title: Fluorescence resonance energy transfer assay for high-throughput screening of ADAMTS1 inhibitors.
abstract: A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1) plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET) assay for high-throughput screening (HTS) of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z' factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.
authors.affiliation: Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China.
authors.forename: Jianhao
authors.initials: J
authors.lastname: Peng
authors.forename: Lili
authors.initials: L
authors.lastname: Gong
authors.forename: Kun
authors.initials: K
authors.lastname: Si
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Bai
authors.forename: Guanhua
authors.initials: G
authors.lastname: Du
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D059748:Proteolysis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D051722:ADAM Proteins