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.
Grant
Stephan K
SK
Department of Human and Animal Infectious Disease Research, Merck and Co., Rahway, NJ, USA.
Sklar
Joseph G
JG
Cummings
Richard T
RT
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