The bradykinin B2 receptor antagonist icatibant (Hoe 140) blocks aminopeptidase N at micromolar concentrations: off-target alterations of signaling mediated by the bradykinin B1 and angiotensin receptors

The N-terminal sequence of icatibant, a widely used peptide antagonist of the bradykinin B(2) receptors, is analogous to that of other known aminopeptidase N inhibitors. Icatibant competitively inhibited the hydrolysis of L-Ala-p-nitroanilide by recombinant aminopeptidase N (K(i) 9.1 microM). In the rabbit aorta, icatibant (10-30 microM) potentiated angiotensin III, but not angiotensin II (contraction mediated by angiotensin AT(1) receptors), and Lys-des-Arg(9)-bradykinin, but not des-Arg(9)-bradykinin (effects mediated by the bradykinin B(1) receptors), consistent with the known susceptibility of these agonists to aminopeptidase N. At concentrations possibly reached in vivo (e.g., in kidneys), icatibant alters physiological systems different from bradykinin B(2) receptors.     

New selective bradykinin receptor antagonists and bradykinin B2 receptor characterization

Substantial progress has been made recently in the field of kinin pharmacology with the identification of sensitive bioassay organs and the discovery of bradykinin B2 receptor antagonists. Data obtained with such compounds in various laboratories support the hypothesis that kinins act on multiple (at least two) receptor types. Domenico Regoli and colleagues review here the basic criteria of receptor characterization as they apply to kinins and present a critical analysis of the bioassay organs and B2 receptor antagonists currently used in kinin pharmacology.     

Pharmacological and functional characterization of bradykinin B2 receptor in human prostate

The objective of this study was to pharmacologically characterize bradykinin receptors, a component of the kallikrein-kinin system, in normal human prostate cells. In primary cultured human prostate stromal cells, bradykinin, but not [des-Arg9]bradykinin or [des-Arg10]kallidin, produced calcium mobilization or inositol phosphates accumulation with potencies (pEC50) of 8.8+/-0.2 and 8.2+/-0.2, respectively. This was consistent with abundance of bradykinin B2 mRNA over bradykinin B1 mRNA in prostate stromal cells. Although the prostate epithelial cells (prostate epithelium, BPH-1, and PC-3) expressed mRNA for bradykinin B2 receptors (albeit in lesser amounts than stromal cells), bradykinin was not functionally efficacious in the epithelial cells. Increasing concentrations of D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahhydro-3-isoquinolinecarbonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE-140), a bradykinin B2-selective peptide antagonist, attenuated bradykinin concentration-response curves in human prostate stromal cells with apparent estimate of affinity similar to that for the human bradykinin B2 receptor. Bradykinin (10 nM) caused proliferation of prostate stromal cells and phosphorylated extracellular signal-regulated kinases (ERK-1 and ERK-2) that were blocked by HOE-140 (1 microM). This study demonstrated that, in primary cultures of normal human prostate stromal cells, bradykinin activates bradykinin B2 receptors that may play a significant role in proliferation via activation of ERK-1/2 pathways.     

Nonpeptide bradykinin B2 receptor antagonists: conversion of rodent-selective bradyzide analogues into potent,orally-active human bradykinin B2 receptor antagonists

