Cardiovascular effects of peptide kinin B2 receptor antagonists in rats

Bradykinin (BK) is a vasoactive peptide reputed to play an important role in cardiovascular homeostasis. In this study, we describe the cardiovascular changes (mean blood pressure (BP) and heart rate (HR)) induced by the i.v. administration (left jugular vein) of two selective kinin B2 receptor antagonist, namely icatibant (0.1-1 micromol/kg as a bolus) and MEN1 1270 (0.1-1 micromol/kg as a bolus or 1 micromol/kg infused in 15 or 60 min), in urethane-anaesthetized or conscious rats with an indwelling catheter implanted in the right carotid artery for BP measurements. In conscious rats, icatibant at 0.1 or 0.3 micromol/kg did not change BP but at 0.1 micromol/kg increased HR at 30 min from administration. MEN1 1270 at 0.1 or 0.3 micromol/kg induced a dose-related increase in BP and a concomitant bradycardia (significant at 0.3 micromol/kg) lasting for 5 or 30 min, respectively. Icatibant at 1 micromol/kg induced a slight (P < 0.05) increase in BP that resolved in 5 min and a biphasic tachycardia (peaks at 30 and 90 min from administration). MEN1 1270 at 1 micromol/kg induced a triphasic change in HR (tachycardia in the first 5 min, bradycardia at 30 min, and tachycardia at 90 and 120 min) and a biphasic change in BP (hypotension at 15 min and hypertension at 30 min). The i.v. infusion of MEN1 1270 (1 micromol/kg in 15 or 60 min) produced hypertension, whereas HR was increased only following the 15-min infusion. In urethane-anaesthetized rats, both icatibant and MEN1 1270 (0.1 micromol/kg as a bolus) increased BP and the onset for this effect was correlated with the time course of the antagonism of BK-induced hypotension, where the effect of MEN1 1270 was more rapid than that of icatibant. These results indicate that kinin B2 receptor antagonists can induce acute cardiovascular effects, and the reason for the different haemodynamic profile between icatibant and MEN1 1270 could be putatively attributed to kinetic characteristics.     

Effects of naloxone benzoylhydrazone on native and recombinant nociceptin/orphanin FQ receptors

We have studied the effects of naloxone benzoylhydrazone (NalBzoH) at recombinant human OP4 receptors expressed in Chinese hamster ovary (CHO) cells (CHOhOP4) and native OP4 sites in isolated tissues from various species. In CHOhOP4 membranes, nociceptin (NC) and NalBzoH displaced [125I]Tyr14-NC with pKi values of 10.1 and 7.3. In the presence of 100 microM GDP, NC stimulated GTPgamma35S binding (pEC50 = 8.5). NalBzoH was ineffective but antagonized the effects of NC (pA2 = 6.9). At 5 microM GDP, there was an increase in potency (pEC50 = 9.3) and efficacy (4.3-fold) of NC. NalBzOH was a partial agonist (pEC50 = 7.0, Emax = 13% relative to NC). In CHOhOP4 cells, NC and NalBzoH inhibited cAMP formation with pEC50 and Emax values of 9.8 and 100% and 6.0 and 44%, respectively. In the rat vas deferens, NalBzoH (10 microM) did not modify electrically induced twitches but competitively antagonized the inhibitory action of NC (pA2 = 6.2). In the mouse vas deferens (mVD) and guinea pig ileum (gpI), NalBzoH inhibited twitches with pEC50 and Emax values of 7.6 and 78% and 8.5 and 77%, respectively. The effect of 3 microM NalBzoH was fully inhibited by 3 microM naloxone in mVD and 30 microM in gpI. Under these conditions, NalBzoH antagonized the actions of NC in both preparations with pA2 values of 6.3 and 6.8, respectively. Collectively, these data demonstrate that NalBzoH is a nonselective OP4 ligand with system-dependent behaviour.     

The design and pharmacology of novel selective muscarinic agonists and antagonists

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.     

The design of novel methoctramine-related tetraamines as muscarinic receptor subtype selective antagonists

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.     

Muscarinic antagonists in development for disorders of smooth muscle function

Compounds with high affinity for muscarinic M3 receptors have been used for many years to treat conditions associated with altered smooth muscle tone or contractility such as urinary urge incontinence, irritable bowel syndrome or chronic obstructive airways disease. M3 selective antagonists have the potential for improved toleration when compared with non-selective compounds. Darifenacin has high affinity (pKi 9.12) and selectivity (9 to 74-fold) for the human cloned muscarinic M3 receptor. Consistent with this profile, the compound potently inhibited M3 receptor mediated responses of smooth muscle preparations (guinea pig ileum, trachea and bladder, pA2 8.66 to 9.4) with selectivity over responses mediated through the M1 (pA2 7.9) and M2 receptors (pA2 7.48). Interestingly, darifenacin also exhibited functional tissue selectivity for intestinal smooth muscle over the salivary gland. The M3 over M1 and M2 selectivity of darifenacin was confirmed in a range of animal models. In particular, in the conscious dog darifenacin inhibited intestinal motility at doses lower than those which inhibit gastric acid secretion (M1 response), increase heart rate (M2 response) or inhibit salivary secretion. Clinical studies are ongoing to determine if darifenacin has improved efficacy and or toleration when compared with non-selective agents.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N-terminal part of the molecule with l-beta-homophenylalanine.

