Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pM, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.
Bibliographical noteFunding Information:
This work was supported in part by European Research Council "Ideas Program" ERC-2013-StG by Contract Grant 336041 (to N. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Incumbent of the Maurizio Pontecorvo Professorial Chair. Supported by the Israeli Science Foundation (1226/13), the European Research Council AdG (THZCALORIMETRY-DLV-695437), and the USA-Israel Binational Science Foundation (712506-01). Supported by National Institutes of Health Grants R01CA154387 and R21CA205471. Supported by Israel Science Foundation Grant 1873/15. We thank Prof. Reuven Reich (Hebrew University of Jerusalem) for providing Matrigel and for consultation on the invasion studies. We thank Dr. Alon Zilka for technical assistance. FACS and Proteon experiments were performed at the NIBN proteomics unit.
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