Francesca Bellucci

Pharmacological profile of the novel mammalian tachykinin, hemokinin 1

The effects of the novel mammalian tachykinin, hemokinin 1 (HEK‐1), have been investigated by radioligand binding and functional in vitro and in vivo experiments. Similar to SP (Ki=0.13 nM), HEK‐1 inhibited in a concentration‐dependent manner and with high affinity [3H]‐substance P (SP) binding to human NK1 receptor (Ki=0.175 nM) while its affinity for [125I]‐neurokinin A (NKA) binding at human NK2 receptor was markedly lower (Ki=560 nM). In isolated bioassays HEK‐1 was a full agonist at tachykinin NK1, NK2 and NK3 receptors. In the rat urinary bladder (RUB) HEK‐1 was about 3 fold less potent than SP. In the rabbit pulmonary artery (RPA) HEK‐1 and in the guinea‐pig ileum (GPI), HEK‐1 was about 500 fold less potent than NKA and NKB, respectively. The responses to HEK‐1 were antagonized by GR 82334 in RUB (pKB=5.6±0.07), by nepadutant in RPA (pKB=8.6±0.04) and by SR 142801 in GPI (pKB=9.0±0.2) with apparent affinities comparable to that measured against tachykinin NK1, NK2 and NK3 receptor‐selective agonists, respectively. Intravenous HEK‐1 produced dose‐related decrease of blood pressure in anaesthetized guinea‐pigs (ED50=0.1 nmol kg−1) and salivary secretion in anaesthetized rats (ED50=6 nmol kg−1) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK1 receptor antagonist, SR 140333. We conclude that HEK‐1 is a full agonist at tachykinin NK1, NK2 and NK3 receptors, possesses a remarkable selectivity for NK1 as compared to NK2 or NK3 receptors and acts in vivo experiments with potency similar to that of SP.

Bradykinin and B2 receptor antagonism in rat and human articular chondrocytes

In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis.

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.

A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis

Binding affinity at the [3H]‐BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide FR190997 were studied at wild‐type or point‐mutated human B2 receptors (hB2R) expressed in CHO cells. The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F. As the wild‐type receptor‐binding affinity of FR190997 was 40‐fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of FR190997 compared with BK. Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400‐ and 150‐fold, and that of FR190997 was reduced by 400‐ and 25‐fold, at the W86A and F259A mutant B2 receptors, respectively. Contrary to BK, the affinity of FR190997 was selectively decreased at I110A, Y295A, and Y295F mutants (>103‐fold), and a different efficacy was measured at the Y295 mutants, FR190997 being devoid of the capability to trigger IP production at Y295A mutant. L114A, F252A, and W256A selectively impaired the efficacy of FR190997, whereas its binding affinity was not affected. As a consequence, FR190997 behaved as a high‐affinity antagonist in blocking the IP production induced by BK. The lack of capability of FR190997 to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand. Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist FR190997 (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only FR190997.

Pharmacological characterization of the bradykinin B2 receptor antagonist MEN16132 in rat in vitro bioassays.

The pharmacological profile of the bradykinin B(2) receptor antagonist MEN16132 at the rat B(2) receptor has been investigated and compared with that of icatibant (formerly Hoe 140). Antagonist affinity has been measured through radioligand binding experiments with membranes prepared from uterine and airway tissue. MEN16132 inhibited [(3)H]bradykinin binding with subnanomolar affinity (pK(i) values 10.4 and 10.1 in the uterus and airways, respectively), and was about 3-fold less potent than icatibant (pK(i) values 10.9 and 10.5). Antagonist potency has been estimated towards bradykinin-induced contractility of uterine and urinary bladder smooth muscle preparations. In these assays MEN16132 (pK(B): 9.7 both in uterus and bladder) was about 10-fold more potent than icatibant [pK(B): 8.8 in uterus, and pK(B) 8.0 in urinary bladder, as from Meini, S., Patacchini, R., Giuliani, S., Lazzeri, M., Turini, D., Maggi, C.A., Lecci, A., 2000a. Characterization of bradykinin B(2) receptor antagonists in human and rat urinary bladder. Eur. J. Pharmacol. 388, 177-182]. Washout experiments conducted in the uterine preparation indicated for MEN16132 (100 nM) a slower reversibility than icatibant (300 nM).Altogether present results indicate that MEN16132 displays high affinity and potency also for the rat bradykinin B(2) receptor, and thus is suitable for further investigations in pathophysiological models in this species.

Characterization of kinin receptors in human cultured detrusor smooth muscle cells

Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor.

Bradykinin B2 and GPR100 receptors: a paradigm for receptor signal transduction pharmacology

The aim of the present report was to investigate the ligand selectivity of the human orphan G‐protein‐coupled receptor GPR100 (hGPR100), recently identified as a novel bradykinin (BK) receptor, as compared with that of the human B2 receptor (hB2R) stably transfected in Chinese hamster ovary cells. BK was able to inhibit the cAMP production induced by forskolin with a potency 100‐fold lower at the hGPR100 (pEC50=6.6) than that measured at the hB2R (pEC50=8.6). Both effects were inhibited by the B2 receptor antagonist Icatibant (1 μM). The nonpeptide B2 receptor agonist FR190997 (8‐[2,6‐dichloro‐3‐[N‐methylcarbamoyl)cinnamidoacetyl]‐N‐methylamino]benzyloxy]‐2‐methyl‐4‐(2‐pyridylmethoxy)quinoline) did inhibit the forskolin‐induced cAMP production (pEC50=7.7) at the hB2R, whereas it was not able to exert any effect at the hGPR100. The human insulin‐like peptide relaxin 3 did inhibit the cAMP production at the hGPR100 (pEC50=7.3) at a greater extent than BK, and was devoid of any effect at the hB2R. FR190997 and relaxin 3 responses at the hB2R and hGPR100, respectively, were not inhibited by Icatibant (1 μM). These data indicate FR190997 and relaxin 3 as selective agonists for hB2R and hGPR100, respectively, and support the concept that different agonists may specifically bias the conformational states of a receptor to result in a final common G protein coupling, which is differentially recognized by antagonists.

Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B2 receptor

BACKGROUND AND PURPOSE Icatibant is a well‐known kinin B2 receptor antagonist currently used for angiooedema attacks. MEN16132 is a non‐peptide B2 receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists.

Multifaceted Approach to Determine the Antagonist Molecular Mechanism and Interaction of Ibodutant ([1-(2-Phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) at the Human Tachykinin NK2 Receptor

Ibodutant (MEN15596, [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK2 receptor (NK2R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK2R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-{[Asn(β-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2β-5β)}) and saredutant [SR48968, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK2R, ibodutant potency measured toward concentration-response curves to neurokinin AaspKB was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [3H]nepadutant and [3H]saredutant binding sites. Determined Kon and Koff values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK2Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK2R complex is proposed.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies.

The aim was to characterize the recently discovered non‐peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists.