Molecular features characterizing non-peptide selectivity to the human B1 and B2 bradykinin receptors

Bradykinin is produced in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases. Actions of this peptide are mediated through two different G-protein coupled receptors, named B1 and B2 that have different pharmacological characteristics. The former is up-regulated during inflammation episodes or tissue trauma whereas, the latter is constitutively expressed in a variety of cell types. In a previous work we have characterized the molecular features that explain the observed structure-activity results for both receptors by means of molecular modeling studies, using diverse ligands for both receptors. These results were summarized in the form of two different pharmacophores that provided new insights to be used for the design of novel molecules with antagonistic profile. In the present work, we compare these pharmacophores to understand the features that characterize ligand selectivity to the two bradykinin receptors. The study shows that most of the residues involved in the binding pocket are similar in both receptors and consequently are the pharmacophores obtained. The main difference between the two pharmacophores remains on point #5 that involves a polar moiety for the B1 receptor and an aromatic ring for the B2 receptor. Accordingly, analysis of the prospective bound conformation of several non-selective small molecule ligands of the bradykinin receptors permits to conclude that fulfilment of point#5 is a requirement to produce selective ligands. However, the study also shows that this is a necessary condition only, since ligands need also to be bulky enough to avoid binding to these receptors in diverse poses. These results provide new insights for a better understanding of the molecular features that ligands are required to exhibit to be selective bradykinin ligands.     

Effects of two novel non-peptide antagonists at the rabbit bradykinin B2 receptor

Large species differences have been previously observed in the pharmacology of bradykinin (BK) B2 receptor antagonists. We investigated the effect of two novel non-peptide antagonists, compound 9 (a benzodiazepine peptidomimetic related to icatibant) and the thiosemicarbazide bradyzide on the rabbit B2 receptor (contractility of the jugular vein, competition of [3H]BK binding to a B2 receptor-green fluorescent protein (B2R-GFP) conjugate, subcellular distribution of B2R-GFP). While compound 9 is about 9000-fold less potent than icatibant, it shares with the latter peptide drug a selective, insurmountable and largely irreversible antagonist behavior against BK and the capacity to translocate B2R-GFP from the membrane into the cells. Bradyzide, reportedly very potent at rodent B2 receptors, was a competitive and reversible antagonist of moderate potency at the rabbit B2 receptor (contractility pA2 6.84, binding competition IC50 5 nM). The C-terminal region of icatibant, reproduced by compound 9, is likely to be important in the non-equilibrium behavior of icatibant. Bradyzide, a non-peptide antagonist developed on different structural grounds, is competitive at the rabbit B2 receptor.     

Formulation of 3D pharmacophore models for bradykinin B2-receptor antagonists

Summary In order to make a further contribution to the elucidation of the essential structural features for bradykinin (BK) antagonism, we extracted 3D pharmacophore models from a training set consisting of nine relatively rigid, small organic, non-peptide molecules, reported to be more or less active, competitive BK B₂-receptor antagonists. This was accomplished by means of the expert system Catalyst^TM. The information contained in one of these models was then used to identify relevant structural features and perform consensus molecular dynamics simulations including, in addition to the non-peptide antagonist set, four prototypical linear and cyclic peptide antagonists, and BK itself. this combined approach allowed us to identify regions of the conformational space shared by this series of compounds, which includes highly flexible, and, hence, otherwise almost inaccessible for conventional conformational sampling techniques, peptide molecules. As a result, we obtained a relatively small number of extended pharmacophore models, whose average, together with the original Catalyst model, could be used to search proprietary 3D databases for potential candidates. The results of such a search are also reported.     

The design of non-peptide human bradykinin B2 receptor antagonists employing the benzodiazepine peptidomimetic scaffold

The Bradykinin B2 receptor antagonist HOE 140 (D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg) has been used as a template for the de novo design and synthesis of a small number of non-peptide lead compounds based on the 1,4-benzodiazepin-2-one framework. Two of the compounds have been found to exhibit moderate K(i) values of 8.9 and 9.2 microM at the human Bradykinin B2 receptor.     

Non-selectivity of new bradykinin antagonists for B1 receptors.

Two new B1 receptor antagonists, [Hyp3,Thi5,DTic7,Oic8]desArg9-BK and DArg[Hyp3,Thi5,DTic7,Oic8]desArg9-BK were tested in vitro on the rabbit jugular vein and the guinea pig ileum (preparations containing B2 receptors) and on the rabbit aorta (preparation containing B1 receptors) for pharmacological characterization. The results indicate that both compounds are antagonists on both B1 and B2 receptors, are competitive and discriminate between B2A and B2B receptor subtypes.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Antagonists of B2 bradykinin receptors

Bradykinin and its active metabolites, produced by kallikreins at their sites of action, potently elicit a variety of biological effects: hypotension, bronchoconstriction, gut and uterine contraction, epithelial secretion in airway, gut, and exocrine glands, vascular permeability, pain, connective tissue proliferation, cytokine release, and eicosanoid formation. These effects are mediated by at least two broad classes of receptors. The most common is the B2 subtype. The availability of competitive antagonists of B2 receptors has provided powerful tools for the study of bradykinin's actions. The significance of kinins in certain human diseases is being explored by using these agents as potential therapeutic agents. Human clinical trials are under way to test the usefulness of bradykinin receptor antagonists to treat symptoms of the common cold and the pain associated with severe burns. Trials are also being comtemplated for use in treatment of asthma.     

