Interaction of linear and cyclic peptide antagonists at the human B(2) kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.     

New conformationally homogeneous beta-turn antagonists of the human B2 kinin receptor.

We have designed and synthesized a conformationally homogeneous series of cyclic pentapeptides of the general structure c[Pro-aa(i)-D-Tic-Oic-aa(i + 3)] which adopt a type-II' beta-turn conformation believed important for high affinity antagonism of the bradykinin (BK) B2 receptor. We incorporated D-Tic and octahydroindole-2-carboxylic acid (Oic) residues (present in known active antagonists) in a cyclic pentapeptide that would place the D-aa in the i + 1 position of the beta-turn and a proline as a bridge between the C- and N-termini sides of the turn. In positions i and i + 3 alkyl, aromatic, polar or charged amino acids could be introduced without dramatically changing the overall structure. Ten analogues were studied using 1H nuclear magnetic resonance (NMR) and evaluated for their binding affinity for the human B2 receptor. The NMR data in dimethylsulfoxide (DMSO) confirmed the structural homogeneity within the class and, on the basis of this, one representative member of the series was chosen for a detailed structure determination using NMR data in sodium dodecylsulphate (SDS) micelles and molecular dynamics calculations. Despite the structural similarity, the binding affinity of the ten analogues was strongly influenced by the nature of the side-chains in positions i and i + 3, with the doubly charged analogue 49 (pKi = 6.2) proving best. This compound may serve as the starting point for the discovery of new non-peptide bradykinin B2 receptor antagonists.     

Identification of a key region of kinin B(1) receptor for high affinity binding of peptide antagonists

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.     

Solid-phase synthesis of MEN 11270, a new cyclic peptide kinin B2 receptor antagonist.

An efficient synthesis of the cyclic decapeptide MEN 11270 [H-DArg1-Arg2 Pro3-Hyp4-Gly5-Thi6-Dab7-DTic8-Oic9-Arg10 c(7gamma - 10alpha)] was developed. Two three-dimensional orthogonal strategies were applied and compared: Fmoc/Tos/Boc (procedure A) and Fmoc/Pmc/Dde (procedure B). Both resulted in a 23-step strategy comprising the stepwise solid-phase chain assembly of the linear protected peptide, partial deprotection, solution-phase cyclization and final full deprotection. The stepwise assembly of the linear peptide was optimized by double coupling and acylation time prolongation for critical residues (Tic, Dab, Thi, Pro). O-(7-azabenzotriazol-1-yl)-N,N,N',N' tetramethyluronium (HATU) was preferred as coupling reagent for Dab. In the cyclization step, the partial racemization of Arg10 (31% using 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide/1-hydroxybenzotriazole (EDC/HOBt) as activation system) was reduced to 3% with HATU. The final deprotection was performed in the presence of dimethylsulfide (procedure A) and thiocresol (procedure B) as scavengers, to avoid the sulfation of Hyp side chain. The final compound and the main by-products were characterized by mass spectroscopy (MS), nuclear magnetic resonance (NMR) and racemization test. Procedure B produced operationally simpler and more efficient results than A (28% overall yield versus 4%).     

Pharmacological characterisation of novel kinin B2 receptor ligands

Peptide and nonpeptide compounds have been shown to interact specifically with B2 receptors of three different species, namely human, rabbit, and pig. Peptide agonists and nonpeptide antagonists show marked differences in potencies and suggest the existence of B2 receptor subtypes. This conclusion is based on data obtained with the modified agonist peptide LF 150943 whose potency (pEC50 9.4) is at least 100-fold higher in rabbit than in humans (7.4) and pig (6.7). The same conclusion can be drawn from data obtained with antagonists that are more potent in humans (LF 160687, pA2 9.2) than in rabbit (8.7) and pig (8.2) or with antagonists (S 1567) that show the opposite potency order, being much weaker in humans (pA2 6.9) than in rabbit (7.6) and pig (9.4). Two other compounds (FR 173657 and FR 172357) show similar pharmacological spectra as S 1567 and differ from LF 160687.     

