Development of high-affinity and high-specificity inhibitors of metalloproteinase 14 through computational design and directed evolution

Citation:

V. Arkadash, G. Yosef, J. Shirian, I. Cohen, Y. Horev, M. Grossman, I. Sagi, E. S. Radisky, J. M. Shifman, and N. Papo. 2017. “Development of high-affinity and high-specificity inhibitors of metalloproteinase 14 through computational design and directed evolution.” J Biol Chem, 292, Pp. 3481-3495.

Abstract:

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 three 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 towards MMP-14 and up to 16,000-fold greater specificity towards 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.

Notes:

Arkadash, ValeriaYosef, GalShirian, JasonCohen, ItayHorev, YuvalGrossman, MoranSagi, IritRadisky, Evette SShifman, Julia MPapo, Niveng2017/01/15 06:00J Biol Chem. 2017 Jan 13. pii: jbc.M116.756718. doi: 10.1074/jbc.M116.756718.