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1.
Bioorg Med Chem ; 20(6): 2091-100, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-22342268

ABSTRACT

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.


Subject(s)
Bradykinin B2 Receptor Antagonists , Glycine/analogs & derivatives , Glycine/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Animals , Bradykinin/metabolism , Bronchoconstriction/drug effects , CHO Cells , Cricetinae , Glycine/chemical synthesis , Guinea Pigs , Humans , Hypotension/drug therapy , Receptor, Bradykinin B2/metabolism , Structure-Activity Relationship , Sulfonamides/chemical synthesis
2.
Expert Opin Investig Drugs ; 18(12): 1843-64, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19938899

ABSTRACT

Tachykinins (TKs) are small peptides widely distributed in the central and peripheral nervous systems where they act as neurotransmitters. Potent and selective TKs antagonists have been developed in the last 20 years and many efforts have been made to prove their efficacy in the treatment of various diseases. Herein the most prominent results in the clinical development are reported and discussed. For aprepitant, the only compound of this class to have been launched to date, results of clinical studies and postmarketing cost-effectiveness data for the treatment of chemotherapy-induced emesis are discussed. The field is still well active, as currently proof-of-concept studies for indications initially missed (i.e., depression) are ongoing and new targets are under investigation.


Subject(s)
Antiemetics/therapeutic use , Neurokinin-1 Receptor Antagonists , Receptors, Neurokinin-2/antagonists & inhibitors , Receptors, Neurokinin-3/antagonists & inhibitors , Aprepitant , Asthma/drug therapy , Clinical Trials as Topic , Gastrointestinal Diseases/drug therapy , Humans , Mental Disorders/drug therapy , Morpholines/therapeutic use , Piperazines/therapeutic use , Piperidines/therapeutic use , Sleep Initiation and Maintenance Disorders/drug therapy
3.
Eur J Pharmacol ; 579(1-3): 350-6, 2008 Jan 28.
Article in English | MEDLINE | ID: mdl-18036587

ABSTRACT

Kinins have been suggested to be involved in human airway diseases such as asthma and rhinitis. MEN16132 is a non-peptide kinin B(2) receptor antagonist able to inhibit the responses produced by intravenous bradykinin into the airways, as bronchoconstriction and microvascular leakage; we tested the effect of MEN16132 on endogenously generated bradykinin through the dextran sulfate-induced contact activation of kinin-kallikrein cascade in guinea-pigs. After dextran sulfate administration (1.5 mg/kg i.v.), the pulmonary insufflation pressure was monitored and the microvascular leakage of upper and lower airways was assessed using Evans blue as tracer of plasma protein extravasation. Our results demonstrated that topical MEN16132 strongly inhibited the dextran sulfate-induced bronchoconstriction (0.3 mM solution aerosol for 5 min) and plasma protein extravasation in both lower airways (3-10 microM solution aerosol for 5 min) and nasal mucosa (0.3 nmol/nostril); Icatibant, the peptide antagonist of kinin B(2) receptor, exerted a 3-30-fold less potent inhibitory effect than MEN16132. We conclude that local application of MEN16132 into the airways abolishes the responses produced by the endogenous generation of bradykinin and it can be useful as new pharmacological tool to check the role of kinins in human diseases.


Subject(s)
Bradykinin B2 Receptor Antagonists , Bradykinin/drug effects , Bronchoconstriction/drug effects , Ornithine/analogs & derivatives , Sulfonamides/pharmacology , Administration, Intranasal , Aerosols , Animals , Blood Proteins/metabolism , Bradykinin/administration & dosage , Bradykinin/analogs & derivatives , Bradykinin/metabolism , Bradykinin/pharmacology , Dextran Sulfate , Dose-Response Relationship, Drug , Guinea Pigs , Kallikrein-Kinin System/drug effects , Male , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Ornithine/administration & dosage , Ornithine/pharmacology , Sulfonamides/administration & dosage
4.
J Med Chem ; 50(3): 550-65, 2007 Feb 08.
Article in English | MEDLINE | ID: mdl-17266207

ABSTRACT

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.


