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Molecules 2014, 19(6), 8571-8588; doi:10.3390/molecules19068571

Evaluation of a Triple-Helical Peptide with Quenched Fluorophores for Optical Imaging of MMP-2 and MMP-9 Proteolytic Activity

1
Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15219, USA
2
Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80208, USA
3
Department of Radiology, Washington University in St Louis, St Louis, MO 63110, USA
4
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA
*
Author to whom correspondence should be addressed.
Received: 18 April 2014 / Revised: 5 June 2014 / Accepted: 11 June 2014 / Published: 23 June 2014
(This article belongs to the Special Issue Fluorescent Probes)
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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. View Full-Text
Keywords: matrix metalloproteinase-2 (MMP-2); MMP-9; gelatinase; triple-helical peptides; optical imaging; fluorescence molecular tomography (FMT) matrix metalloproteinase-2 (MMP-2); MMP-9; gelatinase; triple-helical peptides; optical imaging; fluorescence molecular tomography (FMT)
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MDPI and ACS Style

Zhang, X.; Bresee, J.; Cheney, P.P.; Xu, B.; Bhowmick, M.; Cudic, M.; Fields, G.B.; Edwards, W.B. Evaluation of a Triple-Helical Peptide with Quenched Fluorophores for Optical Imaging of MMP-2 and MMP-9 Proteolytic Activity. Molecules 2014, 19, 8571-8588.

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