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BACKGROUND AND PURPOSE A 3H-labelled derivative of the novel small-molecule bradykinin (BK) B2 receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B2 receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH Equilibrium binding, dissociation and competition studies with various B2 receptor ligands and [3H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B2 receptors of human and animal origin. KEY RESULTS [3H]-JSM10292 bound to B2 receptors at 4°C and at 37°C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B2 receptor agonists or antagonists were added. [3H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B2 receptors, as indicated by the different 'nonspecific' binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [3H]-JSM10292 and competition experiments with unlabelled JSM10292 and [3H]-BK showed a different affinity profile for the wild-type B2 receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B2 receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B2 receptor ligand. CONCLUSIONS AND IMPLICATIONS [3H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B2 receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B2 receptors. [ABSTRACT FROM AUTHOR]

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability in vivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Blockade of the bradykinin B(2) receptor provides therapeutic benefit in hereditary angioedema (HAE) and potentially in many other diseases. Herein, we describe the development of highly potent B(2) receptor antagonists with a molecular weight of approximately 500 g/mol. First, known quinoline-based B(2) receptor antagonists were stripped down to their shared core motif 53, which turned out to be the minimum pharmacophore. Targeted modifications of 53 resulted in the highly water-soluble lead compound 8a. Extensive exploration of its structure-activity relationship resulted in a series of highly potent B(2) receptor antagonists, featuring a hydrogen bond accepting functionality, which presumably interacts with the side chain of Asn-107 of the B(2) receptor. Optimization of the microsomal stability and cytochrome P450 inhibition eventually led to the discovery of the highly potent and orally available B(2) receptor antagonist 52e (JSM10292), which showed the best overall properties.

The authors have developed a class of potent inhibitors against the phosphate specific prolyl isomerase hPin1, which induced apoptosis in transformed cell lines.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

Polypeptides often display proline-mediated conformational substates that are prone to isomer-specific recognition and function. Both possibilities can be of biological significance. Distinct families of peptidyl prolyl cis/trans isomerases (PPIases) evolved proved to be highly specific for proline moieties arranged in a special context of subsites. Structural and chemical features of molecules specifically bound to the active site of PPIases served to improve catalysis of prolyl isomerization rather than ground state binding. For example, results inferred from receptor Ser/Thr or Tyr phosphorylation in the presence of site-directed FKBP12 mutant proteins provided evidence for the crucial role of the enzymatic activity in downregulating function of FKBP12.

The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans isomerase (PPIase) which catalyzes proline-limited protein folding extremely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments which encompass individual domains or combinations of the middle FKBP-like domain (M) with the N-terminal (N) and C-terminal (C) regions, respectively. These fragments appear to be stably folded. They show ordered structure and cooperative urea-induced unfolding transitions, and the far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the native trigger factor shows a modular structure, which is composed of three fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activity in protein folding could not be restored by fusing alternatively the N or the C-terminal regions to the catalytic domain (in NM and MC constructs, respectively). Surprisingly, the high folding activity of the intact trigger factor has been regained partially by functional complementation of the overlapping NM and MC constructs., (Copyright 1997 Academic Press Limited.)

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.