The same moderate mutagenesis scheme was applied for D14 and D16 that are conserved in the ICK family of miniproteins and are involved in stabilization of the oMCoTI scaffold. As the active site of matriptase-1 is negatively charged, it may be beneficial for binding to allow replacement of these residues. Overall, the randomization scheme applied here includes 17 out of 30 residues. However, on average only 6 to 8 of the 17 residues are expected to be changed in each variant and four of these are most likely located within the inhibitor loop. Urokinase-type plasminogen activator causes the degradation of the extracellular BMS 753 matrix and plays a critical role in tumor Eggmanone invasion and metastasis. It was shown that activation of receptor-bound pro-uPA is affected by matriptase-1, which results in a decreased ability of uPAexpressing tumor cells to invade an extracellular matrix layer upon inhibition of membrane-bound matriptase-1. To investigate the inhibitory activity of the newly isolated matriptase-1 inhibitors on pro-uPA activation, a dose-response assay of uPA activity was performed in cell culture with SOTI-based variant and the most potent MCoTIbased inhibitor on human prostate carcinoma cancer cells, as a upregulation of matriptase-1 expression level has been reported for this cell line. For the indirect determination of the IC50 of SOTI Var. 1 and MCoTI Var. 4 on the surface of these cancer cells, the turnover of an uPA substrate was monitored. Pro-uPA is activated through non-inhibited matriptase-1 and substrate turnover was measured and compared to the previously reported small molecule inhibitor S1 of matriptase-1. In this experimental setting, the MCoTI-based inhibitor Var. 4 exhibited an IC50 of 213 nM, while SOTI-III derived inhibitor Var. 1 displayed only minor activity. S1 a small-molecule inhibitor that has been identified recently as potent matriptase-1 inhibitor with an Ki in the single digit nanomolar range was used as reference compound that displayed an tenfold higher IC50 value than MCoTI-based inhibitor Var. 4 in this assay. For control SOTI wt was also applied in this experimental setting at a concentration of 10 mM, displaying no inhibition of either matriptase-1 or uPA. For the isolation of miniprotein-based inhibitors by combinatorial library screening the design of the variant library is a crucial step. We chose a knowledge-based strategy that takes into account the expected contribution to target binding, as well as the natural variability and the contribution to structure and folding of each residue at each position. While we followed a classical variegation scheme for SOTI-III with a full randomization that is restricted to the carboxy terminal loop, a position-specific randomization scheme was applied for oMCoTI-II.