Subject(s)
Bradykinin B2 Receptor Antagonists , Bronchodilator Agents/chemical synthesis , Ornithine/analogs & derivatives , Sulfonamides/chemical synthesis , Animals , Blood Pressure/drug effects , Bronchoconstriction/drug effects , Bronchodilator Agents/chemistry , Bronchodilator Agents/pharmacology , CHO Cells , Cricetinae , Cricetulus , Drug Design , Guinea Pigs , Humans , Ileum/drug effects , Ileum/physiology , In Vitro Techniques , Inositol Phosphates/biosynthesis , Male , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Ornithine/chemical synthesis , Ornithine/chemistry , Ornithine/pharmacology , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology
5.
Curr Drug Targets ; 7(8): 975-92, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16918326

ABSTRACT

In this chapter it is described how, starting from different approaches and through extensive medicinal chemistry studies, several discovery compounds were optimized and reached the development stage. The first tachykinin receptor antagonist to reach the market in 2003 for chemotherapy-induced emesis has been aprepitant. Other clinical candidates (for central nervous system disorders: osanetant, talnetant and saredutant; for irritable bowel syndrome: nepadutant and saredutant) are in advanced clinical phase. The clinical studies reported in the literature and the destiny of the clinical candidates, where available, will be reviewed.


Subject(s)
Randomized Controlled Trials as Topic/methods , Receptors, Tachykinin/antagonists & inhibitors , Technology, Pharmaceutical/methods , Animals , Biomedical Research/methods , Humans , Receptors, Tachykinin/metabolism
6.
J Med Chem ; 49(12): 3602-13, 2006 Jun 15.
Article in English | MEDLINE | ID: mdl-16759102

ABSTRACT

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.


Subject(s)
Bradykinin B2 Receptor Antagonists , Quinolines/chemical synthesis , Sarcosine/analogs & derivatives , Sulfonamides/chemical synthesis , Animals , Bradykinin/metabolism , Bronchoconstrictor Agents/chemical synthesis , Bronchoconstrictor Agents/chemistry , Bronchoconstrictor Agents/pharmacology , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Drug Design , Guinea Pigs , Humans , Hypotension/chemically induced , Inositol Phosphates/biosynthesis , Models, Molecular , Molecular Structure , Piperazine , Piperazines/chemistry , Quinolines/chemistry , Quinolines/pharmacology , Radioligand Assay , Receptor, Bradykinin B2/metabolism , Sarcosine/chemical synthesis , Sarcosine/chemistry , Sarcosine/pharmacology , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology
7.
Eur J Pharmacol ; 528(1-3): 7-16, 2005 Dec 28.
Article in English | MEDLINE | ID: mdl-16324696

ABSTRACT

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.


Subject(s)
Bradykinin B2 Receptor Antagonists , Ornithine/analogs & derivatives , Sulfonamides/pharmacology , Animals , Binding, Competitive , Bradykinin/analogs & derivatives , Bradykinin/metabolism , Bradykinin/pharmacology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Guinea Pigs , Humans , In Vitro Techniques , Inositol Phosphates/metabolism , Middle Aged , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Ornithine/metabolism , Ornithine/pharmacology , Point Mutation , Quinolines/pharmacology , Receptor, Bradykinin B2/genetics , Receptor, Bradykinin B2/metabolism , Sulfonamides/metabolism , Transfection
8.
J Pharmacol Exp Ther ; 315(2): 616-23, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16027229