The 1-(2-nitrophenyl)thiosemicarbazide (TSC) derivative, (S)-1-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl]pyrrolidine-2-carboxylic acid [2-[(2-dimethylaminoethyl)methylamino]ethyl]amide (bradyzide; (S)-4), was recently disclosed as a novel, potent, orally active nonpeptide bradykinin (BK) B2 receptor antagonist. The compound inhibited the specific binding of [3H]BK to NG108-15 cell membrane preparations (rodent neuroblastoma-glioma) expressing B2 receptors with a K(i) of 0.5 +/- 0.2 nM. Compound (S)-4 also demonstrated oral efficacy against Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in rats with an ED50 value of 0.84 micromol/kg. After we optimized the terminal binding determinants projecting from the TSC framework, we found that it was possible to replace the potentially toxicophoric nitro and divalent sulfur moieties with only a 15-fold loss in binding affinity ((S)-14a). However, bradyzide and its congeners were found to have much lower affinities for cloned human B2 receptors, expressed in Cos-7 cells. The hitherto synthesized TSC series was screened against the human B2 receptor, and the dibenzosuberane (DBS) pharmacophore emerged as the key structural requirement for potency. Incorporation of this group resulted in a series of derivatives ((S)-14d,e and 19b-d) with K(i) ranges of 10.7-176 nM in NG108-15 cells (expressing the rodent B2 receptor) and 0.79-253 nM in Cos-7 cells (expressing the human B2 receptor). There was no evidence of agonist activity with any of the nonpeptides in any of the cell lines tested. In vivo, oral administration of compound 19c reversed FCA-induced and turpentine-induced mechanical hyperalgesia in rodents with ED50 values of 0.027 and 0.32 micromol/kg, respectively. The selectivity profiles of compounds (S)-14f and (S)-14g were also assessed to determine the conformational and/or steric preferences of the double-ring arrangement. The affinity of (S)-14 g for the human B2 receptor suggested that it may be a hydrophobic interaction with the ethane bridge of the DBS moiety that accounts for the increased potency of compounds (S)-14d,e and 19b,c at this receptor, by favoring a binding mode inaccessible to the unsubstituted diphenylmethyl derivative, (S)-4.     

Aspirin inhibits human bradykinin B2 receptor ligand binding function

The bradykinin B2 receptor, a member of the G protein-coupled receptors superfamily, is involved in a variety of physiological functions, including vasodilation, electrolyte transfer in epithelia, mediation of pain, and inflammation. The effect of aspirin on bradykinin binding to cell-surface receptor and on signal transduction were studied in CHO-K1 cells, stably expressing the human B2 receptor. Cell-surface organization of the receptor was assessed by immunoprecipitation and Western blot analysis in CHO-K1 cells expressing N-terminally V5-tagged B2 receptor. We found that the widely used analgesic, anti-thrombotic, and anti-inflammatory drug aspirin alters the B2 receptor ligand binding properties. Aspirin reduces the apparent affinity of the receptor for [3H]-bradykinin by accelerating the dissociation rate of [3H]-bradykinin-receptor complexes. In addition, aspirin reduces the capacity of unlabeled bradykinin or the B2 receptor antagonist icatibant to destabilize pre-formed [3H]-bradykinin-receptor complexes. Kinetic and reversibility studies are consistent with an allosteric type of mechanism. Aspirin effect on B2 receptor binding properties is not accompanied by alteration of the cell-surface organization of the receptor in dimers and monomers. Aspirin does not influence the receptor ability to transduce bradykinin binding into activation of G-proteins and phospholipase C. These results suggest that aspirin is an allosteric inhibitor of the B2 receptor, a property that may be involved in its therapeutic actions.     

Functional studies of bradykinin receptors in Chinese hamster ovary cells stably expressing the human B2 bradykinin receptor

Bradykinin B1 and B2 receptors, members of the G-protein coupled receptor superfamily, are involved in inflammation and pain. Chinese hamster ovary (CHO) cells stably expressing the human B2 bradykinin receptor (CHO-B2) were used to characterize the signal transduction pathways associated with this receptor and its regulation. The selective B2 antagonist [3H]NPC17731 but not the selective B1 antagonist [3,4-prolyl-3,4-(3)H(N)]-[des-Arg10,Leu9]kallidin ([3H]DALKD) bound to CHO-B2 cell membranes with a Kd of 0.77 nM and a Bmax of 1087 fmol/mg protein. [3H]NPC17731 binding was inhibited by bradykinin ligands in the order: NPC17731 > bradykinin > kallidin > DALKD > [des-Arg10] kallidin (DAKD), consistent with the pharmacological profile of B2 bradykinin receptors. The B2 agonist bradykinin and the B1/B2 agonist kallidin, but not the B1 agonist DAKD, increased [35S]GTP gamma S binding to the CHO-B2 cell membranes. The B2 bradykinin receptors were co-immunoprecipitated with G alpha q/11. In response to bradykinin stimulation, coupling of the B2 receptors to G alpha q/11 was increased by 10-fold. Bradykinin and kallidin, but not DAKD, induced intracellular calcium release in CHO-B2 cells, which was blocked by NPC17731 but not by DALKD. These results demonstrate that B2 bradykinin receptors directly coupled to G alpha q/11 to regulate intracellular calcium release. CHO-B2 cell is a useful system that can be applied to study the effect of potential agents that may influence the B2 receptor function.     

