A broad matrix metalloproteinase inhibitor with designed loop extension exhibits ultrahigh specificity for MMP-14

Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. While several antibodies against MMPs are in development, our goal is to construct therapeutic anti-MMP inhibitors based on a natural broad MMP inhibitor, tissue inhibitor of metalloproteinases-2 (N-TIMP2). To confer high binding specificity toward one MMP type, we extend one of the N-TIMP2 loops, allowing it to interact with the non-conserved MMP surface. Multiple computational designs of the loop were used to design a focused library for yeast surface display, which was sorted for high binding to the target MMP-14 and low binding to off-target MMP-3. Deep sequencing of the two selected populations followed by comparative data analysis was used to identify the most promising variants, which were expressed, purified, and tested for inhibition of MMP-14 and off-target MMPs. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, 7500-fold more specific than WT N-TIMP2. Furthermore, the variant inhibited cell invasion with increased potency relative to WT N-TIMP2 in two breast cancer cell lines. We obtained the engineered variant high-accuracy model by including NGS data as input to AlphaFold multiple sequence alignment (MSA). Modeling results together with experimental mutagenesis demonstrate that the loop packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates that introduction of loop extensions into inhibitors to stretch to the non-conserved surface of the target proteins is an attractive strategy for conferring high binding specificity in design of MMP inhibitors and other therapeutic proteins.