Xuexun Fang

High-affinity peptide against MT1-MMP for in vivo tumor imaging

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key member of the matrix metalloproteinase (MMP) family. It participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules and is essential for many biological and pathological processes, such as tumor development, angiogenesis and metastasis. A ligand that specifically binds to MT1-MMP may facilitate the labeling of this molecule, allow imaging at the cellular and organism levels, and provide a means for targeted drug delivery specific to MT1-MMP. A non-substrate MT1-MMP binding peptide was identified by screening a Ph.D.-12™ phage display peptide library and conjugated with near-infrared fluorescent (NIRF) dye Cy5.5 for tumor imaging. Peptide HWKHLHNTKTFL (denoted as MT1-AF7p) showed high MT1-MMP binding affinity. Computer modeling verified that MT1-AF7p binds to the MT-loop region of MT1-MMP and interacts with MT1-MMP through hydrogen bonding and hydrophobic interactions. MDA-MB-435 xenografts with high MT1-MMP expression had significantly higher tumor accumulation and better tumor contrast than the low MT1-MMP expressing A549 xenografts after intravenous injection of Cy5.5-MT1-AF7p. Using NIRF imaging, we have demonstrated specific targeting of MT1-AF7p to MT1-MMP-expressing tumors. Thus, MT1-AF7p is an important tool for noninvasive monitoring of MT1-MMP expression in tumors, and it shows great potential as an imaging agent for MT1-MMP-positive tumors.

In vivo optical imaging of membrane-type matrix metalloproteinase (MT-MMP) activity

Herein we demonstrate for the first time that a fluorogenic probe can be used as an in vivo imaging agent for visualizing activities of membrane-tethered, membrane-type matrix metalloproteinases (MT-MMPs). An MT-MMP fluorogenic probe that consisted of an MT1-MMP (MMP-14) substrate and near-infrared (NIR) dye-quencher pair exhibited rapid, efficient boosts in fluorescence upon cleavage by MT1-MMP in tumor-bearing mice. In particular, unlike similar fluorogenic probes designed to target extracellular, soluble-type MMPs (EC-MMPs)--which can be cleared from the bloodstream after activation--the fluorescence signals activated by MT1-MMP enable clear visualization of MT1-MMP-positive tumors in animal models for up to 24 h. The results indicate that a simple form of a fluorogenic probe that is less effective in EC-MMP imaging is an effective probe for imaging MT-MMP activities in vivo. These findings can be widely applied to designing probes and to applications targeting various membrane-anchored proteases in vivo.

Dual-Functional, Receptor-Targeted Fluorogenic Probe for In Vivo Imaging of Extracellular Protease Expressions

Herein, we report a new type of in vivo fluorogenic probe that enables simultaneous and active targeting of overexpressed receptors, α(V)β(3) integrins, and extracellular proteases, matrix metalloproteinases (MMPs), in the tumor regions. This c(RGDyK)-conjugated MMP fluorogenic probe efficiently targets the tumor regions with high retention time while maintaining receptor binding affinity and substrate activity. The probe minimizes nonspecific accumulation, thus demonstrating improved tumor-to-background signal ratio (T/N) in both α(V)β(3) integrin- and MMP-overexpressing U87MG tumor-bearing mouse model. This strategy can be easily tuned for a wide array of applications targeting various receptors and extracellular proteases in vivo.

Locating the Band III protein in quasi-native cell membranes

Band III is a key protein for the structure and function of red blood cell membranes. To date, the distribution and morphology of Band III in cell membranes is still unclear because of limited approaches. We applied Topography and RECognition imaging microscopy (TREC), which extends the application of atomic force microscopy (AFM) to recognize a single molecule in a biological complex, to visually locate a single Band III protein in quasi-native cell membranes by anti-Band III-functionalized AFM tips under physiological conditions. The Band III proteins are well distributed in the inner leaflet of cell membranes. The height of the whole Band III protein in cell membranes is in the range of 9–13 nm. The unbinding force between Band III in the membrane and anti-Band III on the AFM tip is about 70 pN with the loading rate at 40 nN/s. Our result is significant in revealing the location and morphology of Band III in the inner cell membrane at the molecular level.

