Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities q Ivan T.N. Campos, a Melissa M. Silva, a Simone S. Azzolini, a Adriana F. Souza, a Claudio A.M. Sampaio, a Hans Fritz, b and Aparecida S. Tanaka a, * a Departamento de Bioqu  ımica, UNIFESP-EPM, Rua Tres de Maio 100, S~ ao Paulo SP 04044-020, Brazil b Abteilung f€ ur Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik Innenstadt der Ludwig-Maximilians Universit€ at, M€ unchen, Germany Received 2 February 2004, and in revised form 28 February 2004 Abstract A small combinatorial library of LDTI mutants (5.2  10 4 ) restricted to the P1–P4 0 positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor (K i ¼ 0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor (K i ¼ 1.7 nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2–4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Phage display system; Neutrophil elastase inhibitor; Plasmin inhibitor; Combinatorial library; Kazal-type serine proteinase inhibitor; Filamentous phage Serine proteinases seem to be the most versatile proteolytic enzymes and, together with their inhibitors, regulate a great number of physiological and physio- pathological processes [1]. Numerous serine proteinases have been characterized during the last century and identified as key enzymes in processes such as blood coagulation [2], fibrinolysis [3,4], complement system [5], inflammation [6], and cancer [7–9]. These enzymes are expressed as precursors that are activated by limited proteolysis; generation of the active form is one of the ways to control proteinase activity [10]. Proteolytic ac- tivities can be also restricted by proteinase inhibitors which form a complex with the enzyme thus blocking the active site access to the substrates [1,11]. The fol- lowing serine proteinases: neutrophil elastase, plasmin, and thrombin are important active components in some physiological processes: (i) Elastase stored in the azu- rophilic granules of neutrophils is involved in the elim- ination of pathogens and in connective tissue remodeling [6,12]. a1-Proteinase inhibitor (a1-PI) is the primary physiological inhibitor of human neutrophil elastase [6]. Deficiency in a1-PI is the main risk factor for pulmonary emphysema [13]. (ii) Plasmin is a serine proteinase generated from plasminogen by plasminogen activators; it is the central enzyme in the fibrinolytic q This work was supported by: FAPESP—Proc. 95/9256-8, Proc. 99/09097-8, and CNPq (Brazil); SFB 469 A-3 ÔSonderforschungsber- eich der Ludwig-Maximilians-Universit€ at,Õ M€ unchen, and PROBRAL (Germany). * Corresponding author. Fax: +11-55-11-5572-3006. E-mail address: tanaka.bioq@epm.br (A.S. Tanaka). 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.03.004 Archives of Biochemistry and Biophysics 425 (2004) 87–94 ABB www.elsevier.com/locate/yabbi