The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
Hans Fritz - One of the best experts on this subject based on the ideXlab platform.
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evaluation of phage display system and leech derived Tryptase Inhibitor as a tool for understanding the serine proteinase specificities
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ivan T N Campos, Hans Fritz, Melissa Andreia De Moraes Silva, Claudio A. M. Sampaio, Simone S Azzolini, Adriana F Souza, Aparecida S. TanakaAbstract:Abstract A small combinatorial library of LDTI mutants (5.2 × 10 4 ) restricted to the P1–P4 ′ 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 =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin Inhibitor ( K i =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.
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Evaluation of phage display system and leech-derived Tryptase Inhibitor as a tool for understanding the serine proteinase specificities.
Archives of biochemistry and biophysics, 2004Co-Authors: Ivan T N Campos, Hans Fritz, Melissa Andreia De Moraes Silva, Claudio A. M. Sampaio, Simone S Azzolini, Adriana F Souza, Aparecida S. TanakaAbstract:A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' 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) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin Inhibitor ( K(i) =1.7nM) 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.
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Functional phage display of leech-derived Tryptase Inhibitor (LDTI): construction of a library and selection of thrombin Inhibitors
FEBS letters, 1999Co-Authors: Aparecida S. Tanaka, Hans Fritz, Melissa Andreia De Moraes Silva, Ricardo J.s. Torquato, Maria Aparecida Eiko Noguti, Claudio A. M. Sampaio, Ennes A. AuerswaldAbstract: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 5.2×104 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.
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The Three-dimensional Structure of Recombinant Leech-derived Tryptase Inhibitor in Complex with Trypsin IMPLICATIONS FOR THE STRUCTURE OF HUMAN MAST CELL Tryptase AND ITS INHIBITION
Journal of Biological Chemistry, 1997Co-Authors: Milton T. Stubbs, Robert Morenweiser, Gerd Paul Piechottka, Gabriele Matschiner, Margit Bauer, Christian Petro Sommerhoff, Wolfram Bode, Jörg Stürzebecher, Robert Huber, Hans FritzAbstract:Abstract The x-ray crystal structure of recombinant leech-derived Tryptase Inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type Inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase Tryptase. The conformation of the binding region of rLDTI suggests that Tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in Tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the Tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung Tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight Inhibitors of Tryptase.
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Structure-based Design of a Potent Chimeric Thrombin Inhibitor
The Journal of biological chemistry, 1997Co-Authors: Robert Morenweiser, Hans Fritz, Jörg Stürzebecher, Ennes A. Auerswald, A. Van De Locht, Milton T. StubbsAbstract:Abstract Using the three-dimensional structures of thrombin and the leech-derived Tryptase Inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the Inhibitor reactive site loop to fit thrombin’s narrow active site cleft resulted in inhibition constants (K i) in the 10 nmconcentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin’s tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (K i in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best Inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.
Aparecida S. Tanaka - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of phage display system and leech-derived Tryptase Inhibitor as a tool for understanding the serine proteinase specificities.
Archives of biochemistry and biophysics, 2004Co-Authors: Ivan T N Campos, Hans Fritz, Melissa Andreia De Moraes Silva, Claudio A. M. Sampaio, Simone S Azzolini, Adriana F Souza, Aparecida S. TanakaAbstract:A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' 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) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin Inhibitor ( K(i) =1.7nM) 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.
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evaluation of phage display system and leech derived Tryptase Inhibitor as a tool for understanding the serine proteinase specificities
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ivan T N Campos, Hans Fritz, Melissa Andreia De Moraes Silva, Claudio A. M. Sampaio, Simone S Azzolini, Adriana F Souza, Aparecida S. TanakaAbstract:Abstract A small combinatorial library of LDTI mutants (5.2 × 10 4 ) restricted to the P1–P4 ′ 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 =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin Inhibitor ( K i =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.