From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists

The bradykinin (BK) B1 receptor is an attractive target for the treatment of chronic pain and inflammation. Starting from a dual B1 and B2 antagonist, novel antagonists were designed that display low-nanomolar affinity for human B1 receptor and selectivity over B2. Initially, potent imidazoline derivatives were studied, but these compounds suffered from low bioavailability. This issue could be overcome by the use of less basic amino derivatives leading to orally active compounds.     

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts

Background and purpose:

Bradykinin (BK) and B₂ receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B₂ receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells.

Experimental approach:

Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes.

Key results:

[³H]-BK saturation studies indicated receptor density (B_max) and K_d values of 121 550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK_i 8.9) was threefold more potent than icatibant (pK_i 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC₅₀ 0.45 nM), with pK_B values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC₅₀ 216 nM) and IL-8 (EC₅₀ 53 nM) release. Both MEN16132 (IL-6: pIC₅₀ 8.1; IL-8: pIC₅₀ 8.4) and icatibant (IL-6: pIC₅₀ 6.6; IL-8: pIC₅₀ 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002), NF-κb (BAY-117085) and by the glucocorticoid dexamethasone.

Conclusions and implications:

Bradykinin via B₂ receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B₂ receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.     

Two B1 and B2 bradykinin receptor antagonists fail to inhibit the Ca2+ response elicited by bradykinin in human skin fibroblasts

The elevation of intracellular [Ca2+] induced by bradykinin (Bk) was monitored with fura-2 fluorescence in human skin fibroblasts. Neither [des-Arg10][Leu9]kallidin nor D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE140) inhibited the Ca2+ response stimulated by Bk. Moreover, each behaved as a partial agonist causing the elevation of intracellular [Ca2+].     

Intramolecular signal transduction by the bradykinin B2 receptor

To study the intramolecular signal transduction, we performed single point and cassette mutations in transmembranal and intracellular regions of the bradykinin B2 receptor. We studied the influence of the two intramembranal Cys residues at positions 304 and 348, the role of Arg at position 177 in the highly conserved tripeptide sequence Asp-Arg-Tyr, the cytosolic G-protein binding area, and attempted to verify the general hypothesis of an ion tunnel-like interface in GPCRs. Wild type receptor, His-tagged receptor, and His-tagged mutant receptors were expressed in COS-7 cells and functionally compared by bradykinin-induced formation of inositolphosphate and arachidonic acid. To investigate the expression, all mutants were modified at the N-terminus by insertion of two successive His-tags and detected with an anti-poly-His antibody. Replacement of the second and third cytosolic loop by a loop from another membrane protein as well as single replacement of Arg at position 177 by Ala leads to a fully inactive receptor mutant without any ligand binding affinity and stimulatory activity. Mutants with replacement of Cys residues 304 and 348 by Ser showed only moderate effects. Regardless of the replacement of Asp 407 by Ala, the receptor is able to increase the agonist-induced levels of inositolphosphate and of arachidonic acid, indicating that our studies can not verify the postulated ion tunnel hypothesis.     