ABSTRACT

We have tested the activity of 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 (MEN16132), a novel nonpeptide kinin B(2) receptor antagonist, on bradykinin (BK)-induced inflammatory responses, bronchoconstriction, and hypotension in guinea pigs. After i.v. (1-10 nmol/kg i.v.), intratracheal (i.t.) (10-100 nmol/kg i.t.), or aerosol (0.01-0.1 mM/5 min) administration, MEN16132 inhibited in a dose-dependent manner the bronchoconstriction induced by BK (10 nmol/kg i.v.). MEN16132 was more potent and possessed a longer duration of action as compared with the peptide B(2) receptor antagonist icatibant (HOE140; H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH trifluoroacetate). After i.v. administration, its inhibitory effect on bronchoconstriction lasted more than 8 h at 30 nmol/kg. When administered by i.v. or i.t. routes, the dose completely inhibiting bronchoconstriction also partially reduced the hypotensive response to BK, whereas after aerosol administration, the inhibitory effect was limited to respiratory level. Intranasal (i.n.) administration of MEN16132 (0.01-0.3 nmol/nostril) reduced, in a dose-dependent and long-lasting manner, the nasal mucosa plasma protein extravasation induced by BK (100 nmol/nostril), and it exerted a complete inhibition at about 30-fold lower dose than icatibant. At 1 nmol/nostril, MEN16132 activity was significant for at least 6 h with no systemic effect measured as inhibition of BK-induced hypotension, and at 10 nmol/nostril, the inhibitory effect lasted for more than 15 h with only a weak effect on hypotension. These findings indicate that in vivo MEN16132 is a potent kinin B(2) receptor antagonist with long duration of action, both after i.v. and local administration. A complete and prolonged inhibition of BK-induced bronchoconstriction or nasal inflammation can be achieved with MEN16132 topical administration (aerosol or i.n.) at doses devoid of systemic effects.


Subject(s)
Bradykinin B2 Receptor Antagonists , Bradykinin/antagonists & inhibitors , Bronchoconstriction/drug effects , Nasal Mucosa/blood supply , Ornithine/analogs & derivatives , Sulfonamides/pharmacology , Anesthesia , Animals , Blood Pressure/drug effects , Blood Proteins/metabolism , Bradykinin/administration & dosage , Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Capillary Permeability/drug effects , Dose-Response Relationship, Drug , Guinea Pigs , Hypotension/chemically induced , Hypotension/drug therapy , In Vitro Techniques , Injections, Intravenous , Male , Microcirculation/drug effects , Ornithine/pharmacology , Regional Blood Flow/drug effects
9.
Br J Pharmacol ; 143(8): 938-41, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15545289

ABSTRACT

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.


Subject(s)
Receptor, Bradykinin B2/metabolism , Receptors, Bradykinin/physiology , Signal Transduction/physiology , Animals , Bradykinin/metabolism , Bradykinin/pharmacology , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Humans , Signal Transduction/drug effects
10.
Eur J Pharmacol ; 499(3): 229-38, 2004 Sep 24.
Article in English | MEDLINE | ID: mdl-15381044

ABSTRACT

In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK(2) receptor splice variant named beta isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the alpha isoform. Moreover, neurokinin A was unable to inhibit the [(3)H]SR48968 binding to the beta isoform up to microM concentrations. In cells expressing the human tachykinin NK(2) receptor beta isoform, contrary to those expressing the alpha isoform, natural or selective tachykinin receptor agonists (1 microM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca(2+)](i). The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca(2+)](i) increase in cells expressing the alpha or beta isoform of the human tachykinin NK(2) receptor. The present data indicate that the human tachykinin NK(2) receptor beta isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK(2) receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands.


Subject(s)
Alternative Splicing , Receptors, Neurokinin-2/metabolism , Amino Acid Sequence , Animals , Benzamides/metabolism , Binding, Competitive/drug effects , CHO Cells , Calcium/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cricetinae , Cricetulus , Humans , Inositol Phosphates/biosynthesis , Intracellular Space/drug effects , Intracellular Space/metabolism , Iodine Radioisotopes , Molecular Sequence Data , Neurokinin A/metabolism , Neurokinin A/pharmacology , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Piperidines/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Radioligand Assay , Receptors, Neurokinin-2/agonists , Receptors, Neurokinin-2/genetics , Sequence Homology, Amino Acid , Substance P/metabolism , Substance P/pharmacology , Transfection , Tritium
11.
Eur J Pharmacol ; 491(2-3): 121-5, 2004 May 03.
Article in English | MEDLINE | ID: mdl-15140628

ABSTRACT

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.


Subject(s)
Aspartic Acid/metabolism , Bradykinin/analogs & derivatives , Bradykinin/metabolism , Peptide Fragments/metabolism , Receptor, Bradykinin B2/metabolism , Animals , Bradykinin B2 Receptor Antagonists , Cricetinae , Dose-Response Relationship, Drug , Humans , Peptide Fragments/pharmacology , Quinolines/metabolism , Quinolines/pharmacology , Receptor, Bradykinin B2/agonists
12.
Biochem Pharmacol ; 67(4): 601-9, 2004 Feb 15.
Article in English | MEDLINE | ID: mdl-14757160

ABSTRACT

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.


