A Pan Photoaffinity Probe for Detecting Active Forms of Matrix Metalloproteinases

Abstract

A photoaffinity probe based on the scaffold of a potent broad-spectrum phosphinic peptide inhibitor of matrix metalloproteinases (MMPs) has been developed. A photolabile diazirine group for covalent modification of MMP active forms was incorporated at the P(1) ' position, and a tritium radioactive label for the sensitive detection of MMP covalent adducts by radioimaging was attached. The probe was characterized on seven catalytic domains of human MMPs (MMP-2, -3, -8, -9, -12, -13 and -14) and was found to display nanomolar affinities towards this set of MMPs, covalently modifying them with crosslinking yields varying from 12 to 58 %, thus leading to highly sensitive detection of these MMPs. In a complex proteome complemented with four recombinant MMPs (MMP-2, -9, -12 and -13), this probe enabled their simultaneous detection with a threshold of few femtomoles and low background labelling. Those properties should make this new pan-activity-based MMP probe a valuable tool for the detection of MMP active forms from biological fluids or tissue extracts.

Detection of Endogenous Matrix Metalloprotease-12 Active Form with a Novel Broad Spectrum Activity-based Probe*

“…Thus, as expected, probe 2 labeled mMMP-12 by covalently modifying only its free active site, demonstrating that probe 2 can be used as an ABP. By targeting only the free MMP active site, probe 2 will not label either pro-MMP or the TIMP/ MMP complex, as previously shown (24,28). In buffer, a detection threshold for mMMP-12 with probe 2 of 1 fmol was obtained (Fig.…”

“…Thus, efficient MMP ABPs are clearly required to support further MMP drug development and for evaluation of the specificity of these selective inhibitors in animal models. Beyond MMP-12, Probe 2 has also been shown to crosslink in vitro other MMPs (28), and should therefore be useful for the detection of MMPs in various biological systems.…”

“…Acrylamide 40%/bis solution, 37.5:1(2.6% C), molecular markers (Precision Plus Protein Standards, All Blue), and blotting paper (extra thick blot paper) were obtained from BioRad. Probe 2 was prepared as previously described (28).…”

“…Using an internal calibration (involving the spotting of known amounts of radioactivity on the acquisition plate: triplicates of 1, 2, 10, and 50 pCi) and background subtraction; each selected area can be associated with a value expressed in pCi. For the labeling experiment with buffered recombinant MMP-12, a comparison of the cpm obtained for labeled hMMP-12 and for labeled mMMP-12 was sufficient for the assessment of the percentage mMMP-12 crosslinking, as this percentage is known for hMMP-12 (28). In the case of the BALf labeling experimental analysis, we took differences in protein concentration between samples into account by carrying out a second normalization in all cases, to provide counts in pCi per g of total protein, based on the protein concentration determined for each sample.…”

“…Probe 2 also includes radioactive tritium atoms to ensure the sensitive detection of protein covalent adducts by radioimaging and a short polyethylene linker to increase the solubility of the probe in water. This novel probe displays nanomolar affinities toward a large set of human MMPs and was shown to crosslink only active forms of MMPs with very high crosslinking yields, allowing their detection in the femtomolar range in vitro (28). These encouraging results led us to investigate, in the present study, whether probe 2 could be used to detect active form of MMPs in samples from mice.…”