Characterization of bradykinin B(2) receptor antagonists in human and rat urinary bladder

The effect of three selective bradykinin B(2) receptor antagonists, MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-1 0alpha)), Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH), and FR173567 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2, 4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylaminocarbonylmethyl]acrylamide) was evaluated in the human and rat urinary bladder in vitro and in vivo in anaesthetized rats. Bradykinin evoked a concentration-dependent contraction of human (pD(2)=7.2) and rat (pD(2)=7.7) detrusor muscle strips. In human preparations, all the antagonists tested produced a rightward-shift in the concentration-response curve for bradykinin. Schild plot analysis yielded pK(B) values of 8.4, 8.4 and 8.6 for MEN11270, Icatibant, and FR173567, respectively. In the rat preparations the three antagonists (at 100 nM concentration), produced a shift to the right which gave apparent pA(2) values of 8. 2, 8.0 and 8.1 for MEN11270, Icatibant, and FR173567, respectively. In anaesthetized rats, both MEN11270 and Icatibant (1-10 nmol/kg i.v. ) dose dependently reduced the bradykinin (100 nmol/kg i.v.)-induced urinary bladder contraction, their effect being prompt and long-lasting. In contrast, FR173567 (100 nmol/kg i.v.) produced a partial and short-lasting inhibition of bradykinin-induced bladder contractions. The present findings indicate that all the antagonists tested recognize with similar potencies the bradykinin B(2) receptors expressed in the detrusor muscle of both humans and rats. MEN11270 and Icatibant possess a higher potency and longer duration of action in vivo than FR173657, suggesting that the activity of this non-peptide antagonist in vivo is hampered by factors unrelated to its affinity for bradykinin B(2) receptors.     

A non-peptide antagonist unusually selective for the human form of the bradykinin B2 receptor

Analgesic and anti-inflammatory applications for non-peptide bradykinin (BK) B2 receptor antagonists have been documented in rats. However, very large species differences in affinity were also noted within this class of drugs, making the preclinical development of relevant drugs difficult. Bradyzide is a potent antagonist at the rat B2 receptor, but a weak one at the human receptor; a series of analogues in which the diphenylmethyl moiety of this drug has been substituted with dibenzosuberane have been reported to gain potency at the human B2 receptor, with some loss of affinity at the rat receptor. The present experiments have been performed in order to verify that the novel series of dibenzosuberane B2 receptor antagonist optimized for affinity in the human species are effective in the isolated human umbilical vein contractility assay. Bradyzide, its analog compound (S)-14c and the dibenzosuberane compounds (S)-14d and 19c surmountably antagonized BK-induced contraction (pA2 values of 5.42, 6.48, 7.42 and 7.53, respectively). In the rabbit jugular vein contractility assay, the pA2 of compound 19c was smaller than 5. Potency at the recombinant rabbit B2 receptor was generally decreasing in the series of four drugs (Ki in a [3H]BK competition assay to recombinant receptors of 0.78, 0.77, 10.2 and 44.4 nM, respectively); these four compounds did not displace [3H]Lys-des-Arg(9)-BK binding from human B1 receptors expressed by smooth muscle cells. The dibenzosuberane compound 19c, verified to functionally antagonize the vascular B2 receptor, is an example of a drug unusually specific for the human form of the receptor.     

Site-directed mutagenesis at the human B2 receptor and molecular modelling to define the pharmacophore of non-peptide bradykinin receptor antagonists