In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l-beta-homophenylalanine (beta-Hph). Two of them had just this single modification, the next two peptides are analogues of the V2 agonist, namely [3-mercaptopropionic acid (Mpa)1]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)1]AVP, with beta-Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa(1),beta-Hph2]AVP, [Cpa1,beta-Hph2]AVP, [Cpa1,beta-Hph3]AVP, turned out to be uterotonic antagonists with pA2 = 6.3 +/- 0.2, 6.3 +/- 0.1, 6.0 +/- 0.3 respectively. The last one exhibited antipressor properties also (pA2 = 6.4 +/- 0.1).     

Analogues of arginine vasopressin modified in the N-terminal part of the molecule with enantiomers of N-methylphenylalanine.

Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N-methylphenylalanine were synthesized and studied to assess the influence of N-methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N-methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me-d-Phe(2), Me-Phe(3)]AVP and [Me-d-Phe(2,3)]AVP, which showed low antiuterotonic activity (pA(2) = 6.6 and pA(2) = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.     

Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.     

Analogues of a potent oxytocin antagonist with truncated C-terminus or shorter amino acid side chain of the basic amino acid at position 8.

Twelve analogues were synthesized, their structure derived from modifications of [(S)Pmp1, D-Trp2, Pen6, Arg8]oxytocin, PA, in which (S)Pmp = beta,beta-(3-thiapentamethylene-beta-mercaptopropionic acid). PA is a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 8.86) and in the baboon. Truncated analogues of PA from the C-terminus were systematically prepared ending in either the free acid or the amide, i.e. PA1-9 acid, PA1-8 acid, PA1-7 acid, PA1-6 acid, PA1-8 amide, PA1-7 amide and PA1-6 amide. PA1-8 amide was roughly as potent as PA in the rat uterotonic assay in vitro, and the shorter amides were only somewhat weaker antagonists. All four acid analogues were weaker antagonists than PA but still maintained rather high antagonistic potency. These findings suggest that, if these truncated acids form as metabolites in vivo, they may contribute to the overall biological effect of PA and their contribution should be taken into account. Furthermore, using these analogues, the radioimmunoassay measurements of PA may be standardized, as they may cross react with PA antibodies and interfere with the determination. In addition, five analogues were made by substituting Arg8 of PA with Lys, Orn8, Dab8, Dap8 and Cit8. All of these analogues maintained high potency as OTAs in the uterotonic assay, although their activity was only about 1.5-3 times lower than PA. The most potent analogue in the uterotonic assay, [Dap8]PA, pA2 = 8.53, had weak pressor activity (pA2 = 6.90) and no antidiuretic effect. The pressor activity was lower for all tested acids, and for PA1-6 acid it was even below the detection limit. Additionally, PA1-9 acid, PA1-7 acid and PA1-6 acid showed no antidiuretic activity. Hence, the PA1-6 acid is a potent OTA with pA2 = 8.27 and no measurable effect in the pressor or antidiuretic tests and thus it is a pure oxytocin antagonist. This fact makes it an attractive candidate for further studies on inhibition of OT biological effects and on preterm labour.     

Methylene analogues of adenosine 5'-tetraphosphate. Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases

Adenosine 5'-polyphosphates have been identified in vitro, as products of certain enzymatic reactions, and in vivo. Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5'-polyphosphates into the corresponding nucleoside 5'-triphosphates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: alpha,beta-methylene-adenosine 5'-tetraphosphate (pppCH2pA), beta,gamma-methylene-adenosine 5'-tetraphosphate (ppCH2ppA), gamma,delta-methylene-adenosine 5'-tetraphosphate (pCH2pppA), alphabeta,gammadelta-bismethylene-adenosine 5'-tetraphosphate (pCH2ppCH2pA), alphabeta, betagamma-bismethylene-adenosine 5'-tetraphosphate (ppCH2pCH2pA) and betagamma, gammadelta-bis(dichloro)methylene-adenosine 5'-tetraphosphate (pCCl2pCCl2ppA), and tested them as potential substrates and/or inhibitors of three specific nucleoside tetraphosphatases. In addition, we employed these p4A analogues with two asymmetrically and one symmetrically acting dinucleoside tetraphosphatases. Of the six analogues, only pppCH2pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p4A-hydrolysing enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinucleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed lupin. ppCH2ppA and pCH2pppA, inhibited the human enzyme with Ki values of 1.6 and 2.3 nm, respectively, and the lupin enzyme with Ki values of 30 and 34 nm, respectively. They are thereby identified as being the strongest inhibitors ever reported for the (asymmetrical) dinucleoside tetraphosphatases. The three analogues having two halo/methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono- and dinucleoside polyphosphates and on the enzymes involved in their metabolism.     

Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26).     

New conformationally homogeneous beta-turn antagonists of the human B2 kinin receptor.

We have designed and synthesized a conformationally homogeneous series of cyclic pentapeptides of the general structure c[Pro-aa(i)-D-Tic-Oic-aa(i + 3)] which adopt a type-II' beta-turn conformation believed important for high affinity antagonism of the bradykinin (BK) B2 receptor. We incorporated D-Tic and octahydroindole-2-carboxylic acid (Oic) residues (present in known active antagonists) in a cyclic pentapeptide that would place the D-aa in the i + 1 position of the beta-turn and a proline as a bridge between the C- and N-termini sides of the turn. In positions i and i + 3 alkyl, aromatic, polar or charged amino acids could be introduced without dramatically changing the overall structure. Ten analogues were studied using 1H nuclear magnetic resonance (NMR) and evaluated for their binding affinity for the human B2 receptor. The NMR data in dimethylsulfoxide (DMSO) confirmed the structural homogeneity within the class and, on the basis of this, one representative member of the series was chosen for a detailed structure determination using NMR data in sodium dodecylsulphate (SDS) micelles and molecular dynamics calculations. Despite the structural similarity, the binding affinity of the ten analogues was strongly influenced by the nature of the side-chains in positions i and i + 3, with the doubly charged analogue 49 (pKi = 6.2) proving best. This compound may serve as the starting point for the discovery of new non-peptide bradykinin B2 receptor antagonists.     

Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI.

The varicella-zoster virus (VZV) genome contains 70 reading frames (ORF), 5 of which encode the glycoproteins gpI, gpII, gpIII, gpIV, and gpV. ORF 67 and 68 lie adjacent to each other in the unique short region of the VZV genome and code for gpIV and gpI, respectively. These two genes, which are contained within the HindIII C fragment of the VZV genome, were subcloned in the correct orientation downstream from the promoter regions of the eukaryotic expression vectors pCMV5 and pBJ. After transfection, 5 to 20% of the Cos cells bound antibody specific for the given glycoprotein. In this study, it was shown that only the cells transfected with the gpI construct bound to the Fc fragment of human immunoglobulin G. Neither the transfected gpIV gene product nor the vector only bound to the Fc fragment. Thus, VZV gpI is confirmed to be the VZV-encoded Fc-binding glycoprotein. Like the wild-type form of gpI expressed in VZV-infected cells, gpI precipitated from transfected cells contained both N-linked and O-linked glycans and was heavily sialated. In addition, the transfected gpI gene product was phosphorylated both in cell culture and in protein kinase assays by mammalian casein kinases I and II. Extensive computer-assisted analyses of the VZV gpI sequence, as well as those of alphaherpesviral homolog glycoproteins, disclosed properties similar to those of other cell surface receptors; these included (i) exocytoplasmic regions rich in cysteine residues, (ii) membrane-proximal regions with potential O-linked glycosylation sites, and (iii) cytoplasmic domains with consensus phosphorylation sites.     

Design of oxytocin antagonists, which are more selective than atosiban.

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect to VP V1a receptors than Atosiban. Since we have shown that V1a antagonism could be an unwanted side-effect in tocolytics, peptide 4 and some of the OT antagonists reported here have advantages over Atosiban and thus may be suitable candidates for evaluation as potential tocolytic agents for the treatment of preterm labour.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Organophosphate analogs of biologically active compounds. XIV. Halophosphonate analogs of ATP as acetyl CoA carboxylase inhibitors

Halophosphonate ATP analogues pp[CHBr]pA and p[CHBr]ppA synthesised from bromomethylenebisphosphonate and adenosine derivatives, proved to be effective competitive inhibitors of Ac-CoA-carboxylase (CE 6.4.1.2) from rat liver (Ki = 0,2 mM). The inhibitory effects of both analogues were reversible and higher than those of some other ATP analogues. Another enzyme, Ac-CoA-synthetase (CE 6.2.1.1), with a different mode of ATP-cleavage showed wider specificity to ATP-analogues than Ac-CoA-carboxylase.     

