Guinea Pig Lung Membranes Research Articles

Synthesis, structure-activity relationships, and pharmacological evaluation of a series of fluorinated 3-benzyl-5-indolecarboxamides: identification of 4-[[5-[((2R)-2-methyl-4,4,4-trifluorobutyl)carbamoyl]-1-methyl indol- 3-yl]methyl]-3-methoxy-N-[(2-methylphenyl)sulfonyl]benzamide, a potent, orally active antagonist of leukotrienes D4 and E4.

The continued exploration of a series of 3-(arylmethyl)-1H-indole-5-carboxamides by the introduction of fluorinated amide substituents has resulted in the discovery of 4-[[5-[((2R)-2-methyl-4,4,4-trifluorobutyl)carbamoyl]-1-methyli ndol- 3-yl]methyl]-3-methoxy-N-[(2-methyl-phenyl)sulfonyl]benzamide (38p, ZENECA ZD3523), which has been chosen for clinical evaluation. This compound exhibited a Ki of 0.42 nM for displacement of [3H]LTD4 on guinea pig lung membranes, a pKB of 10.13 +/- 0.14 versus LTE4 on guinea pig trachea, and an oral ED50 of 1.14 mumol/kg opposite LTD4-induced bronchoconstriction in guinea pigs. The R enantiomer was found to be modestly more potent than the S enantiomer 38o. Modification of the amide substituent to afford achiral compounds was unsuccessful in achieving comparable levels of activity. Profiling of 38p opposite a variety of functional assays has demonstrated the selectivity of this compound as a leukotriene receptor antagonist. The enantioselective synthesis of 38p, which employed a diastereoselective alkylation of (4R,5S)-3-(1-oxo-4,4,4-trifluorobutyl)-4-methyl-5-phenyl-2-oxazoli dinone (27) as the key step to establish the chirality of the amide substituent, provided an efficient route for generating 38p in > 99% enantiomeric purity.

Development of Novel Photoaffinity Ligands for the β-Adrenergic Receptor

The successful synthesis and use of carrier-free radioiodinated β2-adrenergic receptor competitive antagonist photoaffinity labels (±)-[125I]IABP, (±)-[125I]MAPIT, (−)-[125I]IAPTA, and (±)-[125I]IAPCGP-12177, are described. In addition, the synthesis and use of two carrier-free radioiodinated β-adrenergic receptor agonist photoaffinity labels (±)-[125I]iodoazidoprenalterol ((±)-[125I]IAPr) and (−)-N-(p-azido-m-[125I]iodophenethylamidoisobutyl)norepinephrine ((−)-[125I]NAIN), are described. All antagonist photolabels were capable of highly specific derivatization of the purified recombinant hamster lung β2-adrenergic receptor. Tryptic cleavage of the photolabeled receptor into a 30-kDa radiolabeled fragment (transmembrane 1-5) and an 8-kDa radiolabeled fragment (transmembrane 6,7) showed variable Insertion ratios between the two juxtaposed domains, depending on the structure of the photolabel. Unique synthetic strategies were used for the agonist photolabels. The phenolic hydroxyl of (±)-IAPr was protected as the glucoside and deprotected enzymatically in the final step. The final coupling step in the synthesis of (−)-[125I]NAIN was accomplished by reductive alkylation without protection of the catechol hydroxyls of norepinephrine using sodium cyanoborohydride. (±)-IAPr was found to be a partial agonist for the turkey erythrocyte β-adrenergic receptor and an effective photoaffinity label for the avian β-adrenergic receptor. (−)-NAIN was found to be a full agonist for the β2-adrenergic receptor in guinea pig lung membranes and a highly effective agonist photoaffinity label for the β2-adrenergic receptor. These photolabels will be useful for probing the β-adrenergic receptor binding site In order to "map" this site under nonactivated (antagonist photolabels) or activated states (agonist photolabels).

Binding of a catechol derivative of denopamine (T-0509) and N-tert-butylnoradrenaline (Colterol) to beta 1- and beta 2-adrenoceptors.

