Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates.
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)-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 trade mark collection and natural product extracts despite high levels of fluorescence interference.
Department of Human and Animal Infectious Disease Research, Merck and Co., Rahway, NJ, USA.
Stephan K
SK
Grant
Joseph G
JG
Sklar
Richard T
RT
Cummings
D001418:Baclofen / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D010419:Pentamidine / Q000032:analysis / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010455:Peptides; D011480:Protease Inhibitors; D012235:Rhodamines; C052329:phaclofen; D010419:Pentamidine; D010450:Endopeptidases; D007931:Leucyl Aminopeptidase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D001418:Baclofen