Age-related changes of bradykinin B1 and B2 receptors in rat heart

Aging is a major risk factor for the development of vascular diseases, such as hypertension and atherosclerosis, that leads to end organ damage and especially heart failure. Bradykinin has been demonstrated to have a cardioprotective role by affecting metabolic processes and tissue perfusion under conditions of myocardial ischemia. Its actions are exerted via the bradykinin B1- and B2-type receptors (B1Rs and B2Rs), but the functional status of these receptors during the aging process is poorly understood. This study aims to investigate whether changes in B1R and B2R gene and protein expression in rat heart are associated with the age-related alterations of cardiac structure and function. Using real-time PCR, we found that B1R mRNA expression increased 2.9-fold in hearts of older rats (24 mo of age) compared with younger rats (3 mo of age), whereas B2R gene expression remained unchanged. Western blot analysis showed that expression of B2R at the protein level is approximately twofold higher in young rats compared with old rats, whereas the B1R protein is approximately twofold higher in old rats compared with young rats. The present results provide clear functional and molecular evidence that indicate age-related changes of bradykinin B1Rs and B2Rs in heart. Because the cardioprotective actions of bradykinin are physiologically mediated via the B2Rs, whereas the B1Rs become induced by tissue damage, these results suggest that age-related decreases in B2R protein levels may leave the heart vulnerable to ischemic damage, and increases in B1R expression and activity may represent a compensatory reaction in aging hearts.     

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.     

Quinolinyl- and phenantridinyl-acetamides as bradykinin B1 receptor antagonists

A new series of quinolinyl- and phenantridinyl-acetamides were synthesizer and evaluated against bradykinin B1 receptor. In vitro metabolic stability data were reported for the key compounds.The analgesic effect of compound 20 from the phenantridine series was proved in-vivo.     

Formulation of 3D pharmacophore models for bradykinin B2-receptor antagonists

In order to make a further contribution to the elucidation of the essential structural features for bradykinin (BK) antagonism, we extracted 3D pharmacophore models from a training set consisting of nine relatively rigid, small organic, non-peptide molecules, reported to be more or less active, competitive BK B₂-receptor antagonists. This was accomplished by means of the expert system Catalyst. The information contained in one of these models was then used to identify relevant structural features and perform consensus molecular dynamics simulations including, in addition to the non-peptide antagonist set, four prototypical linear and cyclic peptide antagonists, and BK itself. This combined approach allowed us to identify regions of the conformational space shared by this series of compounds, which includes highly flexible, and, hence, otherwise almost inaccessible for conventional conformational sampling techniques, peptide molecules. As a result, we obtained a relatively small number of extended pharmacophore models, whose average, together with the original Catalyst model, could be used to search proprietary 3D databases for potential candidates. The results of such a search are also reported.     

Role of bradykinin B1 and B2 receptors in normal blood pressure regulation

With inhibition or absence of the bradykinin B2 receptor (B2R), B1R is upregulated and assumes some of the hemodynamic properties of B2R, indicating that both participate in the maintenance of normal vasoregulation or to development of hypertension. Herein we further evaluate the role of bradykinin in normal blood pressure (BP) regulation and its relationship with other vasoactive factors by selectively blocking its receptors. Six groups of Wistar rats were treated for 3 wk: one control group with vehicle alone, one with concurrent administration of B1R antagonist R-954 (70 microg x kg(-1) x day(-1)) and B2R antagonist HOE-140 (500 microg x kg(-1) x day(-1)), one with R-954 alone, one with HOE 140 alone, one with concurrent administration of both R-954 and HOE-140 plus the angiotensin antagonist losartan (5 mg x kg(-1) x day(-1)), and one with only losartan. BP was measured continuously by radiotelemetry. Only combined administration of B1R and B2R antagonists produced a significant BP increase from a baseline of 107-119 mmHg at end point, which could be partly prevented by losartan and was not associated with change in catecholamines, suggesting no involvement of the sympathoadrenal system. The impact of blockade of bradykinin on other vasoregulating systems was assessed by evaluating gene expression of different vasoactive factors. There was upregulation of the eNOS, AT1 receptor, PGE2 receptor, and tissue kallikrein genes in cardiac and renal tissues, more pronounced when both bradykinin receptors were blocked; significant downregulation of AT2 receptor gene in renal tissues only; and no consistent changes in B1R and B2R genes in either tissue. The results indicate that both B1R and B2R contribute to the maintenance of normal BP, but one can compensate for inhibition of the other, and the chronic inhibition of both leads to significant upregulation in the genes of related vasoactive systems.     