Steady-state kinetic studies of binding and catalysis by ribonuclease T2: a microenvironmental survey of the active site by using a series of adenosine-3'- and -5'-alkylphosphates

The effects of a series of adenosine-3'-alkylphosphates, Ap(CH2)n-1CH3 with n = 1-8, on the binding and catalytic activities of RNase T2 were investigated. The inhibition of the RNase T2-catalyzed reaction by a series of adenosine-5'-alkylphosphates, CH3(CH2)n-1pA with n = 1-7, was also studied. Multiple regression analyses of the observed kinetic constants were carried out to determine the contribution of polar and hydrophobic effects to kcat, kcat/Kmi, and cosubstrate and inhibitor bindings (Kmi and Ki). The data show that the pKmi for Ap(CH2)n-1CH3 increases uniformly with n up to n = 4, then remains constant. By contrast pKi for CH3(CH2)n-1pA is independent of n. The data for log kcat correlated well with polar and hydrophobic variables. On the other hand, log kcat/Kmi is independent of a hydrophobic effect, and the data were fitted by a single polar variable equation. The contribution of hydrophobic regions at or near the active site of RNase T2 is discussed. The present results also offer evidence for the existence of an isomerization step of the first formed enzyme-substrate complex.     

DAU 6285: a novel antagonist at the putative 5-HT4 receptor.

The antagonistic properties of DAU 6285, an azabicycloalkyl benzimidazolone derivative, at putative 5-hydroxytryptamine4 (5-HT4) receptors were investigated in in vitro preparations of guinea-pig ileum and human atrium, in comparison to ICS 205-930. DAU 6285 behaved as a competitive antagonist in all the preparations examined. Its affinity (pA2) ranged between 6.50 and 7.12 in the test models considered. The affinity of ICS 205-930 was 2-3 fold lower. At variance with ICS 205-930, DAU 6285 displayed a weak affinity for 5-HT3 receptors (pKi = 6.1, rat cortex; pA2 less than 5, guinea-pig ileum). In the guinea-pig ileum, DAU 6285 (10 microM) did not exert antimuscarinic, antihistaminic, antinicotinic or myolytic activity. Moreover, it did not bind to other 5-HT receptor subtypes, or to adrenergic, dopaminergic, benzodiazepine, nicotine, GABA receptors. DAU 6285 may represent a suitable tool for studies in the field of 5-HT4 receptors.     

Analogues of oxytocin antagonists bearing a ureido group in the amino acid side chain at position 4 or 5.

Substitution of the side chain carboxamido group at position 4 in the potent oxytocin antagonist (OTA) [ThiaPmp(1), D-Trp(2), Cys(6), Arg(8)]-OT, PA, in which ThiaPmp = beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, led to [Orn(Car)(4)]-PA, ([Cit(4)]-PA), which had uterotonic antagonistic activity equal to that of PA. The same modification at position 5, leading to [Cit(5)]-PA, resulted in antagonistic potency more than 10 times lower than that of PA. This paper also describes the same substitutions introduced in the highly potent OTA [Pen(6)]-PA (antioxytocic in vitro pA(2) = 8.72). Analogues of the general formula [U(4)-X(5)-Pen(6)]-PA, in which U = Lys, Orn, Dab, Dap or X = Orn, Dab or Dap, were synthesized by SPPS. Each of these analogues was carbamoylated by treatment with KCNO in DMF-H(2)O, yielding the corresponding U(Car)(4) or X(Car)(5) derivatives. In the uterotonic assay, the substitution with the ureido group at Gln(4) results in retention of high antagonistic potency, albeit somewhat lower than that of PA, e.g. [Orn(Car)(4), Pen(6)]-PA and [Dab(Car)(4), Pen(6)]-PA having pA(2) = 8.52 and pA(2) = 8.42 respectively. In the pressor assay, [Lys(Car)(4), Pen(6)]-PA and [Dab(Car)(4), Pen(6)]-PA were somewhat weaker antagonists of arginine vasopressin than [Pen(6)]-PA; [Dap(Car)(4), Pen(6)]-PA showed only a faint trace of pressor agonistic activity. The substitution with the ureido group at position 5 leads to a significant loss of OTA potency in the in vitro uterotonic assay. The [Orn(Car)(5), Pen(6)]-PA was the most potent of the series (pA(2) = 8.05). An interesting finding is that [Dap(Car)(5), Pen(6)]-PA is equipotent with its precursor [Dap(5), Pen(6)]-PA (potency in the uterotonic test in vitro, pA(2) = 7.71 and pA(2) = 7.68, respectively). Furthermore, neither [Dap(5), Pen(6)]-PA nor [Dap(5), Pen(6), Gly(9)]-PA exhibited activity in the antidiuretic or pressor assays. Although these last two analogues show some decrease in antioxytocin potency, they behave as pure oxytocin antagonists, which makes them attractive candidates for further studies on the development of potent and specific OTAs.