Abstract
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.
Highlights
Matrix metalloproteinases (MMPs) contribute to the degradation of extracellular matrix (ECM) proteins and the processing of proinflammatory cytokines (e.g., IL-1β) and chemokines (e.g., IL-8/CXCL8)
As the Fn-like domain directly contacts the catalytic domain [5], it is preferable to use a bait protein that retains the Fn-like domain in order to obtain inhibitors recognizing the native structure of MMP-9
Proteases labeled by random biotinylation using chemical reagents can be inactive due to blocking of the active-site cleft, so we prepared biotinylated pro-MMP-9_Cat with a site-specific biotin acceptor peptide (BAP) [34] using biotin-protein ligase BirA
Summary
Matrix metalloproteinases (MMPs) contribute to the degradation of extracellular matrix (ECM) proteins (e.g., collagen, elastin, fibronectin, and laminin) and the processing of proinflammatory cytokines (e.g., IL-1β) and chemokines (e.g., IL-8/CXCL8). Most of these active-site chemical inhibitors show high potency by chelating the catalytic zinc ion; they show broad inhibitory activity across the whole family of MMPs due to recognition of the highly homologous activesite cleft Such non-specific MMP inhibitors (e.g., hydroxamate-based inhibitors) achieved promising therapeutic effects in several preclinical studies for cancer; in clinical trials, these inhibitors caused serious side effects such as musculoskeletal pain and inflammation due to the broad inhibition of MMPs, so all of these trials failed [3]. Several assays using MMP-9 mutants suggested that these inhibitors recognized the highly conserved active site and the specific exosite of MMP-9 to achieve the high specificity These specific MMP-9 inhibitors discovered from the engineered SPINK2 library may contribute to clarifying the roles of MMP-9 under physiological and pathological conditions and to the development of therapeutics for MMP9-related diseases
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