Combining site-directed mutagenesis with information obtained from molecular modelling of the bradykinin (BK) human B2 receptor (hB2R) as derived from the bovine rhodopsin crystal structure [Science 289 (2000) 739], we previously defined a putative binding mode for the non-peptide B2 receptor antagonists, FR173657 and LF16-0687 [Can J Physiol Pharmacol 80 (2002) 303]. The present work is aimed to define the specific role of the quinoline moiety in the pharmacophore of these non-peptide antagonists. The effect of the mutations I110A, L114A (TM, transmembrane 3), W256A (TM6), F292A, Y295A and Y295F (TM7) was evaluated. None of the mutations affected the binding interaction of peptide ligands: the agonist BK and the peptide antagonist MEN 11270. The affinities in competing for [3H]-BK binding and in blocking the BK-induced IP production by the non-peptide antagonists LF16-0687 and FR173657 at the wild type and mutant receptors were analysed. While the affinities of LF16-0687 and FR173657 were crucially decreased at the I110A, Y295A, and Y295F mutants, the W256A mutation affected the affinity of the LF16-0687 only. The important contribution of the quinoline moiety was shown by the inability of an analogue of LF16-0687, lacking this moiety, to affect BK binding at the wild type receptor. On the other hand, the benzamidine group did not interact with mutated residues, since LF16-0687 analogues without this group or with an oxidated benzamidine displayed pairwise loss of affinity on wild type and mutated receptors. Further differences between FR173657 and LF16-0687 were highlighted at the I110 and Y295 mutants when comparing binding (pK(i)) and functional antagonist (pKB) affinity. First, the I110A mutation similarly impaired their binding affinity (250-fold), but at a less extent the antagonist potency of FR173657 only. Second, both the hydroxyl and the phenyl moieties of the Y295 residue had a specific role in the LF16-0687 interaction with the receptor, as demonstrated at the Y295F and Y295A mutants, respectively, but not in that of FR173657. Present data identify a receptor binding pocket comprised among TM3, 6, and 7, which concerns the interaction of the non-peptide antagonists FR173657 and LF16-0687, but not that of the peptide agonist or antagonist. Results indicate the quinoline group as the involved pharmacophoric element, and that the studied residues are differently involved in the interaction. The analysis performed by means of the GRID software led us to propose different spatial orientations of the quinoline moieties and partially overlapping binding pockets for the two ligands: that of LF16-0687 is located in the lipophilic environment amongst I110 (TM3), W256 (TM6), and Y295 (TM7) residues, whereas that of FR173657 lies essentially between I110 and Y295.     

The involvement of bradykinin B1 and B2 receptor mechanisms in cytokine-induced mechanical hyperalgesia in the rat.

1. Interleukin-1 beta (IL-1 beta), IL-2 and IL-8 induced a mechanical hyperalgesia following intra-articular (i.artic.) injection into rat knee joints, whereas IL-6 and tumour necrosis factor alpha (TNF-alpha) were without effect. 2. Co-administration of IL-1 receptor antagonist (0.1 micrograms) with IL-1 beta (1 mu), IL-2 (10 mu) or IL-8 (0.1 mu) prevented the subsequent development of the hyperalgesia. 3. Co-administration of desArg9Leu8BK (0.5-5 nmol) with IL-1 beta (1 mu), IL-2 (10 mu) or IL-8 (0.1 mu) reduced the level of hyperalgesia at 1, 4 and 6 h post administration, whereas Hoe 140 (5 pmol) antagonized the hyperalgesia only at the 1 h time point. 4. Intravenous administration of desArg9Leu8BK (10 nmol kg-1) or Hoe 140 (100 pmol kg-1) following IL-1 beta (1 mu), IL-2 (10 mu), or IL-8 (0.1 mu) reversed the subsequent hyperalgesia. 5. Administration of desArg9BK into joints 24 h after pre-treatment with IL-1 beta (1 mu) produced analegsia at low doses (50 pmol) and hyperalgesia at a higher dose (0.5 nmol). Both these effects were blocked by desArg9Leu8BK (0.5 nmol). 6. Administration of desArg9BK (0.5 nmol i.artic.) to animals 24 h after pre-treatment with IL-2 (1-100 mu) or IL-8 (0.1-10 mu) had no effect on the load tolerated by the treated joint. 7. Administration of indomethacin (1 mg kg-1, s.c.) prior to IL-1 beta (1 mu i.artic.) prevented the development of hyperalgesia. Administration of desArg9BK (5 pmol-0.5 nmol, i.artic.) to animals 24 h after indomethacin and IL-1 beta pretreatment had no effect on the load tolerated by the treated joint.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of B2 bradykinin receptor mRNA in the rat retina and sclerocornea

