Directed evolution of metalloproteinase inhibitor TIMP-1 for selective inhibition of MMP-9 exploits catalytic and...

Matrix metalloproteinase-9 (MMP-9) is a critical enzyme involved in extracellular matrix degradation and is strongly implicated in many diseases, including triple-negative breast cancer and other poor prognosis cancers. Selective inhibition of MMP-9 is therefore a promising therapeutic strategy. However, development of MMP inhibitors has been hindered by challenges in achieving specificity, with past efforts failing in clinical trials due to off-target effects and associated toxicity. Here, we present a novel approach to overcoming these challenges by engineering tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural broad-spectrum MMP inhibitor, to achieve enhanced specificity and affinity for MMP-9. We demonstrate that TIMP-1 can be strategically engineered to selectively inhibit MMP-9 through modulating interactions not only with the catalytic domain but also with the unique fibronectin (FN) domains. By leveraging yeast surface display with strategic library design, we identified TIMP-1 variants that exploit multiple surface epitopes to optimize interactions with both the catalytic and FN domains of MMP-9. Molecular dynamics simulations further suggest how modifications in the N-terminal and C-terminal domains of TIMP-1 drive these selective interactions. The top engineered TIMP-1 variant exhibited significantly improved selectivity for MMP-9 in a manner dependent upon novel interactions with the FN domains, as validated through inhibition kinetics. This variant also demonstrated potent inhibition of MMP-9-driven triple-negative breast cancer cell invasiveness, underscoring the therapeutic potential of this approach. Our study highlights the versatility of TIMP-1 as a scaffold that can be optimized for highly selective MMP inhibition, providing new avenues for the development of targeted therapies.