Novel C5a receptor antagonists regulate neutrophil functions in vitro and in vivo.

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1-71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1-71 derivatives with replacements of Q71, C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various lengths (71-74) with Q71C replacements. The majority of C5a1-71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1-71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1-71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, CSaRAM and CSaRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 microM), and chemotaxis (11 and 7 microM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.

Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates.

A nonradioactive, sensitive assay method to evaluate the activity of protein tyrosine kinases is described. This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence. Recombinant c-src was used to demonstrate that substrate phosphorylation was dependent on incubation time, enzyme concentration, and the amount of substrate used to coat the microtiter plate wells. A series of proprietary c-src inhibitors was evaluated in competition assays, and demonstrated a rank order of potency which was identical to that determined by other assay methods. Substrate phosphorylation was also demonstrated to be dependent on the concentration of ATP present during the kinase reaction. Because the kinase assay can be performed with different ATP concentrations (unlike with assays utilizing radioactive ATP analogs), the assay described can be used to distinguish compounds that compete for the ATP or substrate binding sites of the kinase.

Identification of a receptor-binding region in the core segment of the human anaphylatoxin C5a.

In order to identify regions of C5a that contribute to receptor binding and functional activity of the anaphylatoxin, a series of peptides was synthesized in which core segments have been attached to C-terminal segments via native peptidic or disulfide bonds. It has been found that residues Arg40 and Arg46 in the loop-3 region of the core induce a 1000-fold increase in the affinity of the disordered C-terminal segment of C5a. The results obtained from this work lead to the conclusion that the loop-3 region is most likely the core binding site of C5a.

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

Stabilization of C5a receptor--G-protein interactions through ligand binding.

Binding of biotin-C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin-agarose affinity chromatography resulted in the isolation of a receptor complex with associated G-proteins. In contrast, when receptor was detergent-solubilized in the absence of C5a and purified by affinity chromatography with Affigel-C5a, G-proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor--G-protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor-effector interactions. When biotin-C5a-ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both Gialpha2 and Gialpha3 subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor-transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems.

Characterization of the binding of Bolton-Hunter labeled [125I]C5a to human neutrophil, monocyte and U-937 cell membranes.

The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

Synthesis and benzodiazepine binding activity of a series of novel [1,2,4]triazolo[1,5-c]quinazolin-5(6H)-ones.

Investigation of tricyclic heterocycles related to the 2-arylpyrazolo[4,3-c]quinolin-3(5H)-ones, structures with high affinity for the benzodiazepine (BZ) receptor, led to the synthesis of 2-phenyl-[1,2,4]triazolo[1,5-c]quinazolin-5(6H)-one, a compound with 4 nM binding affinity to the BZ receptor. Analogues were prepared to assess the importance of the 2-substituent and ring substitution in modifying activity. Several novel synthetic routes were designed to prepare the target compounds, including a two-step synthesis beginning with an anthranilonitrile and a hydrazide. Of the 34 compounds screened in this series, three compounds were found to be potent BZ antagonists in rat models. The leading compound, 9-chloro-2-(2-fluorophenyl) [1,2,4]triazolo[1,5- c]quinazolin-5(6H)-one (CGS 16228), showed activity comparable to that of CGS 8216 from the pyrazolo[4,3-c]quinoline series.

CGS 20625, a novel pyrazolopyridine anxiolytic.

