Poster Presentation 9th General Meeting of the International Proteolysis Society 2015

New selective internally quenched substrates for the investigation of human matrix metalloproteinases 2 and 9 (#153)

Marcin Poreba 1 , Wioletta Rut 1 , Izabela Rutkowska 2 , Leszek Kaczmarek 2 , Christopher Overall 3 , Marcin Drag 1
  1. Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland
  2. Department of Molecular and Cellular Neurobiology, Nencki Institute, Warsaw, Poland
  3. Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, Canada

The matrix metalloproteinases (MMPs) family consists of over 23 structurally related endopeptidases [1]. These enzymes are known to participate in cancer progression and metastasis which make them very attractive molecular targets for the design of selective chemical probes [2]. Of the various MMPs known to be involved in cancer, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are of special interest since their overexpression is correlated with tumor aggressiveness and a poor prognosis. What is more, the elevated level of these proteins has been found in bladder, brain, breast, colorectal, lung, pancreas and prostate cancers [3].

So far several peptide-based fluorescent substrates for MMP-2 and MMP-9 have been reported in the literature, however none of them displays an absolute specificity toward only one MMP of interest[4,5]. The main limitation of these substrates is that only natural amino acids were use in their structures. Our group has recently demonstrated that the use of a wide range of unnatural amino acids in peptidic substrates can solve the problem regarding proteases overlapping substrate specificity[6].

In the present study we demonstrate a two-step approach for the design of novel and very selective substrates that can distinguish between MMP-2 and MMP-9. In the first step we extracted from the literature and synthesized over 40 "the most specific so far" MMP-2 and MMP-9 sequences (natural protein substrates, synthetic constructs), and by measuring their kinetic parameters we selected two lead structures: one for MMP-2 and one for MMP-9. In the second step, based on these sequences we synthesized two tailored internally quenched fluorogenic libraries containing natural and a wide range of unnatural amino acids. The kinetic analysis of these libraries allowed us to develop the most specific so far MMP-2 and MMP-9 chemical probes.

            This work was supported by the National Science Centre grant 2014/13/B/ST5/00240in Poland

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