The affinities for beta-adrenoceptors, the subtype-selectivity and the agonistic effectiveness of T-0509 (a catechol derivative of denopamine) and colterol (N-tert-butylnoradrenaline; Col) were compared with those of other beta-agonists using a binding assay method. Specific binding of [3H]dihydroalprenolol (3H-DHA) to guinea pig left ventricular and lung membranes was saturable, and Scatchard and Hill analyses suggested that 3H-DHA bound to both membranes with a single population of binding sites with no binding site cooperativity. Addition of 5'-guanylylimidodiphosphate (GppNHp, 30 microM) led to a rightward shift of the 3H-DHA binding displacement curves of T-0509 and Col in both membranes, and the degree of shift was similar to that of full agonists such as isoproterenol (Iso), adrenaline (Adr) and noradrenaline (NA). Both T-0509 and Col were thus considered to be full agonists at both beta 1- and beta 2-adrenoceptors, respectively, unlike denopamine and procaterol. T-0509 and Col showed considerably high affinity for both beta 1- and beta 2-adrenoceptors, and T-0509, like denopamine, was as selective for the beta 1-subtype as NA (4.5-fold compared with Iso as a non-selective agonist), whereas Col was more selective for the beta 2-subtype than Adr (4.5-fold compared with Iso).

Identification of both NK1 and NK2 receptors in guinea-pig airways.

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations. 5. Similar studies using guinea-pig tracheal membranes demonstrated that [125I]-neurokinin A specific binding was composed of a NK1-receptor component (60%), inhibited by both [Sar9Met(02)11]substance P and CP-99,994, and a significant NK2-receptor component, inhibited by both [beta Ala 8]neurokinin A andSR48968.6. In summary, these data demonstrate that guinea-pig lung parenchyma and guinea-pig trachea express both NK1 and NK2 receptors.

Studies on neurokinin antagonists. 3. Design and structure-activity relationships of new branched tripeptides N alpha-(substituted L-aspartyl, L-ornithyl, or L-lysyl)-N-methyl-N-(phenylmethyl)-L-phenylalaninamides as substance P antagonists.

As an extension of our study on discovering a novel substance P (SP) antagonist, we designed new branched tripeptides containing L-aspartic acid (2 and 5), L-ornithine (3 and 6), and L-lysine (4 and 7) by reconstructing the structure of the previously reported tripeptide SP antagonist [Ac-Thr-D-Trp(CHO)-Phe-NMeBzl (1), FR113680]. The strategy for this design was based on the postulate that the dipeptide half D-Trp(CHO)-Phe-NMeBzl in 1 is essential for receptor recognition. Molecular modeling studies implied that these newly designed tripeptides could mimic the spatial orientations of the essential dipeptide structure. As expected, all of these compounds potently inhibited 3H-SP (1 nM) binding to guinea pig lung membranes in the 10(-8) M range. The 1H-indol-3-ylcarbonyl derivatives (5-7) were slightly more potent than the corresponding 1H-indol-2-ylcarbonyl derivatives (2-4), as predicted by the molecular modeling studies. The structure-activity relationships studies on the selected 1H-indol-3-ylcarbonyl derivatives indicated that the threonine moiety at the side chain can be modified into a variety of structures without any significant loss of the activity. Furthermore in the L-lysine series, even dipeptide compounds having nothing or a simple acyl group at the epsilon-amino group, such as N alpha-[N alpha-(1H-indol-3-ylcarbonyl)-L-lysyl]-N-methyl-N-(phenylmethyl)- L-phenylalaninamide (18b), exhibited potent activity. These dipeptides belong to a new structural class of SP antagonist.

Patent Evaluation : EP-528337-A: Fujisawa Pharm Co Ltd: Thiazolylbenzofuran derivatives as anti-asthma agents

Novelty: Novel thiazolylbenzofuran derivatives are disclosed with leukotriene D4 inhibitory activity. They are claimed to be useful in the treatment of inflammatory or allergic conditions such as asthma, bronchitis, inflammatory bowel disease and chronic obstructive lung disease.Biology: [3H]-Leukotriene D4 receptor binding activity in guinea-pig lung membrane preparations was determined by standard radioligand techniques. An IC50 of 38.2 nM was determined for one of the specifically claimed compounds.Chemistry: Fifty-three compounds were exemplified by synthesis and 5(2-[2-(4-tert-butylthiazol-2-yl)benzofuran-5-yloxymethyl]-phenylmeth yl)-1H-tetrazole is specifically claimed.

Photoaffinity labeling of the leukotriene D4 receptor in guinea pig lung.

