Application of chemical arrays in screening elastase inhibitors.
Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).
Gao
Feng
F
National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. dugh@imm.ac.cn
Du
Guan-Hua
GH
D001978:Bromphenol Blue / Q000031:analogs & derivatives / Q000737:chemistry; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013449:Sulfonamides; D001978:Bromphenol Blue; C021155:bromochlorophenol blue; C069195:sivelestat; D010196:Pancreatic Elastase; D005998:Glycine