Sankaran, Banumathi

Matrix metalloproteinases (MMPs) play a pivotal role in progression of several diseases including cancer and fibrosis, which makes these enzymes promising targets for developing protein therapeutics. The challenge in using MMP inhibitors as drugs in MMP‐associated diseases is lack of high selectivity toward specific target MMPs. Therefore, development of selective MMP inhibitors is a critical goal. Tissue inhibitors of metalloproteinases (TIMPs), as natural MMP inhibitors, offer ideal scaffolds for engineering highly selective MMP‐targeted therapeutics. We have previously shown a key role for MMP‐3 in the pathogenesis of pulmonary fibrosis. TIMP‐1 is a natural MMP‐3 inhibitor with picomolar affinity; it also binds to other MMPs with a spectrum of affinities. Engineering of TIMP‐1 for more selective inhibition of MMP‐3 may provide protein therapeutics with applications in treating lung fibrosis. I have used directed evolution and yeast surface display toward development of selective MMP‐3‐targeted biologic agents through protein engineering of TIMP‐1. I have screened a library of TIMP‐1 mutants generated by random mutagenesis of residues comprising the MMP‐interacting loops of TIMP‐1 to select for enhanced MMP‐3 binding on the yeast surface. Diversified residues have been chosen based on structural studies of TIMP‐1/MMP‐3 interactions. TIMP‐1 mutants isolated after rounds of Fluorescent‐Activated Cell Sorting (FACS) showed substantial improvement in MMP‐3 binding compared to the wild‐type TIMP‐1, both in the surface display context and in enzyme inhibition studies using soluble purified proteins. I have also 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 one of the top affinity mutants with the catalytic domain of MMP‐3, and have obtained synchrotron diffraction data at a resolution of 2.2 Å; efforts to solve this crystal structure are underway. I anticipate that this structure will offer insights into the structural basis for improvements in selectivity, and will help to guide future library design for refinement of my directed evolution methodology for generating selective engineered TIMPs. This work provides a promising and innovative strategy to develop protein therapeutics based on natural enzyme inhibitors, critically improving selectivity to eliminate offtarget effects. Support or Funding Information This project is funded through two grants from Department of Defense (DoD) (ID # W81XWH‐16‐2‐003015), and National Institute of Health (NIH) (R21CA205471). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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.