization Strategy for Inhibitor Screening

  • Nathalie Bouquier
  • Sylvie Fromont
  • Anne DebantEmail author
  • Susanne Schmidt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 928)

Abstract

Accumulating work over the past decade has shown that peptide aptamer screening represents a valid strategy for inhibitor identification that can be applied to a variety of different targets. Because of the screening method in cells and the highly combinatorial libraries available, this approach yields rapidly highly specific candidate inhibitors. Once a hit peptide has been identified, its interaction strength and affinity towards its target protein can be optimized even more, in order to increase its inhibition efficiency when subsequently applied in vivo. A condition to a successful optimization is that gain of inhibition strength should not result in loss of specificity.

Here we present a simple method for peptide aptamer optimization, which can be achieved by PCR-based random mutagenesis combined with a selection screen in yeast using a strong selective drug. The rationale of this approach, which has proven valid and efficient, is that stronger interaction in yeast will also lead to stronger inhibition. Our optimization method is effective, without loss of specificity, which is of a great importance for the discovery of inhibitors that target specific protein–protein interactions.

Key words

Peptide aptamers PCR-based mutagenesis Yeast two-hybrid Fluorescent GEF assay Inhibitor screening 

Notes

Acknowledgments

We are grateful to Anne Briançon-Marjollet for her contribution to the library construction. This work was supported by grants from the ANR “Physique et chimie du vivant” grant N° 06-137373. N. B. was supported by a CNRS valorization fellowship from CNRS. All authors are members of the CNRS consortium GDR2823.

References

  1. 1.
    Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 380:548–50PubMedCrossRefGoogle Scholar
  2. 2.
    Crawford M, Woodman R, Ko Ferrigno P (2003) Peptide aptamers: tools for biology and drug discovery. Brief Funct Genomic Proteomic 2:72–9PubMedCrossRefGoogle Scholar
  3. 3.
    Hoppe-Seyler F, Crnkovic-Mertens I, Tomai E, Butz K (2004) Peptide aptamers: specific inhibitors of protein function. Curr Mol Med 4:529–38PubMedCrossRefGoogle Scholar
  4. 4.
    Borghouts C, Kunz C, Groner B (2005) Peptide aptamers: recent developments for cancer therapy. Expert Opin Biol Ther 5:783–97PubMedCrossRefGoogle Scholar
  5. 5.
    Schmidt S, Diriong S, Mery J, Fabbrizio E, Debant A (2002) Identification of the first Rho-GEF inhibitor, TRIPalpha, which targets the RhoA-specific GEF domain of Trio. FEBS Lett 523:35–42PubMedCrossRefGoogle Scholar
  6. 6.
    Bouquier N, Fromont S, Zeeh JC, Auziol C, Larrousse P, Robert B, Zeghouf M, Cherfils J, Debant A, Schmidt S (2009) Aptamer-derived peptides as potent inhibitors of the oncogenic RhoGEF Tgat. Chem Biol 16:391–400PubMedCrossRefGoogle Scholar
  7. 7.
    Yoshizuka N, Moriuchi R, Mori T, Yamada K, Hasegawa S, Maeda T, Shimada T, Yamada Y, Kamihira S, Tomonaga M, Katamine S (2004) An alternative transcript derived from the trio locus encodes a guanosine nucleotide exchange factor with mouse cell-transforming potential. J Biol Chem 279:43998–4004PubMedCrossRefGoogle Scholar
  8. 8.
    Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Nathalie Bouquier
    • 1
  • Sylvie Fromont
    • 2
  • Anne Debant
    • 2
    Email author
  • Susanne Schmidt
    • 3
  1. 1.Centre de Recherche de Biochimie Macromoléculaire, CNRS–UMR 5237Universités Montpellier I et IIMontpellier CedexFrance
  2. 2.Centre de Recherche de Biochimie Macromoléculaire, CNRS–UMR 5237Universités Montpellier I et IIMontpellier Cedex 5France
  3. 3.Centre de Recherche de Biochimie Macromoléculaire, CNRS–UMRUniversités Montpellier I et IIMontpellier CedexFrance

Personalised recommendations