title: Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.
abstract: The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.
authors.affiliation: Departamento de Bioquímica, UNIFESP-EPM, São Paulo, Brazil. tanaka.bioq@epm.br
authors.forename: A S
authors.initials: AS
authors.lastname: Tanaka
authors.forename: M M
authors.initials: MM
authors.lastname: Silva
authors.forename: R J
authors.initials: RJ
authors.lastname: Torquato
authors.forename: M A
authors.initials: MA
authors.lastname: Noguti
authors.forename: C A
authors.initials: CA
authors.lastname: Sampaio
authors.forename: H
authors.initials: H
authors.lastname: Fritz
authors.forename: E A
authors.initials: EA
authors.lastname: Auerswald
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry*; D001483:Base Sequence; D053818:Chymases; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019151:Peptide Library; D011506:Proteins / Q000737:chemistry* / Q000493:pharmacokinetics*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000187:drug effects; D053802:Tryptases
chemical_list: D019151:Peptide Library; D011506:Proteins; C090901:leech-derived tryptase inhibitor C; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D013917:Thrombin; D053802:Tryptases