New insight into the binding mode of peptides at urotensin-II receptor by Trp-constrained analogues of P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-D-Trp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp(7) residue was replaced by the highly constrained L-Tpi and D-Tpi residues. The replacement of the Trp(7) by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation-activity relationships previously reported on UT receptor ligands.

Design and synthesis of novel sulfonamide-containing bradykinin hB(2) receptor antagonists. Synthesis and structure-relationships of α,α-tetrahydropyranylglycine.

A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R'). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.

Booster immunization with a fractional dose of Prevnar 13 affects cell-mediated immune response but not humoral immunity in CD-1 mice.

Achieving durable protective immunity following vaccination is dependent on many factors, including vaccine composition and antigen dose, and it has been investigated for various types of vaccines. Aim of the present study was to investigate the overall immune response elicited by two different booster doses in CD-1 mice, by exploiting the largely used 13-valent pneumococcal conjugate vaccine Prevnar 13® (PCV13). Immunization was performed by two primary doses of PCV13 two weeks apart, and a full or fractional (1/5) booster dose on week 10. Serotype-specific antibody titer, avidity, and opsonophagocytic activity were evaluated one week later, and compared to cell-mediated immunity (CMI) responses determined as the frequency of cytokines producing splenocytes by in vitro recall with the antigens (carrier protein and polysaccharides). Data showed that regardless of the booster dose, a comparable humoral response was produced, characterized by similar amounts of serotype-specific antibodies, with analog avidity and opsonophagocytic properties. On the other hand, when CMI was evaluated, the presence of CRM197-specific IL-5 and IL-2 producing cells was evident in splenocytes from mice immunized with the full dose, while in those immunized with the fractional booster dose, IFN-γ producing cells responsive to both protein and polysaccharide antigens were significantly increased, whereas the number of IL-5 and IL-2 positive cells remained unaffected. Overall the present findings show that PCV13 humoral response in mice is associated to a Th2 predominant response at the full booster dose, while the fractional one favors a mixed Th1/Th2 response, suggesting an important role of CMI besides measurement of functional protective antibodies, as an additional and important key information in vaccine development.

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 NK(2) receptor (NK(2)R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK(2)R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)]) 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 NK(2)R, ibodutant potency measured toward concentration-response curves to neurokinin A as pK(B) 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 [(3)H]nepadutant and [(3)H]saredutant binding sites. Determined K(on) and K(off) 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 NK(2)Rs, 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 NK(2)R complex is proposed.

Cinnamic acids and mono-substituted benzoic acids as useful capping groups for the preparation of hNK2 receptor antagonists.

NK(2) antagonists have been reported to be potentially useful for the treatment of a number of chronic diseases, such as asthma, irritable bowel syndrome, cystitis, and depression. Starting from an in-house prepared library of capped dipeptides, we have identified a series of molecules with subnanomolar binding affinity for the hNK(2) receptor. These molecules are composed by three well-defined regions: a planar aromatic acyl system as N-terminal capping group, a rigid and quite lipophilic core, and a flexible and relatively hydrophilic C-terminal capping group. Here we report how we were able to manipulate the N-terminal capping group to obtain significant in vivo activity after i.v. and i.d. administration.

Discovery of a new series of potent and selective linear tachykinin NK2 receptor antagonists.

Starting from 1 (MEN14268), a selective tachykinin NK2 receptor antagonist with an interesting in vitro pharmacological profile, a family of numerous antagonists was obtained through an optimization process focused on iterated structural modifications. The effects of the introduction of a wide variety of substituents on the lipophilic aromatic part of the molecule and the modulation of the structural constraint through the insertion of different achiral alpha,alpha-dialkylamino acids were investigated. In particular, aromatic and benzofused heteroaromatic moieties were introduced at the pseudo-N-terminal residue to replace the 2-benzothiophene moiety, and a systematic investigation of the best positioning of substituents onto the aromatic platform was reported for the benzothiophene core. Studies on the modulation of the length and the rigidity of the hydrophilic pseudo-C-terminal pendant are presented. Many heteroaliphatic groups are well tolerated by the receptor in this part of the ligand. The product 48f (MEN15596), bearing a methyl substituent on the benzothiophene and a tetrahydropyranylmethylpiperidine pendant, was finally selected for its good in vivo activity after intravenous, intraduodenal, and oral administration in guinea pigs.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 2. Synthesis and structure-activity relationships of alpha,alpha-cycloalkylglycine sulfonamides.

Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist.

The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2

Structure-Activity Study of the Peptides P5U and Urantide by the Development of Analogues Containing Uncoded Amino Acids at Position†9.

Previous modifications of the peptide sequence of human urotensin-II (U-II) led to the identification of two well-known ligands: P5U and urantide. These derivatives are considered to be the most representative agonist and antagonist, respectively, at the human urotensin receptor (UT). Optimization of P5U and urantide was carried out to stabilize specific conformations that may suggest new elements for discriminating agonist versus antagonist activity. We studied novel derivatives containing uncoded amino acids. In particular, the Tyr(9) residue of both P5U and urantide was replaced with nonaromatic hydrophobic bulky residues, as well as conformationally constrained aromatic moieties to generate eight novel derivatives. These analogues further contributed to determining the influence of such residues on binding affinity for and biological activity at UT. One of these eight peptides was also investigated by NMR spectroscopy and docking studies owing to its peculiar conformational properties and mode of interaction with UT. This structure-activity study is aimed at a more thorough examination of the role of tyrosine in modulating the agonism/antagonism of human U-II.

Pharmacology of an original and selective nonpeptide antagonist ligand for the human tachykinin NK2 receptor.

The pharmacological outline of a novel and original antagonist at the human tachykinin NK2 receptor is presented, namely MEN13510 (N-N'-bis-[2-(1H-indol-3-yl)-ethyl]-N,N'-bis-(3-thiomorpholin-4-yl-propyl)-phthalamide). MEN13510 retained nanomolar affinity for the human tachykinin NK2 receptor (Ki 6.4 nM), and micromolar affinity for the human tachykinin NK1 and NK3 receptors. A competitive antagonism is indicated by the Schild analysis (pK(B) 7.8, slope -0.94) of concentration-response curves of NKA induced inositolphosphates accumulation in Chinese hamster ovary (CHO) cells expressing the human NK2 receptor in the presence of MEN13510 (30-300 nM concentration range). The MEN13510 interaction with the human NK2 receptor was evaluated by means of heterologous inhibition binding experiments, by using agonist and antagonist radioligands ([125I]NKA, [3H]nepadutant, [3H]saredutant) at a series of mutant receptors having single aminoacidic substitutions of residues located in transmembrane (TM) segments 3, 4, 5, 6, and 7. MEN13510 affinity was not affected by the mutations in TM 3 and 4 (Q109A, F112A, T171A, C167G), and it was reduced by 10-fold at the I202F mutant, but not at the Y206A (TM4). Amongst the investigated mutants bearing the mutated residues in TM6 (F270A, Y266F, W263A) only F270A decreased the MEN13510 affinity by 7-fold. Even mutations in TM7 did reduce MEN13510 affinity by 32-fold (Y289T, but not Y289F) and 13-fold (F293A). Studied mutations represent the human tachykinin NK2 receptor discriminants involved in the binding of previously reported peptidic and nonpeptidic antagonists, against which results obtained with MEN13510 are compared. Results indicate that the binding site of this antagonist is, at least in part, overlapping to that described for NKA or saredutant. Finally we show that MEN13510 retains nanomolar affinity for the recently discovered splice variant of the human tachykinin NK2 receptor, namely beta isoform, as it has been described for the nonpeptide antagonist saredutant.

MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B2 receptor. In vitro pharmacology and molecular characterization.

The pharmacological characterization of the novel nonpeptide antagonist for the B2 receptor, namely MEN16132 (4-(S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B2 receptor has been investigated by means of competition studies at [3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B2 receptor (pKi 10.5), human lung fibroblasts (pKi 10.5), guinea pig airways (pKi 10.0), guinea pig ileum longitudinal smooth muscle (pKi 10.2), or guinea pig cultured colonic myocytes (pKi 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B2 receptor was checked at the human bradykinin B1 receptor (pKi<5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pKB 10.3) and guinea pig (colonic myocytes: pKB 10.3) B2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pKB 9.9) and guinea pig (ileum longitudinal smooth muscle: pKB 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B2 receptor antagonist.

Monocyclic human tachykinin NK-2 receptor antagonists as evolution of a potent bicyclic antagonist: QSAR and site-directed mutagenesis studies.

