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. EXPERIMENTAL APPROACH: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B2 receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B2 receptors. KEY RESULTS: Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B2 receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B2 receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B2 receptor complex is proposed. CONCLUSIONS AND IMPLICATIONS: MEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

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. While some overlapping of the binding sites exists, the binding modes suggested by this study appear not to allow structural correlation, and therefore general SAR, between these two antagonists.

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. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.

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, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K(d) = 4.8 and 11.5 nM, respectively); Y(266)F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K(d) = 2.8 nM and 1.2 nM, respectively); F(270)A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K(d) = 1.6 nM); Y(289)F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K(d) = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H(198)A and Y(266)F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y(289)F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK(2) receptor.

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. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists.

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. By contrast, deletion of ARE did not appear to affect rhG-CSF production when 3'-UTR sequences from the SV40 early region were used. The most efficient G-CSF transcription unit, fused to a dihydrofolate reductase (DHFR) marker gene and transfected into a CHO cell line, yielded initial transfectant CHO cell lines secreting up to 21 micrograms rhG-CSF/1 x 10(6) cells in 24 h. After two rounds of DHFR gene amplification, a cell line was isolated that contains approx 12 copies of the vector and produces rhG-CSF at a rate of 90 micrograms/1 x 10(6) cells in 24 h.

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. The sequence imposed by the primers did not affect the targeting potential of the antibody. In fact, the affinity of the chimeric antibody for EGF-r was nearly the same as that of the parental murine antibody. Based on previous in vitro and in vivo animal studies. Mint5 was shown to be a good candidate for the targeting of EGF-r overexpressing tumours. chMint5 is expected to be less immunogenic than murine antibody and therefore, could be useful for human treatment.

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. The h2CMV was more active than the other promoters in Bowes human melanoma cells and in Vero African green monkey kidney cells. In human hepatoma (Hep G2) cells, similar levels of CAT synthesis were observed with the h2CMV- and the mCMV-based vectors. In Hep G2 and Bowes cells, 3'-UTR from the SV40 early region resulted in consistently higher levels of cat expression, as compared to the rabbit beta 1-Glb gene, while the converse was true in BALB/3T3 clone A31 and Vero cells. SV40 early region and rabbit beta1-Glb gene 3'-UTR resulted in similar cat expression in CHOdhfr- cells.

[Multiple forms of adenosine deaminase in the thymus of the bovine fetus and calf]. / Forme multiple di adenosina deaminasi nel timo di feti bovini e di vitello.

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. Forms "B" and "C" were compared. The relative amounts of the three forms in 17 weeks old fetus were quite similar to those observed in calf thymus.