Abstract 1331 Engineering Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Variants with Improved Binding Selectivity toward Matrix Metalloproteinase-9 (MMP-9) as potential protein therapeutics

Introduction:Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the function and activity of matrix metalloproteinases (MMPs), enzymes that are known for their involvement in cancer progression. TIMPs possess two domains that pack side by side; the TIMP N-terminal domain inhibits MMP enzymatic activity by binding at the active site and interacting with the catalytic zinc, while our prior work has shown that the C-terminal domain can also contribute to MMP affinity. MMP-9, also known as gelatinase B, plays a crucial role in tumorigenesis of several cancers including triple negative breast cancer (TNBC); tumor cell-produced MMP-9 motivates malignant progression and metastasis of basal-like TNBC. Thus, MMP-9 has been suggested as a potential therapeutic target for breast cancer treatment. Here, we have used full-length TIMP-1 as a scaffold for engineering inhibitors that bind with higher selectivity to MMP-9, for potential therapeutic applications. Methods: We previously developed a yeast surface display library of human TIMP-1, with diversity incorporated at 17 residues within five loops; mutation burden was tuned to 3-5 mutations per clone. Here, we have screened the TIMP-1 library for affinity toward recombinant MMP-9 by using fluorescent-activated cell sorting (FACS). Yeast cells were incubated with an incrementally decreasing concentration of biotinylated MMP-9 (400 nM to 25 nM) in successive rounds of sorting. Samples were analyzed using flow cytometry and highest affinity populations were selected using FACS. After multiple rounds of enrichment, plasmid DNA was extracted from the isolated individual yeast clones for DNA sequencing. Results: Among the TIMP-1 variants with highest enrichment, mutation L34G located in the AB loop, M66W and E67W located in the C-connector loop, L133R in the GH loop, and L152P/E156V in the multiple-turn loop were the most frequent mutations detected. By simultaneously incorporating diversity into both domains, we identified the TIMP-1-C15 variant that was 12-fold enhanced in binding to MMP-9 compared with WT TIMP-1. This variant is highly selective for binding MMP-9 compared to MMP-1, MMP-2 (which has substantial sequence similarity to MMP-9) and MMP-3. Conclusion: We integrated structure-guided library design with directed evolution to create TIMP-1 variants capable of precise discrimination between closely related MMPs. In ongoing work, we will validate the soluble TIMP-1-C15 variant for improved MMP-9 inhibition and selectivity over other MMPs in enzyme assays and conduct structural studies to illuminate the basis for enhanced specificity. Future work will investigate therapeutic efficacy of TIMP variants in models of TNBC. Our recent outcomes are promising because they hold considerable potential to develop a novel strategy for targeted treatment of TNBC via selectively inhibiting MMP-9, an enzyme with crucial roles in tumor growth and metastasis of breast cancer. This work supporting by US National Institutes of Health grants R01 GM132100 and R01 CA258274.