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.

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.

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.

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.

Pharmacological evaluation of alpha and beta human tachykinin NK(2) receptor splice variants expressed in CHO cells.

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.

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.

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.

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.

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).

Expression and characterization of biologically active human Fas ligand produced in CHO cells.

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh- FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 microg/L x 10(6) cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures.

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.

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.

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.

Molecular determinants of peptide and nonpeptide NK-2 receptor antagonists binding sites of the human tachykinin NK-2 receptor by site-directed mutagenesis.

A series of 14 mutants on nine selected residues of the human tachykinin NK(2) receptor was produced and stably transfected into CHO cells to investigate the binding of the peptide MEN 11420 and the nonpeptide SR 48968 antagonists. The main interactions found for MEN 11420 were with Thr171, Tyr206, Tyr266 and Phe270. In the case of SR 48968 crucial residues were Tyr266 and Tyr289.

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms. Pharmacokinetic and activity studies of single-dose administration in mice.

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc.

Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK(2) receptor.

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK(2) receptor, either wild-type or mutated, at four aromatic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [(125)I]neurokinin A (NKA), the peptide antagonist [(3)H]MEN 11420, and the nonpeptide antagonist [(3)H]SR 48968 bound to the wild-type receptor with high affinity (K(d) = 2.4 nM, 0.

Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells.

A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA.

High-level expression of a cDNA for human granulocyte colony-stimulating factor in Chinese hamster ovary cells. Effect of 3'-noncoding sequences.

We compared the production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by Chinese hamster ovary (CHO) cells in a transient expression system, using different analogous vectors carrying a human G-CSF-encoding cDNA under the transcriptional control of the murine cytomegalovirus (CMV) major immediate early promoter. Comparison of two transcription units carrying a human (h)G-CSF cDNA deleted of 3'-untranslated (UTR) sequences containing AT-rich elements (ARE) and using 3'-UTR sequences for processing of transcripts from the SV40 early region or from the rabbit beta 1-globin gene showed that use of the sequences from the rabbit beta 1-globin gene resulted in 7- to 12-fold higher levels of rhG-CSF production. Deletion of ARE of hG-CSF cDNA resulted in increased rhG-CSF synthesis when transcription units using 3'-UTR sequences from the rabbit beta 1-globin gene were compared.

Expression and characterization of a mouse/human chimeric antibody specific for EGF receptor.

Murine antibodies which recognize the epidermal growth factor receptor (EGF-r) are good candidates for therapy and diagnosis of tumors overexpressing this receptor. Here we report the isolation of the variable regions from a murine monoclonal antibody anti-EGF-r (Mint5), the procedure to obtain the mouse/human chimeric antibody (chMint5) and its expression in COS, NS0 and CHO cells. The approach followed to construct chMint5 is based on the use of consensus primers specific for the ends of the variable regions.

Efficiency of different viral promoters in directing gene expression in mammalian cells: effect of 3'-untranslated sequences.

We compared (i) the enhancer/promoter (mCMV promoter) from the murine cytomegalovirus (CMV) major immediate early gene,(ii) the enhancer/promoter from human CMV major immediate early gene, containing a short promoter (h1CMV) or a long stretch of 5' untranslated region (UTR) from the gene promoter (h2CMV) and (iii) the simian virus 40 (SV40) enhancer/early region promoter (SV2) for their ability to direct foreign gene expression in transiently transfected mammalian cell lines. Two series of recombinant plasmids containing the different viral promoters fused to the cat reporter gene and 3'-UTR for processing of transcripts from either the SV40 early region or the rabbit Beta 1-globin-encoding gene (Glb) were also analyzed for their effect on transient gene expression. The mCMV was the most active in dihydrofolate reductase-deficient Chinese hamster ovary (CHOdhfr-) cells and BALB/3T3 clone A31 mouse embryo cells.

[Multiple forms of adenosine deaminase in the thymus of the bovine fetus and calf].

We present data on values of adenosine deaminase (ADA) activity in bovine fetus and calf thymus. Between 13 and 17 weeks of fetal life enzyme activity in thymus increases up to levels observed in older fetuses and calf. ADA activity of thymus is characterized by the presence of three enzymic form ("A", "B", "C") which show molecular weights of greater than 1 x 10(6), 310,000 and 55,000, respectively.