Expression Optimization and Characterization of the Catalytic Domain of Human MT3-MMP

Matrix metalloproteinases (MMPs) are a family of proteases that are required for many biological processes and are also elevated in many pathological conditions. MMP inhibitors (MMPIs) may therefore be useful as therapeutic agents in treating a number of diseases including cancer, cardiovascular diseases and arthritis. Attempts have been made to develop MMPIs. Recombinant MMPs have been used to screening MMPs in vitro assays. In this work, we report the expression of MMP-16 in E. coli and the characterization of the recombinant MMP-16 with a commonly used MMP substrate DQ-gelatin.

Optimization of a MT1-MMP-targeting Peptide and Its Application in Near-infrared Fluorescence Tumor Imaging

Membrane type 1 metalloproteinase (MT1-MMP) is an important regulator of cancer invasion, growth and angiogenesis, thus making it an attractive target for cancer imaging and therapy. A non-substrate peptide (MT1-AF7p) that bonded to the “MT-Loop” region of MT1-MMP was identified by using a phage-displayed peptide library and was used to image the MT1-MMP expression in vivo through optical imaging. However, the substrate in the screening did not have a 3D structure, thus resulting in a loose bonding of MT1-AF7p. To simulate the real conformation of the “MT-Loop” and improve the performance of MT1-AF7p, molecular simulations were performed, because this strategy provides multiple methods for predicting the conformation and interaction of proteinase in 3D. In view of the binding site of the receptor–ligand interactions, histidine 4 was selected for mutation to achieve an increased affinity effect. The optimized peptides were further identified and conformed by atomic force microscopy, isothermal titration calorimetry, cell fluorescence imaging in vitro, and near-infrared fluorescence tumor optical imaging in vivo. The results revealed that the optimized peptide with a mutation of histidine 4 to arginine has the highest affinity and specificity, and exhibited an increased fluorescence intensity in the tumor site in optical imaging.

Quantum Dots Tethered Membrane type 3 matrix metalloproteinase-targeting Peptide for Tumor Optical Imaging

Membrane type matrix metalloproteinases (MT-MMPs) play important roles in malignant tumor progression through the degradation of the extracellular matrix and signal transduction. However, a member of the family, MT3-MMP, has attracted the least concern compared with other MT-MMPs. Here, a novel MT3-MMP-targeting peptide with high affinity and specificity has been developed by a phage-display peptide screening technology and multiple biophysics measurements, including single-molecule recognition force spectroscopy and isothermal titration calorimetry. The binding peptides are conjugated on the surface of CdSe/ZnS quantum dots (QDs) and consequently acted as a ligand that specifically targets MT3-MMP overexpressed tumor cells. The imaging nanoprobes used QDs as the photographic developer for optical imaging in vivo. The nanoprobes exhibited a desirable targeting effect and generated good biodistribution profiles for visualization and imaging of MT3-MMP overexpressed tumor. The peptide could be useful to evaluate the distribution and expression of MT3-MMP. Furthermore, the peptide-functionalized QDs show potential application for cancer diagnosis.

Inhibition of Metalloproteinase Activity by Chinese Formulations Used to Treat Inflammatory Diseases

Matrix metalloproteinases (MMPs) are known to be involved in a number of pathological processes including cancer, atherosclerosis, arthritis and neurodegenerative disease among others. The drive to develop MMP inhibitors as therapeutics has beef put forward for years by pharmaceutical companies as well as academicians. In an attempt to screen for MMP inhibitors from Traditional Chinese Medicines (TCMs), a number of Chinese formulations used to treat inflammatory diseases such as nephritis and hepatitis have been studied. Strong inhibitory effects of three Chinese formulations toward the activity of MMP-16 have been discovered. These results suggest that these anti-inflammatory medicines contain some unknown MMP inhibitory compound(s) and provide reasonable molecular mechanisms for their therapeutic effects.