Abstract
The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. The small molecules that bind to specific proteins can be used as tools to study the functions of proteins and biological processes in cells. We have developed an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes the use of cell lysates that express proteins of interest fused with red fluorescent protein (RFP) and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. To demonstrate large-scale ligand screening for various human proteins of interest, the gene library GLORIA (Gene Library of Osada Laboratory at RIKEN for chemical array analysis) has been established. Using the systematic platform, we detected novel inhibitors of carbonic anhydrase II. We also have shown that this screening method is useful not merely for ligand screening of proteins of interest, but also for gaining insight into structure–affinity relationships (SARs) and for studies of “fragment-based approach.”
Traditional fragment-based ligand discoveries have been demonstrated by using several technologies, such as NMR spectroscopy and X-ray crystallography and mass spectrometry. We present initial studies of fragment-based approach to binding assay by using the chemical array format.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kuruvilla, F. G., Shamji, A. F., Sternson, S. M., Hergenrother, P. J., and Schreiber, S. L. (2002) Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays. Nature 416, 653–657.
Uttamchandain, M., Walsh, D. P., Yao, S. Q., and Chang, Y.-T. (2005) Small molecule microarrays: recent advances and applications. Curr. Opin. Chem. Biol. 9, 4–13.
Koehler, A. N., Shamji, A. F., and Schreiber, S. L. (2003) Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis. J. Am. Chem. Soc. 125, 8420–8421.
Uttamchandain, M.,Walsh, D. P., Khersonsky, S. M., Huang, X., and Yao, S. Q. (2004) Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG. J. Comb. Chem. 6, 862–868.
Hergenrother, P. J., Depew, K. M., and Schreiber, S. L. (2000) Small-molecule microarrays: covalent attachment and screening of alcohol-containing small molecules on glass slides. J. Am. Chem. Soc. 122, 7849–7850.
MacBeath, G., Koehler, A. N., and Schreiber, S. L. (1999) Printing small molecules as microarrays and detecting protein-ligand interactions en masse. J. Am. Chem. Soc. 121, 7967–7968.
Park S., and Shin I. (2002) Fabrication of carbohydrate chips for studying protein-carbohydrate interactions. Angew. Chem. Int. Ed. 41, 3180–3182.
Barnes-Seeman, D., Park, S. B., Koehler, A. N., and Schreiber, S. L. (2003) Expanding the functional group compatibility of small-molecule microarrays: discovery of novel calmodulin ligands. Angew. Chem. Int. Ed. 42, 2376–2379.
Kanoh, N., Kumashiro, S., Simizu, S., Kondoh, Y., Hatakeyama, S., Tashiro, H., and Osada, H. (2003) Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions. Angew. Chem. Int. Ed. 42, 5584–5587.
Kanoh, N., Asami, A., Kawatani, M., Honda, K., Kumashiro, S., Takayama, H., Simizu, S., Amemiya, T., Kondoh, Y., Hatakeyama, S., Tsuganezawa, K., Utata, R., Tanaka, A., Yokoyama, S., Tashiro, H., and Osada, H. (2006) Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions. Chem. Asian J. 1, 789–797.
Bradner, J. E., McPherson, O. M., Mazitschek, R., Barnes-Seeman, D., Shen, J. P., Dhaliwal, J., Stevenson, K. E., Duffner, J. L., Park, S. B., Neuberg, D. S., Nghiem, P., Schreiber, S. L., and Koehler, A. N. (2006) A robust small-molecule microarray platform for screening cell lysates. Chem. Biol. 13, 493–504.
Miyazaki, I., Simizu, S., Ichimiya, H., Kawatani, M., and Osada, H. (2008) Robust and systematic drug screening method using chemical arrays and the protein library: identification of novel inhibitors of carbonic anhydrase II. Biosci. Biotechnol. Biochem. 72, 2739–2749.
He, X. G., Gerona-Navarro, G., Jaffrey, S. R., (2005) Ligand discovery using small molecule microarrays. J. Pharmacol. Exp. Ther. 313, 1–7.
Tomiki, T., Saito, T., Ueki, M., Konno, H., Asaoka, T., Suzuki, R., Uramoto, M., Kakeya, H., and Osada, H. (2006) RIKEN natural products encyclopedia (RIKEN NPEdia), a chemical database of RIKEN natural products depository (RIKEN NPDepo). J. Comp. Aided Chem. 7, 157–162.
Melkko, S., Scheuermann, J., Dumelin, C. E., and Neri, D. (2004) Encoded self-assembling chemical libraries. Nat. Biotechnol., 22, 568–574.
Poker, Y., and Stone, J. T. (1967) The catalytic versatility of erythrocyte carbonic anhydrase. 3. Kinetic studies of the enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. Biochemistry, 6, 668–678.
Miyazaki, I., Okumura, H., Simizu, S., Takahashi, Y., Kanoh, N., Muraoka, Y., Nonomura, Y., and Osada, H. (2009) Structure-affinity relationship study of bleomycins and Shble protein by use of a chemical array. Chembiochem, 10, 845–852.
Miyazaki, I., Simizu, S., Ishida, K., and Osada, H. (2009) On-chip fragment-based approach for discovery of high-affinity bivalent inhibitors. Chembiochem, 10, 838–843.
Shuker, S. B., Hajduk, P. J., Meadow, R. P., and Fesik, S. W. (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Miyazaki, I., Simizu, S., Osada, H. (2010). The Application of the Chemical Array for Biological Study. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 669. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-845-4_8
Download citation
DOI: https://doi.org/10.1007/978-1-60761-845-4_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-844-7
Online ISBN: 978-1-60761-845-4
eBook Packages: Springer Protocols