Evaluation of a Triple-Helical Peptide with Quenched Fluorophores 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.

Collagenase IV plays an important role in regulating hair cycle by inducing VEGF, IGF-1, and TGF-β expression

“…SB-3CT is a selective inhibitor of MMP-2 and MMP-9, and is widely used to investigate the role of the enzymes 17,18. With this inhibitor, we provide the first evidence that collagenase IV plays an important role in hair cycle, which is mediated by VEGF, IGF-1, and TGF-β.…”

Noninvasive Detection and Imaging of Matrix Metalloproteinases for Cancer Diagnosis

“…A THP conjugating NIR fluorescent dye (LS276-THP), sensitive to MMP-2 and MMP-9, enabled the visualization of MMP activity in mice-bearing human fibrosarcoma [29]. Similarly, another THP conjugating 5-FAM was used to visualize the gelatinase activity in human cancer HT1080 cells [30].…”

“…Recently, a self-assembling triple-helical peptide (THP), incorporating a sequence from type V collagen and with high substrate specificity to the gelatinase, was developed to form the ''turn-on'' fluorescence nanoprobes [29,30]. The three peptide chains intertwine and bring the fluorophores into the close proximity, reducing fluorescence via quenching with the efficiency up to 50-70%.…”

“…NIR dyes (excitation 650-900 nm), with deeper tissue penetration ability, low tissue damage, and less nonspecific tissue autofluorescence, are more popular in small animal imaging [23]. The reported quenchers included nanoparticles (NPs) [21,24,25], small molecule quenching agents [26], or triple-helical peptide [29,30].…”

Dissecting MMP P10′ and P11′ subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library

“…Triple-helical peptides (THPs) that model collagen have been developed in two ways to help better define the roles of collagenolytic MMPs in health and disease. First, THP substrates have (a) identified collagen-based sequence specificities of MMPs (17), (b) led to mechanistic proposals for the initial stages of collagenolysis (18 -21), and (c) been developed as in vivo imaging agents (22)(23)(24) and enablers of targeted drug delivery (25)(26)(27). Second, transition state analogs have been integrated into THP assembly to create triple-helical peptide inhibitors that are efficacious in in vivo models of disease (28 -32).…”