Subject(s)
Bradykinin B2 Receptor Antagonists , Oligopeptides/pharmacology , Peptides, Cyclic/pharmacology , Quinolines/pharmacology , Receptor, Bradykinin B2/metabolism , Animals , Binding, Competitive , CHO Cells , Cricetinae , Female , Humans , Models, Molecular , Mutagenesis, Site-Directed , Quinolines/chemistry , Receptor, Bradykinin B2/genetics
13.
J Comb Chem ; 5(6): 760-8, 2003.
Article in English | MEDLINE | ID: mdl-14606803

ABSTRACT

Preliminary studies and synthesis development for the preparation of a bicyclic homodetic peptide library have been carried out using orthogonal protection schemes. The best results have been obtained using two Fmoc/tBu-based strategies, in which the first cycle is carried out in the solid phase through side chain functional groups previously protected with Aloc/Al groups. The second cycle is performed either in the solid phase, which requires side chain anchoring of a trifunctional amino acid and Dmb protection for the C-terminus carboxyl group, or in solution, which requires the use of highly labile resins, such as the 2-chlorotrityl (Barlos) resin. Only when the cycles are formed in a ziplike manner, that is, first the small cycle and then the larger ring, is the desired final product obtained.


Subject(s)
Bridged Bicyclo Compounds/chemistry , Peptide Library , Technology, Pharmaceutical/methods
14.
Br J Pharmacol ; 140(3): 500-6, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12970081

ABSTRACT

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.


Subject(s)
Bradykinin/metabolism , Quinolines/metabolism , Receptor, Bradykinin B2/genetics , Receptor, Bradykinin B2/metabolism , Animals , CHO Cells , Cricetinae , DNA Mutational Analysis/methods , Dose-Response Relationship, Drug , Humans , Mutation , Protein Binding/physiology , Receptor, Bradykinin B2/agonists
15.
Peptides ; 23(8): 1457-63, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12182947

ABSTRACT

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.


Subject(s)
Bradykinin Receptor Antagonists , Bradykinin/analogs & derivatives , Bradykinin/metabolism , Animals , CHO Cells , Cricetinae , Humans , Mutagenesis, Site-Directed , Receptor, Bradykinin B2 , Receptors, Bradykinin/genetics , Receptors, Bradykinin/metabolism , Structure-Activity Relationship
16.
Can J Physiol Pharmacol ; 80(4): 303-9, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12025965

ABSTRACT

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.


Subject(s)
Bradykinin Receptor Antagonists , Point Mutation , Receptors, Bradykinin/genetics , Animals , Binding, Competitive/genetics , Bradykinin/metabolism , CHO Cells , Cricetinae , DNA Mutational Analysis/methods , Humans , Ligands , Models, Molecular , Protein Binding/genetics , Quinolines/metabolism , Quinolines/pharmacology , Receptor, Bradykinin B2 , Receptors, Bradykinin/chemistry , Receptors, Bradykinin/metabolism
17.
Br J Pharmacol ; 135(1): 266-74, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11786503

ABSTRACT

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.


Subject(s)
Protein Precursors/pharmacology , Receptors, Tachykinin/agonists , Tachykinins/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Blood Pressure/drug effects , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Guinea Pigs , Heart Rate/drug effects , Ileum/drug effects , Ileum/physiology , Male , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Pulmonary Artery/drug effects , Pulmonary Artery/physiology , Rabbits , Radioligand Assay , Rats , Rats, Wistar , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/metabolism , Receptors, Neurokinin-2/agonists , Receptors, Neurokinin-2/metabolism , Receptors, Neurokinin-3/agonists , Receptors, Neurokinin-3/metabolism , Receptors, Tachykinin/metabolism , Saliva/metabolism , Salivation/drug effects , Salivation/physiology , Sequence Homology, Amino Acid , Urinary Bladder/drug effects , Urinary Bladder/physiology
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