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purification and characterization of a trypsin like enzyme with fibrinolytic activity present in the abdomen of horn fly haematobia irritans irritans diptera muscidae
Journal of Protein Chemistry, 2000Co-Authors: M Dametto, Simone S Azzolini, A P David, I T N Campos, A M Tanaka, A Gomes, Renato Andreotti, Aparecida S. TanakaAbstract:This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase Inhibitors such as SBTI (soybean trypsin Inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa Inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived Tryptase Inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective Inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease Inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.
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Functional phage display of leech-derived Tryptase Inhibitor (LDTI): construction of a library and selection of thrombin Inhibitors
FEBS letters, 1999Co-Authors: Aparecida S. Tanaka, Hans Fritz, Melissa Andreia De Moraes Silva, Ricardo J.s. Torquato, Maria Aparecida Eiko Noguti, Claudio A. M. Sampaio, Ennes A. AuerswaldAbstract: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 5.2×104 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.
John P. Priestle - One of the best experts on this subject based on the ideXlab platform.
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Structure of the complex of leech-derived Tryptase Inhibitor (LDTI) with trypsin and modeling of the LDTI–Tryptase system
Structure, 1997Co-Authors: Stefania De Marco, John P. PriestleAbstract:Abstract Background: Tryptase is a trypsin-like serine proteinase stored in the cytoplasmic granules of mast cells, which has been implicated in a number of mast cell related disorders such as asthma and rheumatoid arthritis. Unlike almost all other serine proteinases, Tryptase is fully active in plasma and in the extracellular space, as there are no known natural Inhibitors of Tryptase in humans. Leech-derived Tryptase Inhibitor (LDTI), a protein of 46 amino acids, is the first molecule found to bind tightly to and specifically inhibit human Tryptase in the nanomolar range. LDTI also inhibits trypsin and chymotrypsin with similar affinities. The structure of LDTI in complex with an inhibited proteinase could be used as a template for the development of low molecular weight Tryptase Inhibitors. Results: The crystal structure of the complex between trypsin and LDTI was solved at 2.0 A resolution and a model of the LDTI–Tryptase complex was created, based on this X-ray structure. LDTI has a very similar fold to the third domain of the turkey ovomucoid Inhibitor. LDTI interacts with trypsin almost exclusively through its binding loop (residues 3–10) and especially through the sidechain of the specificity residue Lys8. Our modeling studies indicate that these interactions are maintained in the LDTI–Tryptase complex. Conclusions: The insertion of nine residues after residue 174 in Tryptase, relative to trypsin and chymotrypsin, prevents inhibition by other trypsin Inhibitors and is certainly responsible for the higher specificity of Tryptase relative to trypsin. In LDTI, the disulfide bond between residues 4 and 25 causes a sharp turn from the binding loop towards the N terminus, holding the N terminus away from the 174 loop of Tryptase.
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structure of the complex of leech derived Tryptase Inhibitor ldti with trypsin and modeling of the ldti Tryptase system
Structure, 1997Co-Authors: Stefania De Marco, John P. PriestleAbstract:Abstract Background: Tryptase is a trypsin-like serine proteinase stored in the cytoplasmic granules of mast cells, which has been implicated in a number of mast cell related disorders such as asthma and rheumatoid arthritis. Unlike almost all other serine proteinases, Tryptase is fully active in plasma and in the extracellular space, as there are no known natural Inhibitors of Tryptase in humans. Leech-derived Tryptase Inhibitor (LDTI), a protein of 46 amino acids, is the first molecule found to bind tightly to and specifically inhibit human Tryptase in the nanomolar range. LDTI also inhibits trypsin and chymotrypsin with similar affinities. The structure of LDTI in complex with an inhibited proteinase could be used as a template for the development of low molecular weight Tryptase Inhibitors. Results: The crystal structure of the complex between trypsin and LDTI was solved at 2.0 A resolution and a model of the LDTI–Tryptase complex was created, based on this X-ray structure. LDTI has a very similar fold to the third domain of the turkey ovomucoid Inhibitor. LDTI interacts with trypsin almost exclusively through its binding loop (residues 3–10) and especially through the sidechain of the specificity residue Lys8. Our modeling studies indicate that these interactions are maintained in the LDTI–Tryptase complex. Conclusions: The insertion of nine residues after residue 174 in Tryptase, relative to trypsin and chymotrypsin, prevents inhibition by other trypsin Inhibitors and is certainly responsible for the higher specificity of Tryptase relative to trypsin. In LDTI, the disulfide bond between residues 4 and 25 causes a sharp turn from the binding loop towards the N terminus, holding the N terminus away from the 174 loop of Tryptase.
