Engineering protein therapeutics for cancer based on the natural matrix metalloproteinase inhibitor TIMP‐1

Abstract

Enzymes of the matrix metalloproteinase (MMP) family have been shown to play critical roles in cancer progression and metastasis, making them ideal targets for developing cancer therapeutics. MMP‐9 plays a significant role in tumor progression and metastasis of triple negative breast cancer (TNBC), while MMP‐3, the biological activator of MMP‐9, can also promote mammary carcinoma through inducing epithelial‐mesenchymal‐transition (EMT) in tumor cells. These enzymes are attractive as therapeutic targets; however, broad spectrum MMP inhibitors have proven ineffective, so inhibitors with greater selectivity are desired. Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) is a natural inhibitor of MMP‐3 and MMP‐9 with picomolar affinity, and offers a scaffold for engineering of selective MMP‐targeted therapeutics. To overcome the challenges of wide multispecificity of TIMP‐1 for different classes of MMPs, we used state‐of‐the‐art directed evolution and yeast surface display techniques recruiting high‐throughput library screening technology to fully evolve novel protein‐based drugs, based on the TIMP‐1 scaffold, with high selectivity for MMP‐3 or MMP‐9. We have developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized the top affinity and selectivity mutants with the catalytic domain of MMP‐3, and have solved the structure. These structures provide insights into the structural basis for improvements in binding affinity and selectivity. These studies and methodology developed in this project, will lay the foundation for developing novel therapeutic strategies for MMP‐related diseases and understanding protein‐protein interaction between MMPs and their inhibitors.