title: A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates.
abstract: We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.
authors.affiliation: Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Nakata
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohtsuki
authors.forename: Masahiko
authors.initials: M
authors.lastname: Sisido
mesh_terms: D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D004151:Dipeptides / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006877:Hydroxamic Acids / Q000032:analysis / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D010445:Peptide Elongation Factors / Q000032:analysis / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D061945:Caspase Inhibitors; D004151:Dipeptides; D005456:Fluorescent Dyes; D006877:Hydroxamic Acids; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010445:Peptide Elongation Factors; D011480:Protease Inhibitors; C023512:elongation factor Ts; D010447:Peptide Hydrolases; D053148:Caspase 3; D020780:Matrix Metalloproteinase 9; C010531:actinonin