Membrane type-1 matrix metalloproteinase (MT1-MMP) has been shown by many studies to play a critical role in cancer cell invasion and metastasis since it can recognize and digest a wide range of extracellular matrix components and plays a role in activating other MMPs. Since MT1-MMP is a membrane associated protease, it warrants further investigation as a plausible biomarker in therapeutic and diagnostic applications. The objective of this study was to identify MT1-MMP substrates with improved first order rate constants (kcat/KM) by quantitatively screening cellular libraries of peptide substrates (CLiPs) using FACS. The substrate consensus sequence resulting from screening a 5-mer random peptide library was P-X-G↓L. In order to identify substrates with higher kcat/KM, a focused library with the sequence X-X-X-P-X (G/P)-(L/M)-X-X-X was screened under increasingly stringent conditions to identify an extended substrate consensus sequence. These substrates showed a 4-fold higher kcat/KM when compared with previously reported substrates and were not cleaved in human plasma. Finally, the substrates were effectively cleaved when incubated with human tumor cells known to over-express MT1-MMP. These results demonstrate that extended protease substrates can be kinetically optimized for therapeutic and diagnostic applications.

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