Abstract
Fluorescence polarization (FP) has become a powerful technique to quantitatively analyze the binding of a small soluble fluorescence-labeled probe to a larger soluble protein and its displacement by other molecules. Here, we describe a detailed protocol to identify small molecules capable of targeting p53-MDM2/MDMX interactions using a fluorescence polarization assay with Rhodamine-labeled p53 peptides.
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References
Czarna A, Popowicz GM, Pecak A, Wolf S, Dubin G, Holak TA (2009) High affinity interaction of the p53 peptide-analogue with human Mdm2 and Mdmx. Cell Cycle 8:1176–1184
Pazgier M, Liu M, Zou G, Yuan W, Li C, Li J, Monbo J, Zella D, Tarasov SG, Lu W (2009) Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX. Proc Natl Acad Sci USA 106:4665–4670
Phan J, Li Z, Kasprzak A, Li B, Sebti S, Guida W, Schonbrunn E, Chen J (2010) Structure-based design of high affinity peptides inhibiting the interaction of p53 with MDM2 and MDMX. J Biol Chem 285:2174–2183
Ding K, Lu Y, Nikolovska-Coleska Z, Qiu S, Ding Y, Gao W, Stuckey J, Krajewski K, Roller PP, Tomita Y, Parrish DA, Deschamps JR, Wang S (2005) Structure-based design of potent non-peptide MDM2 inhibitors. J Am Chem Soc 127:10130–10131
Shangary S, Qin D, McEachern D, Liu M, Miller RS, Qiu S, Nikolovska-Coleska Z, Ding K, Wang G, Chen J, Bernard D, Zhang J, Lu Y, Gu Q, Shah RB, Pienta KJ, Ling X, Kang S, Guo M, Sun Y, Yang D, Wang S (2008) Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proc Natl Acad Sci USA 105:3933–3938
Reed D, Shen Y, Shelat AA, Arnold LA, Ferreira AM, Zhu F, Mills N, Smithson DC, Regni CA, Bashford D, Cicero SA, Schulman BA, Jochemsen AG, Guy RK, Dyer MA (2010) Identification and characterization of the first small molecule inhibitor of MDMX. J Biol Chem 285:10786–10796
Lee JH, Zhang Q, Jo S, Chai SC, Oh M, Im W, Lu H, Lim HS (2011) Novel pyrrolopyrimidine-based alpha-helix mimetics: cell-permeable inhibitors of protein-protein interactions. J Am Chem Soc 133:676–679
Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28
Ding K, Lu Y, Nikolovska-Coleska Z, Wang G, Qiu S, Shangary S, Gao W, Qin D, Stuckey J, Krajewski K, Roller PP, Wang S (2006) Structure-based design of spiro-oxindoles as potent, specific small-molecule inhibitors of the MDM2-p53 interaction. J Med Chem 49:3432–3435
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Zhang, Q., Lu, H. (2013). Identification of Small Molecules Affecting p53-MDM2/MDMX Interaction by Fluorescence Polarization. In: Deb, S., Deb, S. (eds) p53 Protocols. Methods in Molecular Biology, vol 962. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-236-0_8
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DOI: https://doi.org/10.1007/978-1-62703-236-0_8
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