The leukotriene (LT)D4 receptor has been defined as a G-protein-coupled receptor. In order to characterize this receptor, an iodinated, photoactivatable azido derivative of LTD4 (125I-azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of 125I-azido-LTD4 specific binding to guinea pig lung membranes were directly comparable to those of [3H]LTD4 specific binding to this tissue. 125I-Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. 125I-Azido-LTD4 specific binding was also strongly inhibited by the nonhydrolyzable GTP analog, GTP gamma S, with ATP gamma S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G-protein. The cysteinyl leukotrienes competed for 125I-azido-LTD4 specific binding to guinea pig lung membranes with the following rank order of potency: LTD4 > LTE4 > LTC4, while the non-cysteinyl LTB4 was virtually inactive. Two structurally different LTD4 receptor antagonists, MK-571 and ICI 204,219, also competed for 125I-azido-LTD4 specific binding with nanomolar potency, whereas the leukotriene synthesis inhibitor, MK-886, was 10,000-fold less active. These data are in agreement with 125I-azido-LTD4 binding specifically to a G-protein-coupled LTD4 receptor. Photolysis of 125I-azido-LTD4 under equilibrium binding conditions resulted in the selective radiolabeling of a 45-kDa guinea pig lung membrane protein. The photolabeling of this 45-kDa protein was saturable, modulated by cations and inhibited by nucleotide analogs in an analogous way to 125I-azido-LTD4 specific binding. In addition, the photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the 125I-azido-LTD4 binding assay. It is proposed, therefore, that this novel 45-kDa protein is the guinea pig lung LTD4 receptor.

Absence of functional inhibition of cloned human β2‐adrenergic receptors by autoantibodies in asthmatic subjects

Using guinea-pig lung membranes and cloned human beta 2-receptor adrenergic receptors the effects of whole serum, plasma, purified immunoglobulins and cellular activation products on beta 2-adrenergic receptor ligand binding and function were investigated. Sera from 24 non-asthmatic subjects and 115 asthmatics in different clinical categories were studied. There were no significant differences between antagonist ([125I] cyanopindolol) inhibition mediated by serum, plasma or by purified IgG when the asthmatics were compared with non-asthmatics. There was also no inhibition of 10(-6) M isoproterenol stimulated cAMP release from L cells expressing human beta 2-adrenergic receptors, by plasma, DEAE purified IgG fractions from asthmatics and non-asthmatics, or by products of activated platelets or lymphocytes. Since we have no evidence that immunoglobulins from asthmatic subjects exert functional inhibition of human beta 2-adrenergic receptors we conclude that autoantibodies to the beta 2-adrenergic receptors do not play an important functional role in the pathophysiology of asthma.

Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs.

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-Cys6,D-Cys11,Lys12,Nle17,Val26,Th r28]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys12,Cys17,Val26,Cys28]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (> 40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor-active conformation of VIP.

Characterization of the leukotriene D 4 receptor in dimethylsulphoxide-differentiated U937 cells: comparison with the leukotriene D 4 receptor in human lung and guinea-pig lung

The leukotriene D 4 receptor has been fully characterized by radioligand binding in membrane preparations from dimethyl sulphoxide-differentiated U937 cells, a human monocyte leukemia cell line, and, in parallel experiments, compared with leukotriene D 4 receptor found in human lung and guinea-pig lung preparations. [ 3H]Leukotriene D 4 specific binding in differentiated U937 cell membranes is of high affinity (K D = 0.35 nM), saturable (B max = 287 fmol/mg protein), with differentiation resulting in a 3–5-fold increase in the number of detectable binding sites. [ 3H]Leukotriene D 4-specific binding in differentiated U937 cell membranes displays several features of G-protein-cupled receptors, being inhibited by GTP analogues and sodium ions, but increased by divalent cations. These characteristics are shared with [ 3H]leukotriene D 4-specific binding in human and guinea-pig lung preparations. However, differences between these leukotriene D 4 receptor types were observed. [ 3H]Leukotriene D 4 equilibrium binding to differentiated U937 cell membranes could be dissociated to non-specific binding levels by 1000-fold excess of competing ligand, whereas binding to guinea-pig lung membranes was only partially dissociated under these conditions. In addition, differences in potency were demonstrated in competition studies with leukotriene E 4 and leukotriene C 4, although leukotriene D 4 and the leukotriene D 4-receptor antagonists MK-571 and ICI 204,219 were equipotent in competing for [ 3H]leukotriene D 4-specific binding in all three membranes preparations. In conclusion, the leukotriene D 4 receptor in differentiated U937 cell membranes resembles that in human lung, validating the use of this cell line as a suitable source of receptor in the development of potent specific antagonists.

Effect of eosinophil peroxidase on beta-adrenergic receptor density on guinea pig lung membrane

We studied whether eosinophil peroxidase (EPO), an eosinophil granule basic protein, can alter beta-adrenergic receptor (BAR) density on the guinea pig lung membrane. Lung membrane was first preincubated with 1–10 U/ml EPO and then incubated with 10 −4 M NaI and 10 −4 or 10 −6 M H 2O 2 for 2 hours. BAR density was determined using (-) 125 I-cyanopindolol. EPO combined with 10 −4 M H 2O 2 and I decreased the BAR density in a concentration-dependent manner. When only 10 −4 M H 2O 2 was used, the decrease in BAR density was small but significant. When compared to I, bromide was less effective and chloride alone was not effective. These results suggest that EPO is one of the factors responsible for beta-adrenergic blockade in asthma.