Antitumor activity of cell-penetrant kinin B1 receptor antagonists in human triple-negative breast cancer cells

High nuclear expression of G protein-coupled receptors, including kinin B1 receptors (B1R), has been observed in several human cancers, but the clinical significance of this is unknown. We put forward the hypothesis that these “nuclearized” kinin B1R contribute to tumorigenicity and can be a new target in anticancer strategies. Our initial immunostaining and ultrastructural electron microscopy analyses demonstrated high B1R expression predominantly located at internal/nuclear compartments in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line as well as in clinical samples of patients with TNBC. On the basis of these findings, in the present study, we evaluated the anticancer therapeutic potential of newly identified, cell-permeable B1R antagonists in MDA-MB-231 cells (ligand–receptor binding/activity assays and LC-MS/MS analyses). We found that these compounds (SSR240612, NG67, and N2000) were more toxic to MDA-MB-231 cells in comparison with low- or non-B1R expressing MCF-10A normal human mammary epithelial cells and COS-1 cells, respectively (clonogenic, MTT proliferative/cytocidal assays, and fluorescence-activated cell-sorting (FACS)-based apoptosis analyses). By comparison, the peptide B1R antagonist R954 unable to cross cell membrane failed to produce anticancer effects. Furthermore, the putative mechanisms underlying the anticancer activities of cell-penetrant B1R antagonists were assessed by analyzing cell cycle regulation and signaling molecules related to cell survival and apoptosis (FACS and western blot). Finally, drug combination experiments showed that cell-penetrant B1R antagonists can cooperate with suboptimal doses of chemotherapeutic agents (doxorubicin and paclitaxel) to promote TNBC death. This study provides evidence on the potential value of internally acting kinin B1R antagonists in averting growth of breast cancer.     

Modulation of kinin B1 receptor expression by endogenous angiotensin II in hypertensive rats

We investigated the expression and localization of B1 receptor in tissues of rats submitted to a renin-dependent model of hypertension (2K-1C), and analyzed the influence of endogenous Ang II in modulating the in vivo expression of these receptors. B1 mRNA levels in the heart, kidney and thoracic aorta were quantified by real time PCR, B1 receptor protein expression was assessed by immunohistochemistry, plasma Ang II levels were analyzed by radioimmunoassay and the effects of AT1 receptor blockade were determined after losartan treatment. 2K-1C rats presented a marked increase in Ang II levels when compared to sham-operated rats. In parallel, cardiac- (but not renal and aortic) B1 mRNA levels were 15-fold higher in 2K-1C than in sham rats. In 2K-1C, B1 expression was detected in the endothelium of small cardiac arteries and in cardiomyocytes. Losartan completely reverted the increased B1 mRNA levels and significantly decreased the protein expression observed in 2K-1C rats, despite reducing, but not normalizing blood pressure. We conclude that in the 2K-1C rat, induction of cardiac B1 receptor might be tightly linked to AT1 receptor activation. These data suggest the existence of a new site of interaction between kinins and angiotensins, and might provide important contributions for a better understanding of the pathophysiology of hypertension.     

Effects of alpha-lipoic acid on kinin B1 and B2 receptor binding sites in the spinal cord of chronically angiotensin-treated rats

A quantitative autoradiographic study was performed to determine whether kinin receptors are altered in the rat spinal cord in an experimental model of arterial hypertension under antioxidant therapy with alpha-lipoic acid. Sprague-Dawley rats were fed for 4 weeks with a normal chow diet or with an alpha-lipoic acid supplemented diet (1000 mg/kg feed), and treated for the last 2 weeks with angiotensin II (AT II) (200 ng/kg/min with an osmotic pump implanted s.c.). Control rats received either diet but not AT II. A 2-week administration of AT II increased significantly systolic blood pressure, the production of superoxide anion in the aorta and B1 receptor binding sites in the thoracic spinal dorsal horn. This treatment did not affect spinal B2 receptor binding sites, glycemia and insulinemia. The diet supplemented with alpha-lipoic acid reduced significantly the increase in systolic blood pressure, the production of aortic superoxide anion and prevented the increases of B1 receptor binding sites. Results show an association between the oxidative stress and the increases of B1 receptors and arterial blood pressure induced by AT II. Data also exclude the possibility that arterial hypertension is a primary mechanism leading to an increase of B2 receptor binding sites in the rat spinal cord.     

Cardiovascular effects of neuropeptide FF antagonists

The neuropeptide FF (NPFF) antagonist desaminotyrosyl-Phe-Leu-Phe-Gln-Pro-Gln-Arg-NH2 dose-dependently reversed NPFF-induced elevation of blood pressure in anesthetized rats after intravenous injection without causing a significant change of blood pressure and heart rate by itself. However, another antagonist dansyl-Pro-Gln-Arg-NH2 produced a significant drop of the mean arterial pressure only at a large dose (10 micromol/kg body weight), but reversal of the NPFF-induced hypertension was modest. Consequently and contrary to the conclusions of a previous study, NPFF antagonists cannot be identified simply by measuring the changes in the hemodynamic parameters upon the injection of the compounds alone and without a subsequent NPFF challenge.     

Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: discussion of a model proposing receptor precoupling to G protein

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [3H]BK and the antagonist [3H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for [3H]BK and a Kd of 3.8 nM for the antagonist [3H]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [3H]NPC17731 binding unaffected, but reduced the receptor affinity for [3H]BK to a Kd of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C. The rank order of the guanosine nucleotides for [3H]BK binding reduction was GTP[gammaS] = Gpp[NH]p > GTP = GDP > GDP[betaS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.     

Cardiovascular expression of inflammatory signaling molecules, the kinin B1 receptor and COX2, in the rabbit: effects of LPS, anti-inflammatory and anti-hypertensive drugs

We first aimed to test the effect of anti-inflammatory drugs, etanercept and dexamethasone sodium phosphate (DSP), on the expression of inducible inflammatory signaling molecules (the bradykinin [BK] B(1) receptor [B(1)R], cyclooxygenase [COX]-2) in lipopolysaccharide (LPS)-treated rabbits. Preliminary experiments mostly based on a novel cellular model, rabbit dermis fibroblasts, showed that etanercept inhibited TNF-alpha-induced B(1)R expression ([(3)H]Lys-des-Arg(9)-BK binding), but that DSP also inhibited cytokine-induced B(1)R upregulation with less selectivity. LPS (100 microg/kg i.v.) induced the expression of the B(1)R (aortic contractility ex vivo, mRNA in hearts) and COX2 (immunoblots, heart extracts). However, the function of the BK B(2) receptor was unchanged (jugular vein contractility ex vivo). DSP pre-treatment profoundly reduced the induction of the B(1)R and COX2 whereas etanercept significantly inhibited only COX2 expression. The second aim was to verify whether chronic angiotensin converting enzyme (ACE) blockade in rabbits would induce B(1)R expression, as reported in other species. 14-Day enalapril oral dosing, but not treatment with the angiotensin receptor antagonist losartan, significantly increased aortic contractions mediated by B(1)Rs, however much less than LPS. Enalapril treatment did not increase COX2 expression but increased the ex vivo relaxation of the mesenteric artery mediated by endogenous prostaglandins. Chronic ACE inhibition recruits inflammatory signaling systems.     

MCH receptor peptide agonists and antagonists

Melanin-concentrating hormone (MCH) is an important neuropeptide hormone involved in multiple physiological processes. Peptide derivatives of MCH have been developed as tools to aid research including potent radioligands, receptor selective agonists, and potent antagonists. These tools have been used to further understand the role of MCH in physiology, primarily in rodents. However, the tools could also help elucidate the role for MCHR1 and MCHR2 in mediating MCH signaling in higher species.     

Effect of novel selective non-peptide kinin B(1) receptor antagonists on mouse pleurisy induced by carrageenan

Two novel selective non-peptide kinin B(1) receptor antagonists, the benzodiazepine antagonist and SSR240612, were evaluated in carrageenan-induced mouse pleurisy. The peptide R-715 (0.5 mg/kg, i.p.) and the non-peptide benzodiazepine (3 mg/kg, i.p.) antagonists significantly decreased cellular migration (predominantly neutrophils), without altering plasma exudation. SSR240612 (1 mg/kg, i.p.) diminished total cells and neutrophils, besides exudation. Oral administration of SSR240612 (10 mg/kg) also reduced total cell and neutrophil counts. Only the benzodiazepine antagonist inhibited the lung myeloperoxidase activity. No tested antagonist significantly altered the lung and pleural TNFalpha and IL-1beta production. We provide interesting evidence on the anti-inflammatory in vivo effects of non-peptide B(1) receptor antagonists.     

Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors

Bradykinin (BK) and des-Arg⁹-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B₂ (B₂R) and B₁ (B₁R) receptors, respectively. It was already shown that the deletion of kinin B₁R or of B₂R induces upregulation of the remaining receptor subtype [10], [12], [16], [28], [36]. However studies on overexpression of B₁R or B₂R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17], [19], [33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B₁R (TGR(Tie₂B₁)) exclusively in the endothelium. In another study, mice overexpressing B₁R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B₂R was investigated in the thoracic aorta isolated from TGR(Tie₂B₁) rats overexpressing the B₁R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B₂R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie₂B₁) rats could be due to the enhanced expression of B₂R associated with the overexpression of the B₁R.     

