Abstract
Peptide aptamers have primarily been used as research tools to manipulate protein function and study regulatory networks. However, they also find multiple applications in therapeutic research, from target identification and validation to drug discovery. Because of their unbiased combinatorial nature, peptide aptamers interrogate the biological significance of numerous molecular surfaces on target proteins. Their use enables the identification and validation of some of these surfaces as interesting therapeutic targets to pursue. Peptide aptamers can subsequently be used to guide the discovery of small molecule drugs specific for these molecular surfaces.
Here, we present a high-throughput screening assay that identifies small molecules that displace interactions between proteins and their cognate peptide aptamers. AptaScreen is a duplex yeast two-hybrid assay featuring two luciferase reporter genes. It can be performed in 96- or 384-well plates and can be fully automated.
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Notes
- 1.
Carine Bardou and Christophe Borie contributed equally to this work
References
Colas, P., Cohen, B., Jessen, T., Grishina, I., McCoy, J. and Brent, R. (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin- dependent kinase 2. Nature 380, 548–550.
Hoppe-Seyler, F., Crnkovic-Mertens, I., Tomai, E. and Butz, K. (2004) Peptide aptamers: specific inhibitors of protein function. Curr. Mol. Med. 4, 529–538.
Baines, I.C. and Colas, P. (2006) Peptide aptamers as guides for small molecule drug discovery. Drug Disc. Today 11, 334–341.
Colas, P. (2006) Peptide aptamers. In Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine (Ganten, D. and Ruckpaul, K., eds.), Springer, Berlin Hedeilberg, pp. 1368–1372.
Abed, N., Bickle, M., Mari, B., Schapira, M., Sanjuan-Espana, R., Robbe Sermesant, K., et al. (2007) A comparative analysis of perturbations caused by a gene knockout, a dominant negative allele, and a set of peptide aptamers. Mol. Cell. Prot. 6, 2110–2121.
Xu, X., Leo, C., Jang, Y., Chan, E., Padilla, D., Huang, B.C., et al. (2001) Dominant effector genetics in mammalian cells. Nat. Genet. 27, 23–29.
de Chassey, B., Mikaelian, I., Mathieu, A-L., Bickle, M., Olivier, D., Nègre, D., et al. (2007) An antiproliferative genetic screening identifies a peptide aptamer that targets Calcineurin and upregulates its activity. Mol. Cell. Prot. 6, 451–459.
Borghouts, C., Kunz, C. and Groner, B. (2005) Peptide aptamers: recent developments for cancer therapy. Expert Opin. Biol. Ther. 5, 783–797.
Bickle, M., Dusserre, E., Moncorgé, O., Bottin, H. and Colas, P. (2006) Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures. Nat. Protoc. 1, 1066–1091.
Pamonsinlapatham, P., Hadj-Slimane, R., Raynaud, F., Bickle, M., Corneloup, C., Barthelaix, A., et al. (2008) A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death, PLoS ONE 3, e2902.
Nieuwenhuijsen, B.W., Huang, Y., Wang, Y., Ramirez, F., Kalgaonkar, G. and Young, K.H. (2003) A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions. J. Biomol. Screen 8, 676–684.
Mukhopadhyay, K., Kohli, A. and Prasad, R. (2002) Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob. Agents Chemother. 46, 3695–3705.
Acknowledgments
We are grateful to Jérôme Pansanel for the design and implementation of the screening result analysis method, and to Matthieu Schapira for his suggestions. We thank Eric Dusserre for his contribution to the first two-hybrid luminescence assays, Philippe Crouzeix and Guy-Nestor N’Gambai for their contributions to the screening result analysis method, Sandra Dollet for her contribution to the implementation of AptaScreen, and Hélène Bottin and Olivier Moncorgé for plasmid constructions. We are grateful to Marc Blondel for the gift of a erg6 deletion strain, which we used to disrupt erg6 in our own strain background. We are indebted to Christine Pernelle for her most useful advice and constant support. This work was supported by an OSEO /ANVAR innovation grant.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Bardou, C., Borie, C., Bickle, M., Rudkin, B.B., Colas, P. (2009). Peptide Aptamers for Small Molecule Drug Discovery. In: Mayer, G. (eds) Nucleic Acid and Peptide Aptamers. Methods in Molecular Biology™, vol 535. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-557-2_21
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DOI: https://doi.org/10.1007/978-1-59745-557-2_21
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