Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

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 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 ( ) 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 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.

Therapeutic Potential of Matrix Metalloproteinase Inhibition in Breast Cancer

“…More recent efforts have employed the yeast surface display platform to evolve variants of the N-terminal domain of TIMP-2 with high affinity and selectivity toward MMP-14 [Arkadash et al 2017]. This recent study combined the strengths of structure-based design with directed evolution, by using computational predictions of the most promising residues for optimization to guide design of the yeast surface display library.…”

Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP‐9 and MMP‐14

“…It is known that WT N‐TIMP2 does not undergo glycosylation in humans ; our mutants do not create any new predictable glycosylation sites, further establishing them as good candidates for in vivo targeting of MMPs. As several of our previously reported MMP‐14‐specific N‐TIMP2 variants have already showed high potency in inhibiting cancer‐related MMP activity in various cellular assays , the MMP‐14‐ and MMP‐9‐specific mutants reported in this study present interesting candidates for further testing in cellular and in vivo studies. Overall, our results help to understand the molecular origins of binding specificity in TIMP/MMP interactions and facilitate development of cancer drugs and diagnostic tools targeting MMPs.…”

“…A). This arrangement is crucial since the N terminus of N‐TIMP2 mediates binding to the MMP active site and thereby facilitates proper inhibition of MMPs, as could be inferred from the crystal structure of N‐TIMP2/MMP complexes and was confirmed in previous experiments . The c‐Myc tag was used to monitor protein expression, through binding of an anti‐c‐Myc antibody and a fluorescently labeled secondary antibody against the anti‐c‐Myc.…”

“…Varying concentrations of N‐TIMP2 were incubated with 0.5 n m MMP‐1 CAT , 6 n m MMP‐2 CAT , 0.1 n m MMP‐9 CAT , 0.5 n m MMP‐10 CAT , and 0.2 n m MMP‐14 CAT under conditions similar to that described in Ref. . Fluorescence resulting from cleavage of fluorogenic substrate was measured at 395 nm, at least every 10 s for at least 6 min, immediately after addition of fluorogenic substrate with irradiation at 325 nm on a Biotek Synergy H1 plate reader (BioTek, Winooski, VT, USA).…”

“…Growth of yeast (EBY100) cells, FACS analyses, and sorting of libraries were performed as in Arkadash et al [48]. For the detection of binding to MMP-14 CAT , the cells were Residues located in the WT N-TIMP2:MMP interface are color coded according to chemical character of their side chains.…”