This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (19) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Grant, S. K. Articles by Cummings, R. T. Search for Related Content PubMed PubMed Citation Articles by Grant, S. K. Articles by Cummings, R. T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Social Bookmarking                         What's this?

Development of Novel Assays for Proteolytic Enzymes Using Rhodamine-Based Fluorogenic Substrates

Department of Human & Animal Infectious Disease Research, Merck & Co., Rahway, NJ.

Joseph G. Sklar

Department of Human & Animal Infectious Disease Research, Merck & Co., Rahway, NJ.

Richard T. Cummings

Department of Molecular Design and Diversity, Merck & Co., Rahway, NJ.

Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)₂-Rhod110 and bis-(Ala-Pro)₂-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC^TM collection and natural product extracts despite high levels of fluorescence interference.

Journal of Biomolecular Screening, Vol. 7, No. 6, 531-540 (2002)
DOI: 10.1177/1087057102238627


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				C. A. Sasiela, D. H. Stewart, J. Kitagaki, Y. J. Safiran, Y. Yang, A. M. Weissman, P. Oberoi, I. V. Davydov, E. Goncharova, J. A. Beutler, et al. Identification of Inhibitors for MDM2 Ubiquitin Ligase Activity from Natural Product Extracts by a Novel High-Throughput Electrochemiluminescent Screen J Biomol Screen, March 1, 2008; 13(3): 229 - 237. [Abstract] [PDF]

				F. Ausseil, A. Samson, Y. Aussagues, I. Vandenberghe, L. Creancier, I. Pouny, A. Kruczynski, G. Massiot, and C. Bailly High-Throughput Bioluminescence Screening of Ubiquitin-Proteasome Pathway Inhibitors from Chemical and Natural Sources J Biomol Screen, February 1, 2007; 12(1): 106 - 116. [Abstract] [PDF]

				M. A. O'Brien, W. J. Daily, P. E. Hesselberth, R. A. Moravec, M. A. Scurria, D. H. Klaubert, R. F. Bulleit, and K. V. Wood Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model J Biomol Screen, March 1, 2005; 10(2): 137 - 148. [Abstract] [PDF]