Cardiovascular effects of newly discovered peptide intermedin/adrenomedullin 2

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP). The present study aimed to investigate the cardiovascular effects of IMDs (IMD1-47 and IMD8-47) in rats. Intravenous administration of 150 nmol IMDs continuously decreased mean arterial pressure and inhibited cardiac function. Administration with IMDs decreased left ventricular end-systolic pressure (LVESP) and maximal rate of left-ventricle pressure development (+/-LVdp/dt(max)), and elevated left ventricular end-diastolic pressure (LVEDP). Changes with IMD1-47 treatment were close to that with IMD8-47 (P>0.05). Perfusion of isolated rat hearts in vitro with IMD8-47 (10(-8) and 10(-7)mol/L) resulted in lower LVSP, by 40 and 56% (P<0.01); lower +LVdp/dt (max), by 33 and 47% (P<0.01); lower -LVdp/dt(max), by 25 and 39% (P<0.01); but higher coronary perfusion flow (CPF), by 25% (P<0.05) and 33% (P<0.01), respectively, than controls. However, both IMD8-47 and IMD1-47 (from 10(-13) to 10(-7)mol/L) relaxed preconstricted aortic rings in a dose-dependent manner. Intravenous administration of IMD1-47 and IMD8-47 (10(-7)mol/L) in vivo increased the cyclic adenosine monophosphate (cAMP) content by 68 and 150% (both P<0.01), respectively, in myocardia and 320 and 281% (both P<0.01), respectively, in aortas, compared with controls. Perfusion of isolated hearts with IMD1-47 and IMD8-47 (10(-7)mol/L) enhanced cAMP content by 24% (P<0.05) and 73% (P<0.01), respectively, compared with controls. IMDs inhibited 3H-Leucine incorporation in cardiomyocytes in a concentration-dependent manner. IMD1-47 and IMD8-47 (10(-7) and 10(-8)mol/L) decreased 3H-Leucine incorporation by 12-25% (P<0.01) and 14-18% (P<0.01), respectively. IMD mRNA was detected in cultured neonatal cardiomyocytes and isoproterenol-induced hypertrophic myocardia but not normal myocardia of adult rats. These results suggest that IMD might be a regulatory factor for cardiovascular function and myocardial hypertrophy as a cardiovascular active peptide.     

New spiro-piperidines as 5-HT2B receptor antagonists

A functional screening highlighted a series of spiro-piperidines as 5-HT2B receptor antagonists. Preliminary structure-activity relationship has been explored driving to potent antagonists (IC50 = 1 nM) and indicating directions for further explorations.     

Oxamides as novel NR2B selective NMDA receptor antagonists

A novel series of oxamides derived from indole-2-carboxamides was identified as potent NR2B selective NMDA receptor antagonists. Several members of this group showed good analgesic activity in the mouse formalin test.     

Indole-2-carboxamides as novel NR2B selective NMDA receptor antagonists

A novel series of indole-2-carboxamide derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of the number and position of OH groups on the indole skeleton as well as the substitution of the piperidine ring on the biological activity of the compounds was studied.     

Preliminary mutational analysis of the human kinin B2 receptor for nonpeptide antagonist ligands recognition

FR173657, LF16,0335, and LF16,0687 are nonpeptide antagonists, endowed with high affinity and selectivity for the human kinin B2 receptor. The kinin B2 receptor belongs to the family of G-protein-coupled receptors with seven transmembrane (TM) helices. In the present study, we aimed, through computer-assisted modeling and mutagenesis, to identify residues in the human B2 receptor (hB2R) amino acid sequence that are involved in nonpeptide antagonist binding in order to build up experimental data as a first step towards a molecular model of nonpeptide ligands binding site. Fourteen amino acid residues within the TM segments were mutated to alanine. The wild type and mutant receptors were stably expressed in Chinese hamster ovary (dhfr-) cells and tested for their ability to bind agonist ([3H]bradykinin) and peptide antagonist ([3H]MENI 1270) radioligands. The affinity of nonpeptide ligands was determined by heterologous competition experiments using the above radioligands. We found that some mutations in TM2 (W86A) and TM7 (Y295A, N297A) impair the binding affinity of the three nonpeptide antagonists. On the other hand, some mutated residues in TM3 (S1 17A) and TM6 (W256A) reduce the affinity of LF16,0335 and LF16,0687 only. Results are discussed in order to build up a hypothesis for the likely different interactions of various nonpeptide ligands with the B2 receptor.     

Indole-2-carboxamidines as novel NR2B selective NMDA receptor antagonists

A novel series of indole-2-carboxamidine derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of the substituents on the indole skeleton as well as the substitution of the benzyl moiety on the biological activity of the compounds was studied. Compound 5a was po active in the formalin test in mouse.