The expression of the B2 bradykinin receptor (BKR) mRNA in the adult rat eye was investigated by RNA blot and in situ hybridization analyses. Blot hybridization analysis of poly(A)+ RNA from the whole eye identified RNA species of 6000 and 4000 nucleotides, consistent with those observed in brain, lung, kidney and uterus. In situ hybridization analysis using digoxigenin-labeled riboprobes revealed intense labeling in the retinal ganglion cell layer and in a population of cells adjacent to the sclerocorneal junction. These data suggest that the B2 BKR is involved in biological processes in both retina and sclerocornea.     

The therapeutic potential of bradykinin B2 receptor agonists in the treatment of cardiovascular disease

The nonapeptide bradykinin (BK) is a Janus-faced hormone, which exerts pathophysiological as well as pronounced beneficial physiological effects, mainly by stimulation of BK B(2) receptors. In various animal models and in humans it has been shown that the stimulation of BK B(2) receptors is not only implicated in the pathogenesis of inflammation, pain and tissue injury but also in powerful cardioprotective mechanisms. Either exogenous administration of BK or locally increased BK concentrations as a consequence of the inhibition of its metabolic breakdown by angiotensin-converting enzyme inhibitors, reveal the significant contribution of BK in powerful cardioprotective mechanisms. These are mainly triggered by the synthesis and release of the vasorelaxant, anti-hypertrophic and anti-atherosclerotic endothelial mediators nitric oxide, prostaglandins and tissue-type plasminogen activator, by ischaemic preconditioning and by an increase in insulin sensitivity. Consequently, BK B(2) receptor agonists may have important clinical value in the treatment and prevention of various cardiovascular disorders such as hypertension, ischaemic heart disease, left ventricular hypertrophy, ventricular remodelling and congestive heart failure as well as diabetic disorders by mimicking the reported beneficial effects of BK. However, none of the currently known potent and selective peptide and non-peptide agonists of BK B(2) receptors--RMP-7 (lobradamil, Cereport; Alkermes), JMV-1116 (Fournier), FR-190997 (Fujisawa) and FR-191413 (Fujisawa)--have been selected for a clinical assessment in cardiovascular indications. One major challenge of this approach is the still unanswered question of whether there is a sufficient safe therapeutic window between potential cardioprotective and pro-inflammatory effects following BK B(2) receptor agonism.     

The therapeutic potential of bradykinin B2 receptor agonists in the treatment of cardiovascular disease

The nonapeptide bradykinin (BK) is a Janus-faced hormone, which exerts pathophysiological as well as pronounced beneficial physiological effects, mainly by stimulation of BK B₂ receptors. In various animal models and in humans it has been shown that the stimulation of BK B₂ receptors is not only implicated in the pathogenesis of inflammation, pain and tissue injury but also in powerful cardioprotective mechanisms. Either exogenous administration of BK or locally increased BK concentrations as a consequence of the inhibition of its metabolic breakdown by angiotensin-converting enzyme inhibitors, reveal the significant contribution of BK in powerful cardioprotective mechanisms. These are mainly triggered by the synthesis and release of the vasorelaxant, antihypertrophic and antiartherosclerotic endothelial mediators nitric oxide, prostaglandins and tissue-type plasminogen activator, by ischaemic preconditioning and by an increase in insulin sensitivity. Consequently, BK B₂ receptor agonists may have important clinical value in the treatment and prevention of various cardiovascular disorders such as hypertension, ischaemic heart disease, left ventricular hypertrophy, ventricular remodelling and congestive heart failure as well as diabetic disorders by mimicking the reported beneficial effects of BK. However, none of the currently known potent and selective peptide and non-peptide agonists of BK B₂ receptors – RMP-7 (lobradamil, Cereport^®; Alkermes), JMV-1116 (Fournier), FR-190997 (Fujisawa) and FR-191413 (Fujisawa) – have been selected for a clinical assessment in cardiovascular indications. One major challenge of this approach is the still unanswered question of whether there is a sufficient safe therapeutic window between potential cardioprotective and pro-inflammatory effects following BK B₂ receptor agonism.     