Ennes A. Auerswald - One of the best experts on this subject based on the ideXlab platform.
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Functional phage display of leech-derived Tryptase Inhibitor (LDTI): construction of a library and selection of thrombin Inhibitors
FEBS letters, 1999Co-Authors: Aparecida S. Tanaka, Hans Fritz, Melissa Andreia De Moraes Silva, Ricardo J.s. Torquato, Maria Aparecida Eiko Noguti, Claudio A. M. Sampaio, Ennes A. AuerswaldAbstract: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 5.2×104 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.
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Structure-based Design of a Potent Chimeric Thrombin Inhibitor
The Journal of biological chemistry, 1997Co-Authors: Robert Morenweiser, Hans Fritz, Jörg Stürzebecher, Ennes A. Auerswald, A. Van De Locht, Milton T. StubbsAbstract:Abstract Using the three-dimensional structures of thrombin and the leech-derived Tryptase Inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the Inhibitor reactive site loop to fit thrombin’s narrow active site cleft resulted in inhibition constants (K i) in the 10 nmconcentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin’s tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (K i in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best Inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.
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Structure of leech derived Tryptase Inhibitor (LDTI-C) in solution
FEBS letters, 1994Co-Authors: Peter Mühlhahn, Robert Morenweiser, Christian Petro Sommerhoff, Michael Czisch, Bianca Habermann, Richard A. Engh, Ennes A. Auerswald, Tad A. HolakAbstract:The three-dimensional solution structure of the leech derived Tryptase Inhibitor form C (LDTI-C), an Inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 φ angles, supplemented by 3 ϕ and 15χ1 angles. The core of LDTI-C is very well defined and consists of a short 310-helix-loop and a short two-stranded antiparallel β-sheet between residues 13–14 and 20–21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the β-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase Inhibitors.
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a kazal type Inhibitor of human mast cell Tryptase isolation from the medical leech hirudo medicinalis characterization and sequence analysis
Biological chemistry Hoppe-Seyler, 1994Co-Authors: Christian P. Sommerhoff, Gerd Paul Piechottka, Ennes A. Auerswald, Christa Sollner, Reinhardt Mentele, Hans FritzAbstract:Human Tryptase, a tetrameric proteinase expressed by mast cells, is virtually unique among the serine proteinases as it is not inhibited by any proteinaceous Inhibitor tested so far. We have now isolated, sequenced, and characterized an Inhibitor of human Tryptase from the medical leech Hirudo medicinalis. LDTI (Leech-Derived Tryptase Inhibitor) was purified to apparent homogeneity by cation exchange and affinity chromatography. Amino acid sequencing of the protein consisting of 46 residues (M(r) 4738) revealed a high degree of similarity to the non-classical Kazal-type Inhibitors bdellin B-3 and rhodniin, Inhibitors isolated from the medical leech and the insect Rhodnius prolixus, respectively. LDTI is a tight-binding and relatively specific Inhibitor of human Tryptase; it inhibits only trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with similar affinities. Inhibition studies using small chromogenic substrates revealed that LDTI inhibits the amidolytic activity of Tryptase by approximately 50%, suggesting that most likely due to steric hindrance LDTI binds to and inhibits only 2 of 4 active sites of Tryptase. LDTI appears useful as a prototype of Inhibitors of human Tryptase and as a pharmacological tool for the investigation of the role of Tryptase in health and disease.