A new series of monocyclic pseudopeptidic tachykinin NK-2 receptor antagonists has been derived from nepadutant with the help of site-directed mutagenesis studies and QSAR models. MEN11558 is the lead compound which is evaluated on a series of 13 new human tachykinin NK-2 receptor mutants (Tyr107Ala, Gln109Ala, Asn110Ala, Phe112Ala, Ser164Phe, Cys167Gly, Phe168Ala, Tyr169Ala, Ile202Phe, Trp263Ala, Tyr269Phe, Tyr269Ala, and Phe293Ala) and 8 mutants on which data from nepadutant were already available (Gln166Ala, Ser170Ala, Thr171Ala, His198Ala, Tyr206Phe, Tyr266Phe, Tyr289Phe, and Tyr289Thr). The results show that the two compounds share most of their binding sites, in agreement with their hypothesized binding modes. This allows us to transfer the structural knowledge we already had for nepadutant to the new series of compounds. At the same time, a sound QSAR model is developed to assist the prioritization of new chemical syntheses. The result is the discovery of receptor antagonists with a higher affinity than nepadutant for the hNK-2 receptor.

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 B(2) receptor (hB(2)R) 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 (pEC(50) = 6.6) than that measured at the hB(2)R (pEC(50) = 8.6). Both effects were inhibited by the B(2) receptor antagonist Icatibant (1 microM). The nonpeptide B(2) 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 (pEC(50) = 7.7) at the hB(2)R, 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 (pEC(50) = 7.3) at a greater extent than BK, and was devoid of any effect at the hB(2)R. FR190997 and relaxin 3 responses at the hB(2)R and hGPR100, respectively, were not inhibited by Icatibant (1 microM). These data indicate FR190997 and relaxin 3 as selective agonists for hB(2)R 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.

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 profile of the novel mammalian tachykinin, hemokinin 1.

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. 2. Similar to SP (K(i)=0.13 nM), HEK-1 inhibited in a concentration-dependent manner and with high affinity [(3)H]-substance P (SP) binding to human NK(1) receptor (K(i)=0.175 nM) while its affinity for [(125)I]-neurokinin A (NKA) binding at human NK(2) receptor was markedly lower (K(i)=560 nM). 3. In isolated bioassays HEK-1 was a full agonist at tachykinin NK(1), NK(2) and NK(3) 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. 4. The responses to HEK-1 were antagonized by GR 82334 in RUB (pK(B)=5.6+/-0.07), by nepadutant in RPA (pK(B)=8.6+/-0.04) and by SR 142801 in GPI (pK(B)=9.0+/-0.2) with apparent affinities comparable to that measured against tachykinin NK(1), NK(2) and NK(3) receptor-selective agonists, respectively. 5. Intravenous HEK-1 produced dose-related decrease of blood pressure in anaesthetized guinea-pigs (ED(50)=0.1 nmol kg(-1)) and salivary secretion in anaesthetized rats (ED(50)=6 nmol kg(-1)) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK(1) receptor antagonist, SR 140333. 6. We conclude that HEK-1 is a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors, possesses a remarkable selectivity for NK(1) as compared to NK(2) or NK(3) receptors and acts in vivo experiments with potency similar to that of SP.

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.

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.

The N-terminal of icatibant and bradykinin interact with the same Asp residues in the human B2 receptor.