High-affinity binding of peptide agonists to the human B1 bradykinin receptor depends on interaction between the peptide N-terminal L-lysine and the fourth extracellular domain of the receptor

The aim of this study was to identify the location of the N terminus of peptide agonist ligands when bound to the human B1 bradykinin (BK) receptor. To reach this aim, we exploited the fact that high-affinity binding of kinin peptides to the human B1 receptor subtype requires a peptide N-terminal L-Lys, whereas high-affinity binding to the B2 receptor subtype does not require this residue. This was done by comparing the affinities of BK, a B2 receptor-selective peptide, and kallidin or Lys-BK, a less receptor-selective peptide, for chimeric proteins in which each B1 receptor domain had been substituted in the human B2 receptor and expressed in HEK293 cells. Individual substitution of transmembrane domains 1-7 (TM-I-VII) and extracellular domains 1-4 (EC-I-IV) of the B1 receptor in the B2 receptor influenced the affinities of BK and Lys-BK approximately equally. In contrast, substitution of B1 EC-IV dramatically reduced the affinity and potency of BK, whereas these parameters for Lys-BK were essentially unaltered. Substitution of either the N- or C-terminal half of B1 EC-IV in the B2 receptor only had a limited effect on the peptide binding constants, indicating the involvement of multiple residues throughout this domain. Complementary mutations of the N-terminal residue in Lys-BK revealed that both the positive charge and the proper spatial orientation of this residue were required for interaction with B1 EC-IV. Thus, the N-terminal residue of peptide agonists when bound to the human B1 receptor is positioned extracellularly and interacts with EC-IV.     

Non-peptide antagonists and agonists of the bradykinin B(2) receptor

Stimulation of the bradykinin (BK) B(2) receptor by kinins is associated with pathophysiological as well as pronounced beneficial effects. Consequently, interference with BK B(2) receptors by either antagonism or agonism offers promising therapeutic approaches for the development of drugs for the treatment of various human diseases. BK B(2) receptor antagonists may prove useful for the treatment of pathological situations caused by excessively increased local kinin concentrations, such as inflammation, tissue injury and pain. Beneficial effects of peptide BK B(2) receptor antagonists in perennial rhinitis, asthma and brain edema have already been demonstrated in clinical trials. On the other hand, kinins have also been identified as potent vasodilatory and organ-protective peptides. Therefore, BK B(2) receptor agonists may have the potential to become valuable therapeutics in the treatment of cardiovascular diseases such as hypertension, myocardial hypertrophy, myocardial infarction and arrhythmias as well as diabetic disorders. For both approaches, potent, selective and even orally active non-peptide compounds have been discovered recently. Prototypes of these novel third generation classes of compounds are the alkylphosphonium salt WIN-64338, the pseudopeptide NPC-18884, the thiosemicarbazide bradyzide and especially the imidazo[1,2-a]pyridine FR-167344 and the quinolines FR-173657 and LF-16.0687 as non-peptide BK B(2) receptor antagonists, whereas the 4-(2-pyridylmethoxy)-substituted quinoline FR-190997 and the 3-(2-pyridylmethyl)-substituted benzimidazole FR-191413 emerged as non-peptide BK B(2) receptor agonists. These antagonists and agonists of the BK B(2) receptor have already demonstrated efficacy a various animal models of human diseases, which offers promising therapeutic approaches for the development of drugs for the treatment or even prevention of a variety of severe human diseases either via stimulation or via blockade of BK B(2) receptors.