Formula omitted]-alkoxyphenol leukotriene B 4 receptor antagonists

A series of ortho -alkoxyphenols containing a tetrazole acid sidechain have been prepared as antagonists of leukotriene B 4 receptors. These compounds were tested as receptor antagonists of human neutrophil and guinea pig lung membrane leukotriene B 4 receptors. Compounds in this series were found to be up to 18-fold more potent than LY255283. These results indicate that the acyl group of the 1,2,4,5 substituted hydroxyacetophenone class of LTB 4 antagonists is not critical to antagonist potency.

Pharmacological profile of a high affinity dipeptide NK1 receptor antagonist, FK888.

1. In our search for compounds that inhibit the binding of [3H]-substance P (SP) to guinea-pig lung membranes, the dipeptide SP antagonist, FK888, was developed by chemical modification of the parent compound, (D-Pro4, D-Trp7,9,10, Phe11)SP4-11. 2. In a [3H]-SP binding assay using guinea-pig lung membranes and rat brain cortical synaptic membranes, FK888 displaced [3H]-SP binding with a Ki value of 0.69 +/- 0.13 nM and 0.45 +/- 0.17 microM, respectively, in a competitive manner. 3. FK888 inhibited the contraction of guinea-pig isolated ileum induced by SP in the presence of atropine and indomethacin (a NK1 receptor bioassay) with a pA2 value of 9.29 (8.60-9.98). 4. FK888 inhibited contractions of rat vas deferens by NKA (a NK2 receptor bioassay) and of rat portal vein by NKB (a NK3 receptor bioassay) at concentrations at least 10,000 times greater than that required to inhibit contractions of guinea-pig ileum. 5. FK888 also inhibited SP-induced airway oedema in guinea-pig after both intravenous and oral administration. 6. These data demonstrate that FK888 is a potent and selective NK1 antagonist which is active both in vitro and in vivo.

Pharmacologic characterization of the novel ligand [4,5- 3H-Leu 9]Neurokinin-A binding to NK-2 receptors on hamster urinary bladder membranes

We synthesized a novel ligand [4,5- 3H-Leu 9]-Neurokinin A ( 3H-NKA, S.A 117–144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 ± 4%) and protein-dependent manner. Binding was rapid (k 1 = 0.037 nM −1∗min −1) and saturable (B max = 1210 ± 177 fmol/mg protein), to a single population of high-affinity sites (K D = 2.41 ± 0.15 nM, nH = 0.99 ± 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [β-Ala8]-NKA(4–10) > [Nle10]-NKA(4–10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar 9-Met (O2) 11]-SP, (±) CP96,345 and Senktide respectively, did not inhibit binding at 10 μM, whereas, the selective NK-2 antagonists: (±) SR-48,968 ⪢ L-659,877 > R396 ⪢ MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (<30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1–10 μM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.

Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+-)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid.

Based on LTD4 receptor antagonist activity of 3-(2-quinolinyl-(E)-ethenyl)pyridine (2) found in broad screening, structure-activity studies were carried out which led to the identification of 3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (1, MK-571) as a potent and orally active LTD4 receptor antagonist. These studies demonstrated that a phenyl ring could replace the pyridine in 2 without loss of activity, that 7-halogen substitution in the quinoline group was optimal for binding, that the (E)-ethenyl linkage was optimal, that binding was enhanced by incorporation of a polar acidic group or groups in the 3-position of the aryl ring, and that two acidic groups could be incorporated via a dithioacetal formed from thiopropionic acid and the corresponding styrylquinoline 3-aldehyde to yield compounds such as 20 (IC50 = 3 nM vs [3H]LTD4 binding to the guinea pig lung membrane). It was found that one of the acidic groups could be transformed into a variety of the amides without loss of potency and that the dimethylamide 1 embodied the optimal properties of intrinsic potency (IC50 = 0.8 nM on guinea pig lung LTD4 receptor) and oral in vivo potency in the guinea pig, hyperreactive rat, and squirrel monkey. The evolution of 2 to 1 involves the increase of > 6000-fold in competition for [3H]LTD4 binding to guinea pig lung membrane and a > 40-fold increase in oral activity as measured by inhibition of antigen-induced dyspnea in hyperreactive rats.

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.

FR 113680: a novel tripeptide substance P antagonist with NK1 receptor selectivity.

