Design, characterization, and evaluation of peptide arrays allowing the direct monitoring of MMP activities

Abstract

Characterization of matrix metalloprotease (MMP) activities is of increasing interest for cancer prognosis or treatment follow-up. Indeed, MMP-1, -2 and -9 are widely involved in the growth of many tumors and progression steps such as angiogenesis, invasion, and metastasis. Fluorogenic peptide MMP substrates were previously synthesized with the aim of detecting MMP activities. One of their drawbacks is their limited solubility in biological media. Grafting them onto a solid support represented a novel way to yield efficient analysis devices whilst at the same time decreasing the quantities of peptides used. Novel peptide arrays were designed in order to detect MMP activities in biological fluids. Silicon plates were used as the solid support for the design of these novel tools. These were functionalized by organic self-assembled monolayers (SAMs) on which fluorogenic peptides were covalently coupled. SAM and peptide grafting on silicon plates were confirmed by epifluorescence, ellipsometry, and FT-IR analysis. Enzymatic assays were monitored by fluorescence spectrometry and showed that immobilized linear peptides were recognized and cleaved by MMPs.

Enzymatic reactions on immobilised substrates

“…235 Proteases such as trypsin, lys-C and chymotrypsin are often used for protein digestion in peptide-based experiments, routinely used in proteomics. 107,108,216 Peptide bond hydrolysis often results in large structural changes that can be monitored directly by AFM or SPR. 107,116 There are several types of proteases, classified by their hydrolysis mechanism (Fig.…”

Supramolecular Peptide/Surface Assembly for Monitoring Proteinase Activity and Cancer Diagnosis

“…To overcome some of these drawbacks, we developed a solid sensor based on covalent grafting of fluorogenic substrates on a solid support as a proof-of-concept that MMPs activities can be also monitored with solid assay. 10 However, some points such as the behavior and stability of this sensor with tumor biopsy or tumor cells, the ease of synthesis and the regeneration of the device biosensing area remained to be addressed in order to obtain an innovative and relevant device for MMP activity screening. Some of these points are also relevant for other scientific communities involved in biosensor or lab-on-chip development.…”

“…However, this screening strategy presents some drawbacks, in particular, the lack of selectivity and the poor solubility of linear peptide substrates and the screening peptide quantity used for tests. To overcome some of these drawbacks, we developed a solid sensor based on covalent grafting of fluorogenic substrates on a solid support as a proof-of-concept that MMPs activities can be also monitored with solid assay . However, some points such as the behavior and stability of this sensor with tumor biopsy or tumor cells, the ease of synthesis and the regeneration of the device biosensing area remained to be addressed in order to obtain an innovative and relevant device for MMP activity screening.…”

Interaction of Fluorescently Labeled Triethyleneglycol and Peptide Derivatives with β‐Cyclodextrin

“…7 The first approach was the covalent binding of the fluorogenic peptides to a solid support made of silicon plates functionalized with organic self‐assembled monolayers. Enzymatic assays showed that immobilized linear peptides were recognized and cleaved by MMPs 8. Subsequently, a non‐covalent approach was explored with the aim of developing a highly versatile and reactivatable biosensor.…”