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Recombinant leech-derived Tryptase Inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.
Biological chemistry Hoppe-Seyler, 1994Co-Authors: Ennes A. Auerswald, Robert Morenweiser, Gerd Paul Piechottka, Christian Petro Sommerhoff, Christoph Eckerskorn, Lutz G. Gürtler, Hans FritzAbstract:A synthetic gene coding for leech-derived Tryptase Inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant Inhibitor is typical for a small folded protein. rLDTI-C is Inhibitorily fully active, its complexes with bovine trypsin and human mast cell Tryptase display equilibrium dissociation constants which are nearly identical to those with the natural Inhibitor. Remarkably, the Inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.
William M Abraham - One of the best experts on this subject based on the ideXlab platform.
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potent small molecule Inhibitors of human mast cell Tryptase antiasthmatic action of a dipeptide based transition state analogue containing a benzothiazole ketone
Journal of Medicinal Chemistry, 2003Co-Authors: Michael J. Costanzo, Stephen C. Yabut, Lawrence De Garavilla, William M Abraham, Harold R Almond, Patricia Andradegordon, Thomas W Corcoran, Jack A Kauffman, Rosario Recacha, And Debashish ChattopadhyayAbstract:Inhibitors of human mast cell Tryptase (EC 3.4.21.59) have therapeutic potential for treating allergic or inflammatory disorders. We have investigated transition-state mimetics possessing a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6 (RWJ-56423) as a potent, reversible, low-molecular-weight Tryptase Inhibitor with a Ki value of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the stereochemistry. Analogues 12 and 15−17 are also potent Tryptase Inhibitors. Although RWJ-56423 potently inhibits trypsin (Ki = 8.1 nM), it is selective vs other serine proteases, such as kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with bovine trypsin (1.9-A resolution), which depicts inter alia a hemiketal involving Ser-189, and hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ-58643) to allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70−75% blockad...
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Inhibition of allergen-induced pulmonary responses by the selective Tryptase Inhibitor 1,5-bis-{4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-phenoxy}-pentane (AMG-126737)
Biochemical pharmacology, 1999Co-Authors: Clifford D. Wright, Mohammad Abul Kashem, Scot Middleton, Andy M. Havill, David Dripps, William M Abraham, David S Thomson, Laurence E BurgessAbstract:Abstract Emerging evidence suggests that mast cell Tryptase is a therapeutic target for the treatment of asthma. The effects of this serine protease are associated with both pathophysiologic pulmonary responses and pathologic changes of the asthmatic airway. In this study, the Tryptase Inhibitor 1,5-bis-{4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-phenoxy}-pentane (AMG-126737) was evaluated for its pharmacologic effects against allergen-induced airway responses. AMG-126737 is a potent Inhibitor of human lung mast cell Tryptase ( K i = 90 nM), with greater than 10- to 200-fold selectivity versus other serine proteases. Intratracheal administration of AMG-126737 inhibited the development of airway hyperresponsiveness in allergen-challenged guinea pigs with an ed 50 of 0.015 mg/kg. In addition, the compound exhibited oral activity in the guinea pig model. The in vivo activity of AMG-126737 was confirmed in a sheep model of allergen-induced airway responses, where the compound inhibited early and late phase bronchoconstriction responses and the development of airway hyperresponsiveness. These results support the proposed role of Tryptase in the pathology of asthma and suggest that AMG-126737 has potential therapeutic utility in this pulmonary disorder.