1. We have discovered a novel tripeptide substance P (SP) antagonist, FR 113680 [N alpha-[N alpha-(N alpha-acetyl-L-threonyl)-N'-formyl-D- tryptophyl]-N-methyl-N-phenylmethyl-L-phenylalaninamide]. In binding experiments, FR 113680 inhibited [3H]-SP binding to guinea-pig lung membranes (NK1) in a competitive manner but had not effect on [3H]-SP binding to rat cerebral cortical membranes (NK1), [3H]-neurokinin A ([3H]-NKA) binding to rat duodenum smooth muscle membranes (NK2) and [3H]-eledoisin (Ele) binding to rat cerebral cortical membranes (NK3). 2. In bioassay experiments, FR 113680 dose-dependently inhibited SP-induced guinea-pig ileum contraction (NK1), but did not inhibit either NKA-induced rat vas deferens contraction (NK2) or neurokinin B (NKB)-induced contraction of rat portal vein (NK3). According to Schild plot analysis, the inhibitory effect of FR 113680 on SP-induced guinea-pig ileum contraction is competitive and the pA2 value is 7.53. 3. The inactivity of FR 113680 on NK1 receptors in rat compared to guinea-pig may represent species-specific forms of the NK1 receptor. 4. These findings suggest that FR 113680 interacts selectively with the NK1 neurokinin receptor.

Inhibition of Radiolabeled Leukotriene-Binding by AS-35 in Guinea Pig Lung Membrane Fraction

The inhibitory effects of 9-[(4-acetyl-3-hydroxy-2-n-propylphenoxy)-methyl]-3-(lH-tetrazol-5-yl)-4H-pyrido[l,2-α]pyrimidin-4-one (AS-35), a peptide leukotriene (LT) antagonist, on specific bindings of radiolabeled LTC4 and LTD4 in guinea pig lung membrane were investigated to clarify the mechanism by which this agent inhibited LT-induced physiological responses. Binding assays were performed at 20’C in 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2, 10 mM MgCl2 and 10 mM cysteine in the absence (LTD4 binding assay) or presence (LTC4 binding assay) of 80 mM L-serine borate. Scatchard analysis of each LT specific binding indicated a single and high affinity binding site with a Kj of 0.21 ± 0.05 nM and Bmax of 808 ± 71 fmol/mg protein for [3H]-LTD4, and with a Kd of 21.6 ± 3.8 nM and Bmax of 74.9 ± 2.6pmol/mg protein for [3H]-LTC4. Competition binding studies showed that AS-35 antagonized [3H]-LTD4 specific binding with a Ki value of 92.7 nM. In contrast, AS-35 was 100 times less effective in inhibiting [3H]-LTC4 specific binding, compared with [3H]-LTD4 specific binding. These results indicate that AS-35 interacts directly with peptide LTs receptors, especially the LTD4 specific binding site to produce its pharmacological effects.

Inhibition of radiolabeled leukotriene-binding by AS-35 in guinea pig lung membrane fraction.

The inhibitory effects of 9-[(4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (AS-35), a peptide leukotriene (LT) antagonist, on specific bindings of radiolabeled LTC4 and LTD4 in guinea pig lung membrane were investigated to clarify the mechanism by which this agent inhibited LT-induced physiological responses. Binding assays were performed at 20 degrees C in 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2, 10 mM MgCl2 and 10 mM cysteine in the absence (LTD4 binding assay) or presence (LTC4 binding assay) of 80 mM L-serine borate. Scatchard analysis of each LT specific binding indicated a single and high affinity binding site with a Kd of 0.21 +/- 0.05 nM and Bmax of 808 +/- 71 fmol/mg protein for [3H]-LTD4, and with a Kd of 21.6 +/- 3.8 nM and Bmax of 74.9 +/- 2.6 pmol/mg protein for [3H]-LTC4. Competition binding studies showed that AS-35 antagonized [3H]-LTD4 specific binding with a Ki value of 92.7 nM. In contrast, AS-35 was 100 times less effective in inhibiting [3H]-LTC4 specific binding, compared with [3H]-LTD4 specific binding. These results indicate that AS-35 interacts directly with peptide LTs receptors, especially the LTD4 specific binding site to produce its pharmacological effects.

WS9326A, a novel tachykinin antagonist isolated from Streptomyces violaceusniger No. 9326. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

Data from several studies suggest that tachykinins may play an important role in the pathophysiology of airway diseases, especially asthma. Our aim is to discover tachykinin antagonists which exhibit therapeutically useful anti-asthmatic activity. In our search for activities inhibiting the binding of [3H]substance P to guinea-pig lung membrane preparations, we have found that the fermentation product, WS9326A, isolated from Streptomyces violaceusniger, is a potent tachykinin receptor antagonist.