title: Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1.
abstract: Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.
authors.affiliation: Clemens-Schöpf-Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Glotzbach
authors.forename: Michael
authors.initials: M
authors.lastname: Reinwarth
authors.forename: Niklas
authors.initials: N
authors.lastname: Weber
authors.forename: Sebastian
authors.initials: S
authors.lastname: Fabritz
authors.forename: Michael
authors.initials: M
authors.lastname: Tomaszowski
authors.forename: Heiko
authors.initials: H
authors.lastname: Fittler
authors.forename: Andreas
authors.initials: A
authors.lastname: Christmann
authors.forename: Olga
authors.initials: O
authors.lastname: Avrutina
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D002460:Cell Line; D057771:Cystine-Knot Miniproteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: D057771:Cystine-Knot Miniproteins; D004791:Enzyme Inhibitors; D019151:Peptide Library; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases; C485595:matriptase; D014568:Urokinase-Type Plasminogen Activator