Research Papers:

Combinatorial engineering of N-TIMP2 variants that selectively inhibit MMP9 and MMP14 function in the cell

Valeria Arkadash, Evette S. Radisky and Niv Papo _

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Oncotarget. 2018; 9:32036-32053. https://doi.org/10.18632/oncotarget.25885

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Valeria Arkadash1, Evette S. Radisky2 and Niv Papo1

1Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel

2Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA

Correspondence to:

Niv Papo, email: [email protected]

Keywords: binding specificity; directed evolution; matrix metalloproteinase; protease inhibitor; protein engineering

Received: March 07, 2018     Accepted: July 21, 2018     Published: August 10, 2018


Developing selective inhibitors for proteolytic enzymes that share high sequence homology and structural similarity is important for achieving high target affinity and functional specificity. Here, we used a combination of yeast surface display and dual-color selective library screening to obtain selective inhibitors for each of the matrix metalloproteinases (MMPs) MMP14 and MMP9 by modifying the non-specific N-terminal domain of the tissue inhibitor of metalloproteinase-2 (N-TIMP2). We generated inhibitor variants with 30- to 1175-fold improved specificity to each of the proteases, respectively, relative to wild type N-TIMP2. These biochemical results accurately predicted the selectivity and specificity obtained in cell-based assays. In U87MG cells, the activation of MMP2 by MMP14 was inhibited by MMP14-selective blockers but not MMP9-specific inhibitors. Target specificity was also demonstrated in MCF-7 cells stably expressing either MMP14 or MMP9, with only the MMP14-specific inhibitors preventing the mobility of MMP14-expressing cells. Similarly, the mobility of MMP9-expressing cells was inhibited by the MMP9-specific inhibitors, yet was not altered by the MMP14-specific inhibitors. The strategy developed in this study for improving the specificity of an otherwise broad-spectrum inhibitor will likely enhance our understanding of the basis for target specificity of inhibitors to proteolytic enzymes, in general, and to MMPs, in particular. We, moreover, envision that this study could serve as a platform for the development of next-generation, target-specific therapeutic agents. Finally, our methodology can be extended to other classes of proteolytic enzymes and other important target proteins.

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