Potent inhibitors of a type II transmembrane serine protease

Herein, we report the design, total chemical synthesis and activity profile of new inhibitors of a type II transmembrane serine protease (matriptase/hepsin/corin family) that plays an important role in tumor growth and progression. A structure-based library of potential protease binders was designed using the open-chain McoTI-II miniprotein scaffold as a structural template. The extremely stable scaffold of McoTI-II from the seeds of squash Momordica cochinchinensis comprises approximately 30 residues and is held together by three disulfide bonds forming a cystine knot.^[1] Such a robust architecture allows manipulating the amino acid composition without the loss of structural integrity.^[2] Randomization of amino acid sequences within the inhibitor loop as well as neighboring regions resulted in a library of 2 ∙ 107 miniprotein variants established in S. cerevisiae in a cell surface display format using the aga1p/aga2p system.^{[3, 4, 5]} Four selection rounds of ultra-high throughput screening via FACS using indirectly fluorescently labeled target protein were performed to enrich binders of the type II transmembrane serine protease. After analysis of single clones with respect to their binding properties, the most potent variants were synthesized on a multimilligram scale by microwave-assisted Fmoc-SPPS, followed by oxidative folding. Kinetic assays revealed inhibitory activity of isolated miniproteins, the best of them showing affinity in low-nanomolar range. Molecules of this type may hold great promise for applications in tumor diagnostics and therapy.^[6]