Novel series of O-substituted 8-quinolines and 4-benzothiazoles as potent antagonists of the bradykinin B2 receptors

The synthesis and the SAR study of novel O-substituted 8-quinolines and 4-benzothiazoles as highly potent non-peptide bradykinin B2 receptor antagonists are described. Several members of this series of antagonists efficiently inhibited the BK-induced vasoconstriction on different isolated organ preparations.     

Alpha-hydroxy amides as a novel class of bradykinin B1 selective antagonists

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.     

Activation of B2 bradykinin receptors by neurotensin

Many previous reports suggested that relatively high concentrations of neurotensin were required to exert its effects on neurotransmitter secretion. The neurotensin binding sites, which recognize high concentrations of neurotensin, were characterized in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with neurotensin, [3H]norepinephrine secretion and elevation of cytosolic calcium were evoked at EC(50) values of 59+/-4 and 37+/-7 microM, respectively. Both calcium release and inositol 1,4,5-trisphosphate (IP(3)) production induced by neurotensin suggested involvement of phospholipase C. Experiments with simultaneous or sequential treatment with neurotensin and bradykinin suggested that neurotensin and bradykinin act on the same binding sites. Furthermore, both inhibition of bradykinin- and neurotensin-induced calcium rises by bradykinin receptor antagonists with similar IC(50) values and receptor binding analysis using [3H]bradykinin confirmed that neurotensin directly binds to B2 bradykinin receptors. The data suggest that neurotensin binds and activates the B2 bradykinin receptors.     

Mechanisms regulating the expression,self-maintenance,and signaling-function of the bradykinin B2 and B1 receptors

Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Galphai and Galphaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg(10)-kallidin or desArg(9)-BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-alpha. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self-maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980, Pharmacol Rev 32:1-46; Regoli et al., 1990, J Cardiovasc Pharmacol 15(Suppl 6):S30-S38; Regoli et al., 1993, Can J Physiol Pharmacol 71:556-557; Marceau, 1995, Immunopharmacology 30:1-26; Regoli et al., 1998, Eur J Pharmacol 348:1-10).     

Solubilization and characterization of B2 bradykinin receptors from cultured human fibroblasts.

Active B2 bradykinin (BK) receptors were solubilized in high yields from intact monolayers or particulate fractions of cultured human foreskin fibroblasts using 4 mM of the non-denaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Other detergents showed only minor (digitonin) or no (Triton X-100, n-octyl glucopyranosid) efficacy at all. The stability of CHAPS-solubilized BK binding activity was temperature dependent being reduced to 30% of initial binding after 3 days of storage at 4 degrees C. CHAPS extracts, however, retained BK binding activity for at least several days when they were stored at -20 degrees C in the presence of 10% glycerol. The pharmacological characterization gave a rank order of potency for unlabeled BK, BK agonists, and antagonists to compete with [3H]BK for specific binding very similar to that observed in intact fibroblasts. Association and dissociation kinetics demonstrated that the binding of [3H]BK to the soluble CHAPS extracts was time dependent and reversible. Scatchard analysis of equilibrium binding data exhibited saturable binding of a single class of high affinity BK-binding sites with a Kd of 1.68 +/- 0.8 nM. Gel filtration revealed an apparent molecular weight of 250,000 for the solubilized BK receptor complex in CHAPS extracts. The ability to solubilize the B2 BK receptor in an active and stable form should allow for its future purification and for the characterization of its chemical properties.     

Design of non-peptide agonists and antagonists at neuropeptide receptors starting from the chemical structure of neuropeptides

Summary Non-peptide small-molecule antagonists for cholecystokinin (CCK)-A and-B receptors, tachykinin NK-1, NK-2 and NK-3 receptors and bombesin BB-1 receptors have been designed and synthesized starting from the chemical structure of the endogenous mammalian neuropeptides cholecystokinin, substance-P and bombesin, respectively. A non-peptide CCK-A agonist, with weak potency but high efficacy, was also identified from the same strategy.     

Design of non-peptide agonists and antagonists at neuropeptide receptors starting from the chemical structure of neuropeptides

Non-peptide small-molecule antagonists for cholecystokinin (CCK)-A and -B receptors, tachykinin NK-1, NK-2 and NK-3 receptors and bombesin BB-1 receptors have been designed and synthesized starting from the chemical structure of the endogenous mammalian neuropeptides cholecystokinin, substance-P and bombesin, respectively. A non-peptide CCK-A agonist, with weak potency but high efficacy, was also identified from the same strategy.     

1-Benzylbenzimidazoles: the discovery of a novel series of bradykinin B(1) receptor antagonists

The design, synthesis, and structure-activity studies of a novel series of BK B(1) receptor antagonists based on a 1-benzylbenzimidazole chemotype are described. A number of compounds, for example, 38g, with excellent affinity for the cynomolgus macaque and rat bradykinin B(1) receptor were discovered.