Engineering high-affinity and high-specificity inhibitors of MMP-9

Matrix metalloproteinases (MMPs) are family of zinc-dependent proteases that degrade extracellular matrix proteins. While MMPs are known to have an important role in many physiological processes, they are also involved in cancer progression and metastasis and in other pathological conditions. Therefore, MMPs present great targets for drug design, yet specific inhibition is important to avoid serious side effects. This project aims to develop a high-affinity and high-specificity inhibitor of MMP-9 based on Tissue Inhibitor of Metalloproteinase -2 (TIMP-2), a natural MMP inhibitor with broad activity against several MMPs. In previous studies done by our laboratory, we used computational methods along with yeast surface display (YSD) technique to obtain TIMP2 mutants with high affinity and high specificity toward two different MMPs, MMP-9 and MMP-14. Those studies yielded two different TIMP-2 mutants that exhibited 1000-fold better affinity for MMP-9 vs. MMP-14. However, the binding affinity of the engineered TIMP2 mutants for MMP-9 was relatively weak. This project aims on further optimizing one of these TIMP-2 mutants to improve its affinity to MMP-9. For this purpose, we designed a focused library of TIMP-2 mutants that randomizes three additional positions on the TIMP2 surface that were chosen based on their potential to increase the affinity toward MMP-9. Using this library, YSD technology was applied to obtain new TIMP-2 mutants with high-affinity toward MMP-9 and high specificity against alternative targets. These mutants will be assayed for their ability to inhibit cancer progression in various cancer cells.