Matrix metalloproteinases (MMPs) are zinc‐dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small‐molecule inhibitors of MMPs have remained elusive. In a tour‐de‐force peptide engineering approach combining phage‐display selections, rational design of enhanced zinc chelation, and d‐amino acid screening, we succeeded in developing a first synthetic MMP‐2 inhibitor that combines high potency (Ki=1.9±0.5 nm), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallowsubstrate binding sites,selective,small-molecule inhibitors of MMPs have remained elusive.I natour-de-force peptide engineering approach combining phage-displays elections,rational design of enhanced zinc chelation, and d-amino acid screening,w es ucceeded in developing af irst synthetic MMP-2 inhibitor that combines high potency (K i = 1.9 AE 0.5 nm), high target selectivity,a nd proteolytic stability,a nd thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics.Matrix metalloproteinases (MMPs) are af amily of more than 20 zinc-dependent endopeptidases that play pivotal roles in physiological processes,s uch as morphogenesis,a ngiogenesis,tissue repair, and immunity,aswell as in pathological conditions,such as cancer, arthritis,and inflammation. While much progress has been made in the past few decades in understanding the roles of MMPs using powerful tools,s uch as MMP knockout mice, [1] proteomic approaches, [2,3] and partially selective inhibitors, [4] many important questions remain unanswered, such as the exact contribution of individual MMP proteases to the different physiological and pathologic processes. [5] Ad ifficulty in the study of MMPs is the lack of selective inhibitors for this class of proteases.With hundreds attempts by both industry and academia at designing selective inhibitors,t here is still no selective smallmolecule inhibitor for an MMP family protein except MMP-13. [17] Highly selective MMP inhibitors would first provide an invaluable research tool, and depending on the MMP,potentially offer drug candidates. [6] Peptides represent ap romising underexplored modality for developing small, selective MMP inhibitors because they can interact with extended surfaces and thus bind selectively to protein targets with shallow binding pockets while maintaining molecular weights close to those of small molecules. Thep otential for generating peptide inhibitors with high affinities and target specificities was demonstrated by the MMP-2 inhibitor APP-IP,adecapeptide with the ISYGN-DALMP sequence derived from the amyloid precursor protein (APP) that displays ah igh affinity (IC 50 = 30 nm) and good selectivity for MMP-2. [7] Unfortunately,t he linear peptide has al ow proteolytic stability in cultured cells (t 1/2 = 30 min), [8] preventing its use in cellular assays and in vivo. Genetic fusion of APP-IP to at issue inhibitor of metalloproteinase (TIMP) improved affinity and stability but also substantially increased the size (23 kDa), [8] hindering efficient diffusion into tissues ...