Bivalent Inhibition of -Tryptase: Distance Scan of Neighboring Subunits by Dibasic Inhibitors Norbert Schaschke, a, * Andreas Dominik, b Gabriele Matschiner c and Christian P. Sommerhoff c a Max-Planck-Institut fu ¨r Biochemie, D-82152 Martinsried, Germany b Byk Gulden Lomberg Chemische Fabrik GmbH, D-78467 Konstanz, Germany c Abteilung fu ¨r Klinische Chemie und Klinische Biochemie, Klinikumsstandort Innenstadt der LMU Mu ¨nchen, D-80336 Mu ¨nchen, Germany Received 22 November 2001; revised 14 January 2002; accepted 22 January 2002 Abstract—Based on bifunctional diketopiperazines as templates and m-aminomethyl-phenylalanine as arginine mimetic, we have synthesized a new class of structurally related dibasic tryptase inhibitors with systematically increasing spacer length. These com- pounds were used to scan the distance between the active sites of two neighboring subunits of the b-tryptase tetramer. The K i -values obtained are a function of the distance between the terminal amino groups and indicate optimal binding of inhibitors with 29–31 bonds between the amino groups. These experimental data are in full agreement with predictions derived from a novel modeling program that allows the docking of bivalent ligands. # 2002 Elsevier Science Ltd. All rights reserved. Human b-tryptase is a mast cell-specific serine protease that exhibits trypsin-like activity by hydrolyzing peptide bonds C-terminally of arginine and lysine residues. 1,2 A growing number of biological and immunological data suggests that tryptase plays a key role in the pathogen- esis of diverse allergic and inflammatory disorders, most prominently asthma, 3,4 and thus is an interesting thera- peutic target. 5 In particular, the enzyme acts as a (neu- ro)peptidase and is responsible for enhancing the contractility of the airway smooth muscle. 6 The X-ray structure of human b-tryptase 7,8 revealed that the enzyme consists of four quasi-identical subunits (A, B, C, and D) whose active sites are directed towards a central pore. Therefore, the four negatively charged S1 binding pockets are displayed in a defined spatial arrangement that should allow inhibition of the enzyme by dibasic ligands of appropriate length interacting simultaneously with two neighboring S1 subsites as depicted schematically in Figure 1. Compared to a monovalent binding, the advantage of such a bivalent interaction is the gain in affinity and selectivity achieved by exploiting the entropy effect. 9 For human b-tryptase, a variety of dibasic inhibitors has been described that take advantage of this particular feature of the enzyme. 10 15 In this study, we have systematically investigated the acceptor properties of b-tryptase by performing a dis- tance scan using a set of structurally related dibasic probing compounds. In addition, the ability of the compounds to bind in a mono- or bivalent fashion was analyzed using a novel modeling program that allows the docking of bivalent ligands. Due to entropic reasons, 9 we have focused our scanning approach on the shortest distance between neighboring S1 pockets, that is those of subunits A and D (and their 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00063-X Bioorganic & Medicinal Chemistry Letters 12 (2002) 985–988 Figure 1. Schematic representation of the b-tryptase tetramer inter- acting with a bivalent inhibitor. The inter-S1 subsite distances are indicated and the side-chain carboxylate of Asp-189 at the bottom of each S1 pocket is shown. *Corresponding author. Tel.: +49-89-8578-3910; fax: +49-89-8578- 2847; e-mail: schaschk@biochem.mpg.de