The pharmacology of peptide and non-peptide bradykinin B2 receptor ligands was evaluated in the inositol phosphate (IP) production assay in CHO cells expressing the human bradykinin B2 receptor. The effect of single and double alanine mutation of D266 and D284 residues at the human bradykinin B2 receptor was evaluated on the agonist profile of bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) and the synthetic agonist FR190997 (8-[2,6-dichloro-3-[N-methylcarbamoyl)cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline). Bradykinin potency (EC50 0.5 nM at the wild-type receptor) was reduced by 16-fold at D266A and D284A mutants and by 2300-fold at the D266A/D284A double mutant. None of the mutants affected the potency or the efficacy of FR190997. Peptide antagonists, Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH) and MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10alpha)) (100 nM) similarly antagonized the concentration-response curve to bradykinin or FR190997 (pA2 values 8.5 and 8.4 versus bradykinin and 8.2 and 8.4 versus FR190997) at the wild-type receptor. Non-peptide antagonists FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl]phenyl]-N-methylaminocarbonyl methyl]acrylamide) and LF16-0687 (1-[[2,4-dichloro-3-[(2,4-dimethylquinolin-8-yl)oxy] methyl]-phenyl]sulfonyl]-N-[3-[[4-(aminoiminomethyl)-phenyl]carbonylamino]propyl]-(S)-pyrrolidine carboxamide) (100 nM) showed an equivalent potency values in blocking the IP production induced by bradykinin or FR190997 (pA2 values 8.7 and 8.8 versus bradykinin and 8.8 and 8.6 versus FR190997). Whilst the antagonist potency of FR173657 and LF16-0687 was not affected by D266A/D284A double mutation (IP production induced by the synthetic agonist), that of Icatibant and MEN11270 was reduced by 50- and 200-fold. The antagonist potency of [Ala1]-Icatibant and [Ala2]-Icatibant (pA2 values at wild-type 7.7 and 6.4) was significantly less reduced (20-fold and 13-fold, respectively) by the D266A/D284A double mutation. Our results highlight a crucial role for two aspartic residues, D266 and D284, located at the top of transmembrane segments 6 and 7, in the high-affinity interaction of peptide antagonists with the human bradykinin B2 receptor. An interaction of these receptor residues with the N-terminal basic residues of Icatibant is hypothesized.

Mutagenesis at the human tachykinin NK(2) receptor to define the binding site of a novel class of antagonists.

The pharmacological profile of novel antagonists endowed with high affinity for the human tachykinin NK(2) receptor is presented. MEN13918 (Ngamma[Nalpha[Nalpha(benzo[b]thiophen-2-yl)carbonyl]-1-aminocyclohexan-1-carboxy]-d-phenylalanyl]-3-cis-aminocyclohexan-1-carboxylic-acid-N-(1S,2R)-2-aminocyclohexyl)amide trifluoroacetate salt) and MEN14268 (Nalpha[Nalpha(benzo[b]thiophen-2-yl)carbonyl)-1-aminocyclopentane-1-carboxyl]-d-phenylalanine-N-[3(morpholin-4-yl)propyl]amide trifluoroacetate salt) were more potent in blocking neurokinin A (NKA, His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH(2)) induced contraction in human, which induced greater contraction in human (pK(B) 9.1 and 8.3) than rat (pK(B) 6.8 and <6) urinary bladder smooth muscle preparation in vitro. In agreement with functional data, in membrane preparations of CHO cells stably expressing the human NK(2) receptors, both MEN13918 and MEN14268 potently inhibited the binding of agonist ([(125)I]NKA, K(i) 0.2 and 2.8 nM) and antagonist ([(3)H]nepadutant, K(i) 0.1 and 2.2 nM, [(3)H]SR48968 K(i) 0.4 and 6.9 nM) radioligands. Using site-directed mutagenesis and radioligands binding we identified six residues in the transmembrane (TM) helices that are critical determinants for the studied antagonists affinity. To visualize these experimental findings, we constructed a homology model based on the X-ray crystal structure of bovine rhodopsin and suggested a possible binding mode of these newly discovered antagonist ligands to the human tackykinin NK(2) receptor. Both MEN13918 and MEN14268 bind amongst TM4 (Cys167Gly), TM5 (Tyr206Ala), TM6 (Tyr266Ala, Phe270Ala), and TM7 (Tyr289Phe, Tyr289Thr). MEN13918 and MEN14268 diverging binding profile at Y289 mutations in TM7 (Tyr289Phe, Tyr289Thr) suggests a relation of their different chemical moieties with this residue. Moreover, the different influence on binding of these two ligands by mutations located deep along the inner side of TM6 (Phe270Ala, Tyr266Ala, Trp263Ala) indicates a nonequivalent positioning, although occupying the same binding crevice. Furthermore, binding data indicate the Ile202Phe mutation, which mimics the wild-type rat NK(2) receptor sequence, as a species selectivity determinant. In summary, data with mutant receptors describe, for these new tachykinin NK(2) receptor antagonists, a binding site which is partially overlapping either with that of the cyclized peptide antagonist nepadutant (cyclo-[[Asn(beta-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)] or the nonpeptide antagonist SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide).