Discovery of potent and specific inhibitors targeting the active site of MMP-9 from the engineered SPINK2 library

• Published in: PloS one Vol. 15; no. 12; p. e0244656
• Main Authors: Yano, Hidenori, Nishimiya, Daisuke, Kawaguchi, Yoshirou, Tamura, Masakazu, Hashimoto, Ryuji
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 29.12.2020; Public Library of Science (PLoS)
• Subjects: Binding sites; Biological products; Biological research; Biology and Life Sciences; Biology, Experimental; Cancer; Catalytic Domain - drug effects; Cell adhesion & migration; Collagen; Drug Discovery; Extracellular matrix; Gelatinase B; Glutamic Acid - metabolism; Health aspects; Humans; Identification and classification; Inflammation; Laboratories; Libraries; Matrix metalloproteinase; Matrix Metalloproteinase 9 - chemistry; Matrix Metalloproteinase 9 - genetics; Matrix Metalloproteinase 9 - metabolism; Matrix Metalloproteinase Inhibitors - chemistry; Matrix Metalloproteinase Inhibitors - pharmacology; Matrix metalloproteinases; Metalloenzymes; Metastasis; Models, Molecular; Mutation; Peptide Library; Peptides; Physical Sciences; Physiological aspects; Physiology; Protease inhibitors; Protein Conformation; Protein engineering; Proteinase inhibitors; Proteins; Proteolysis; Research and Analysis Methods; Residues; Serine; Serine Peptidase Inhibitors, Kazal Type - chemistry; Serine proteinase; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0244656

Matrix metalloproteinases (MMPs) contribute to many physiological and pathological phenomena via the proteolysis of extracellular matrix components. Specific blocking of the active site of each MMP sheds light on its particular role. However, it remains difficult to acquire an active-site inhibitor with high specificity for only the target MMP due to the highly conserved structure around the active site of MMPs. Recently, we reported that potent and specific inhibitors of serine proteases were obtained from our proprietary engineered serine protease inhibitor Kazal type 2 (SPINK2) library. In this research, using this library, we succeeded in obtaining potent and specific MMP-9 inhibitors. The obtained inhibitors bound to the active site of MMP-9 and inhibited MMP-9 with low nanomolar Ki values. The inhibitors did not cross-react with other MMPs that we tested. Further analysis using MMP-9 mutants demonstrated that the inhibitors recognize not only the residues around the conserved active site of MMP-9 but also different and unique residues in exosites that are distant from each other. This unique recognition manner, which can be achieved by the large interface provided by engineered SPINK2, may contribute to the generation of specific active-site inhibitors of MMPs.