CGS 20625 (2-(4-methoxyphenyl)2,3,5,6,7,8,9,10-octa hydrocyclohepta[b]pyrazolo-[3,4-d]pyridin-3-one) is a potent and selective ligand for the central benzodiazepine receptor (IC50 = 1.3 nM), with little or no affinity to several other neurotransmitter receptor binding sites in vitro. CGS 20625 had a gamma-aminobutyric acid ratio of 0.9 and increased t-[35S]butylbicyclophosphorothionate binding by 20% in vitro, a profile indicative of a partial agonist or mixed agonist/antagonist. In vivo, CGS 20625 blocked a pentylenetetrazol discriminative cue with an ED50 = 1.7 mg/kg p.o. The compound selectively increased conflict responding in the Cook-Davidson paradigm with a minimal effective dose of 0.3 mg/kg p.o., as compared with 3.0 mg/kg p.o. for diazepam. At doses as high as 100 mg/kg p.o., CGS 20625 had no effect on variable interval responding, suggesting minimal sedation. Unlike diazepam, CGS 20625 had no effect on rotorod performance at doses up to 100 mg/kg p.o. indicating no overt muscle relaxation, and did not potentiate the action of ethanol in this behavioral paradigm. Also, CGS 20625 had no marked effect on locomotor behavior, did not potentiate hexobarbital sleep time and had no sedative activity at doses up to 300 mg/kg p.o. CGS 20625 was efficacious in preventing pentylenetetrazol-induced seizures (ED50 = 0.7 mg/kg p.o.), had less efficacy with no clear dose-response relationship against picrotoxin-induced seizures and had no effect on either strychnine or electroshock-induced convulsions at doses up to 300 mg/kg p.o.(ABSTRACT TRUNCATED AT 250 WORDS)

The N-methyl-D-aspartate receptor complex.

The effects of agonists of the N-methyl-D-aspartate (NMDA) receptor can be blocked by dissociative anesthetics such as phencyclidine (PCP) in a non-competitive manner. This finding together with the fact that ligand binding to the PCP receptor is dependent on the presence of L-glutamate has led to the suggestion that there may exist an NMDA/PCP receptor complex in mammalian brain tissue. This concept has been extended to the inclusion of a cation channel based on the inhibitory actions of the divalent cation, magnesium. Evaluation of the binding of tritiated TCP (thienylcyclohexylpiperidine) a high affinity ligand for the PCP receptor, under four conditions: in basal, well washed rat cortical membranes; in the presence of L-glutamate; in the presence of magnesium; and in the presence of both magnesium and L-glutamate, with NMDA antagonists and dissociative anesthetics showed that these agents had distinct profiles of activity at the PCP receptor. Furthermore, while both classes of compound could modulate TCP binding, only NMDA receptor antagonists inhibited the binding of tritiated CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) which labels central NMDA recognition sites. The present data support the existence of an NMDA/PCP receptor complex in mammalian brain tissue. The data currently available would suggest however, that the interface is sequentially NMDA to PCP with the latter site affecting NMDA-mediated responses at a step intermediate between receptor activation and physiological response.

Interaction of L-glutamate and magnesium with phencyclidine recognition sites in rat brain: evidence for multiple affinity states of the phencyclidine/N-methyl-D-aspartate receptor complex.

Biochemical and electrophysiological studies have provided evidence that a complex comprising the N-methyl-D-aspartate (NMDA)-type excitatory amino acid (EAA) receptor and the phencyclidine (PCP) recognition site exists in mammalian brain. This complex, which has been compared to that established for the inhibitory amino acid, gamma-aminobutyric acid, and the benzodiazepine anxiolytic, diazepam, is sensitive to the effects of the divalent cation Mg2+, which has suggested the presence of a third, ion channel component. Using a radioreceptor assay for the PCP receptor, L-glutamate (L-Glu) produced a concentration-dependent increase in the binding of [3H]thienyl cyclohexylpiperazine ([3H]TCP) in well washed membranes from rat forebrain. The EAA produced a maximal increase in specific binding of 400%, with an EC50 value of 340 nM. The ability of L-Glu to enhance [3H]TCP binding was 10-fold more potent in the presence of 30 microM Mg2+, which inhibits NMDA-evoked responses in intact tissue preparations and produces a 50% increase in [3H]TCP binding on its own. Analysis of saturation curves indicated that the effect of both L-Glu and Mg2+ could be attributed to an increase in receptor affinity as well as increases in the proportion of a high affinity state of the PCP-binding site. Assessment of the effect of a number of EAAs on basal [3H]TCP binding (well washed membranes in the absence of either L-Glu or Mg2+) showed that the EAA recognition site involved in the effects of L-Glu was the NMDA subtype. Further studies examined a series of compounds thought to interact with either the NMDA or PCP components of the receptor complex under four binding conditions: basal, +Mg2+; +L-Glu; and +Mg2+/L-Glu. These results showed that dissociative anesthetics, such as dexoxadrol and PCP, as well as the novel anticonvulsant MK-801, selectively interact with the high affinity state of the PCP receptor. NMDA antagonists, such as CPP, were also found to inhibit binding to the high affinity state of the PCP receptor, although not as potently as the dissociative anesthetics. Interestingly, the NMDA antagonists did not inhibit any of the binding to the low affinity state of the receptor. The sigma ligands (+/-)-SKF 10,047 and haloperidol recognized two components of [3H]TCP binding only in the presence of L-Glu. The results of the present study are consistent with the finding that agonists of the NMDA receptor induce a high affinity state of the PCP receptor.

