title: Evaluation of a triple-helical peptide with quenched FluorSophores for optical imaging of MMP-2 and MMP-9 proteolytic activity.
abstract: Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.
authors: Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15219, USA. xuanzh2001@hotmail.com.
authors: Xuan
authors: X
authors: Zhang
authors: Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15219, USA. breseeje@upmc.edu.
authors: Jamee
authors: J
authors: Bresee
authors: Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80208, USA. pcheney@du.edu.
authors: Philip P
authors: PP
authors: Cheney
authors: Department of Radiology, Washington University in St Louis, St Louis, MO 63110, USA. baogangx@mir.wustl.edu.
authors: Baogang
authors: B
authors: Xu
authors: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA. mbhowmick@tpims.org.
authors: Manishabrata
authors: M
authors: Bhowmick
authors: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA. mcudic@tpims.org.
authors: Mare
authors: M
authors: Cudic
authors: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA. gfields@tpims.org.
authors: Gregg B
authors: GB
authors: Fields
authors: Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15219, USA. edwardsw@upmc.edu.
authors: Wilson Barry
authors: WB
authors: Edwards
mesh_terms: D045744:Cell Line, Tumor; D024062:Collagen Type V / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D061986:MCF-7 Cells; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D041622:Tomography, Optical / Q000379:methods*
chemical_list: D024062:Collagen Type V; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010455:Peptides; C045132:4-carboxyfluorescein; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9