Engineered Cystine Knot Miniproteins as Potent Inhibitors of Human Mast Cell Tryptase β Christian P. Sommerhoff 1 , Olga Avrutina 2 , Hans-Ulrich Schmoldt 2 , Dusica Gabrijelcic-Geiger 1 , Ulf Diederichsen 3 and Harald Kolmar 2 ⁎ 1 Division of Clinical Chemistry and Clinical Biochemistry, Surgical Department, Ludwig-Maximilians- University Munich, Nußbaumstraße 20, D-80336 Munich, Germany 2 Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstraße 22, D-64287 Darmstadt, Germany 3 Institute of Organic and Biomolecular Chemistry, Georg-August Universität Göttingen, Tammannstraße 2, D-37077 Göttingen, Germany Received 21 August 2009; received in revised form 12 October 2009; accepted 15 October 2009 Available online 21 October 2009 Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase β, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis.A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase β tetramer with an overall equilibrium dissociation constant K i of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase β described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors. © 2009 Elsevier Ltd. All rights reserved. Edited by R. Huber Keywords: human mast cell tryptase; protein inhibitor; knottins; cystine knot miniproteins; macrocyclic peptides Introduction The family of human tryptases comprises a group of sequence homologous serine proteases expressed almost exclusively in mast cells and to a smaller extent in basophils. 1–7 Tryptase β is the prototypic and by far best characterized member of this group, as well as the predominant protease and protein component of mast cells. The activa- tion of mast cells causes the release of tryptase β, which is stored in fully active form in secretory granules, together with other preformed mediators and proteases, such as histamine, chymase, and heparin. Both in vitro and in vivo studies have demonstrated that tryptase β promotes inflamma- tion, matrix destruction, and tissue remodelling by acting on soluble proteins and peptides and on cells via PAR-2-dependent and -independent mechanisms. Tryptase β thus contributes to the pathogenesis of allergic and inflammatory disor- ders, most notably asthma and rheumatoid arthri- tis, and has caused considerable interest in it as a potential drug target. 8–11 Due to its peculiar molecular architecture— human tryptase β is active as a heparin-stabilized tetramer 12–14 (Fig. 1a), the elucidation of inhibitors acting against tryptase β has focused both on *Corresponding author. E-mail address: Kolmar@Biochemie-TUD.de. Abbreviations used: LDTI, leech-derived tryptase inhibitor; TdPI, tick-derived protease inhibitor; EETI-II, Ecballium elaterium trypsin inhibitor II; MCoTI-II, Momordica cochinchinensis trypsin inhibitor II; MCoEETI, linear hybrid of MCoTI-II and EETI-II. doi:10.1016/j.jmb.2009.10.028 J. Mol. Biol. (2010) 395, 167–175 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.