title: Extra- and intracellular imaging of human matrix metalloprotease 11 (hMMP-11) with a cell-penetrating FRET substrate.
abstract: Matrix metalloprotease 11 (MMP-11), a protease associated with invasion and aggressiveness of cancerous tissue, was postulated as a prognostic marker for pancreatic, breast, and colon cancer patients. Expression analysis, however, did not reveal localization and regulation of this protease. Thus, cellular tools for the visualization of MMP-11 are highly desirable to monitor presence and activity and to elucidate the functional role of MMP-11. Therefore, fluorescein-Dabcyl-labeled Foerster resonance energy transfer (FRET) substrates were developed. The design focused on enhanced peptide binding to human MMP-11, employing an unusual amino acid for the specificity pocket P1'. The addition of several arginines resulted in a cell-permeable FRET substrate SM-P124 (Ac-GRRRK(Dabcyl)-GGAANC(MeOBn)RMGG-fluorescein). In vitro evaluation of SM-P124 with human MMP-11 showed a 25-fold increase of affinity (k(cat)/K(m) = 9.16 × 10(3) m(-1) s(-1), K(m) = 8 μm) compared with previously published substrates. Incubation of pancreatic adenocarcinoma cell line MIA PaCa-2 and mamma adenocarcinoma cell line MCF-7 with the substrate SM-P124 (5 μm) indicated intra- and extracellular MMP-11 activity. A negative control cell line (Jurkat) showed no fluorescent signal either intra- or extracellularly. Negative control FRET substrate SM-P123 produced only insignificant extracellular fluorescence without any intracellular fluorescence. SM-P124 therefore enabled intra- and extracellular tracking of MMP-11-overexpressing cancers such as pancreatic and breast adenocarcinoma and might contribute to the understanding of the activation pathways leading to MMP-11-mediated invasive processes.
authors.affiliation: Department of Organic Chemistry, Freie Universität Berlin, Berlin 14195, Germany.
authors.forename: B Sina
authors.initials: BS
authors.lastname: Meyer
authors.forename: Jörg
authors.initials: J
authors.lastname: Rademann
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004151:Dipeptides / Q000494:pharmacology; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology; D005452:Fluoresceins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000201:enzymology; D019169:Jurkat Cells; D007700:Kinetics; D061986:MCF-7 Cells; D053507:Matrix Metalloproteinase 11 / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D057054:Molecular Imaging / Q000379:methods*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D004151:Dipeptides; D005452:Fluoresceins; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D053507:Matrix Metalloproteinase 11