LAUSR

MMP‐14 and MMP‐9 are two well‐established cancer targets for which no specific clinically relevant inhibitor is available. Using a powerful combination of computational design and yeast surface display technology, we engineered such an inhibitor starting from a nonspecific MMP inhibitor, N‐TIMP2. The engineered purified N‐TIMP2 variants showed enhanced specificity toward MMP‐14 and MMP‐9 relative to a panel of off‐target MMPs. MMP‐specific N‐TIMP2 sequence signatures were obtained that could be understood from the structural perspective of MMP/N‐TIMP2 interactions. Our MMP‐9 inhibitor exhibited 1000‐fold preference for MMP‐9 vs. MMP‐14, which is likely to translate into significant differences under physiological conditions. Our results provide new insights regarding evolution of promiscuous proteins and optimization strategies for design of inhibitors with single‐target specificities.