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Lactoferrin, a Potent Tryptase Inhibitor, Abolishes Late-Phase Airway Responses in Allergic Sheep
American Journal of Respiratory and Critical Care Medicine, 1997Co-Authors: Kyle C. Elrod, William M Abraham, William R. Moore, Richard D. TanakaAbstract:Tryptase, a serine protease released exclusively from activated mast cells, has been implicated as a potential causative agent in asthma. Enzymatically active Tryptase is comprised of four subunits, and heparin stabilizes the associated tetramer. Lactoferrin, a cationic protein released from activated neutrophils, binds tightly to heparin, therefore we investigated lactoferrin as an Inhibitor of Tryptase and found that it is both a potent (Ki' is 24 nM) and selective Inhibitor. Size exclusion chromatography studies revealed that lactoferrin disrupted the quaternary structure of active Tryptase. Lactoferrin was tested in an allergic sheep model of asthma; aerosolized lactoferrin (10 mg in 3 ml phosphate-buffered saline, 0.5 h before as well as 4 and 24 h after inhalation challenge by Ascaris suum) abolished both late-phase bronchoconstriction (no significant increase in specific lung resistance 4 to 8 h following provocation, p < 0.05 versus vehicle treatment) and airway hyperresponsiveness (no detectable increase in airway sensitivity to carbachol challenge 24 h after antigen challenge, p < 0.05 versus vehicle). These data suggest Tryptase involvement in both late-phase bronchoconstriction and airway hyperreactivity and furthermore suggest that a physiological function of neutrophil lactoferrin is the inhibition of Tryptase released from mast cells.
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inhaled Tryptase causes bronchoconstriction in sheep via histamine release
American Journal of Respiratory and Critical Care Medicine, 1996Co-Authors: Jussara F. Molinari, William R. Moore, Richard D. Tanaka, Mario Scuri, James M Clark, William M AbrahamAbstract:Allergen-induced bronchoconstriction involves mast cell activation. Tryptase is a mast cell serine protease that is released during this process, but little is known about the action of Tryptase in the airway. The purpose of this study was to determine: (1) if aerosolized Tryptase causes bronchoconstriction, and (2) the mechanism by which this occurs. We measured mean pulmonary flow resistance (RL) in five allergic sheep before and after consecutive inhalations of 100 and 500 ng Tryptase (in 2 ml total volume). Inhaled Tryptase at 100 and 500 ng increased RL (mean +/- SE) by 33 +/- 12 and 122 +/- 8% (p < 0.05) over baseline. The response was reproducible upon repeat challenges. These studies were repeated in the same animals after pretreatment with aerosolized APC 366 (9 mg/3 ml), a specific Tryptase Inhibitor. In APC-366-treated sheep, Tryptase increased RL by 10 +/- 3 and 6 +/- 2% (p < 0.05 versus control values) at 100 and 500 ng, respectively. The response to Tryptase was also blocked by pretreating the sheep intravenously with the histamine H1-antagonist chlorpheniramine (2 mg/kg), in which RL increased only 5 +/- 4 and 7 +/- 6% after 100 and 500 ng Tryptase. APC 366, however, did not block histamine-induced bronchoconstriction. Consistent with these findings was the observation that segmental bronchial challenge with Tryptase (1 microgram) resulted in a significant increase in histamine levels in bronchoalveolar lavage. Inhaled Tryptase (500 ng) also caused airway hyperresponsiveness to aerosolized carbachol 2 h after Tryptase challenge. This Tryptase-induced airway hyperresponsiveness could be blocked either by pretreating the sheep with APC 366 (30 min before challenge) or by treating the sheep 30 min after challenge. These results indicate that inhaled Tryptase causes bronchoconstriction and airway hyperresponsiveness in allergic sheep by an event that may involve mast cell activation.
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Role of Tryptase in immediate cutaneous responses in allergic sheep
Journal of applied physiology (Bethesda Md. : 1985), 1995Co-Authors: Jussara F. Molinari, W.r. Moore, J. Clark, R. Tanaka, J. H. Butterfield, William M AbrahamAbstract:In this study, we used a specific Tryptase Inhibitor, APC-366 [N-(1-hydroxy-2-napthoyl)-L-arginyl-L- prolinamide hydrochloride] to investigate the effect of intradermally administered Tryptase and ...