Benzodiazepine receptor modulation of [35S]TBPS binding to the chloride channel. Noncompetitive inhibition of classical benzodiazepines and competitive inhibition of the partial agonist, CGS 9895, by CGS 8216.

Benzodiazepine receptor ligands are known to modulate the binding of [35S]TBPS to the chloride channel via an allosteric action. In the cases of diazepam, zopiclone and CGS 9895, the enhanced binding of [35S]TBPS induced by these benzodiazepine agonists was antagonized by CGS 8216. This antagonism was characterized both by a shift to the right and a decrease in the maximal stimulation, for the dose-response curves of diazepam and zopiclone. In the case of CGS 9895, the maximal response was not decreased. These data indicate that CGS 8216 is a noncompetitive antagonist of classical benzodiazepine receptor agonists but is a competitive inhibitor of the partial agonist, CGS 9895.

Biochemical characterization of the triazoloquinazoline, CGS 15943, a novel, non-xanthine adenosine antagonist.

CGS 15943A, a triazoloquinazoline, is a potent and selective adenosine receptor antagonist as assessed by its effects on radioligand binding and adenosine-stimulated adenylate cyclase activity in guinea pig synaptoneurosomes. At the adenosine A-1 receptor labeled with [3H]cyclohexyladenosine, CGS 15943A had an IC50 value of 20 nM. At the striatal A-2 receptor labeled with [3H]5'-N-ethylcarboxamidoadenosine in the presence of a low concentration of cyclopentyladenosine to block A-1 receptors labeled by this nonselective adenosine agonist, CGS 15943A had an IC50 value of 3 nM, indicating that the compound had some degree of selectivity for the A-2 receptor. Analysis of the effect of the compound on the saturation isotherms for each of the receptors indicated that it was a competitive antagonist at the brain A-1 receptor but that it was noncompetitive at the striatal A-2 receptor. CGS 15943A was a potent adenosine antagonist in the adenosine-stimulated adenylate cyclase system in guinea pig synaptoneurosomes, where the compound was found to have an IC50 value of 30 to 70 nM against the increase in cyclic AMP evoked by 5 microM adenosine. CGS 15943A had no effect on the binding of [3H]nitrobenzylthioinosine, a ligand thought to bind to adenosine uptake sites, and, at a concentration of 10 microM, had no effect on beef heart type III phosphodiesterase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of the in vivo binding of [3H]flunitrazepam by the atypical neuroleptic, clozapine.

Modulation of the benzodiazepine receptor/GABA receptor/chloride ionophore complex in vivo involves a number of intricate regulatory interactions between the three components of the receptor complex. One way to assess these potential interactions involves the in vivo labelling of the benzodiazepine receptor with [3H]flunitrazepam. In these studies, we used this approach to demonstrate that the neuroleptic, clozapine, increases [3H]flunitrazepam binding in mouse brain in a bicuculline-reversible manner. This potentiation of benzodiazepine binding was not antagonized by picrotoxin and was found to result from a slower dissociation of [3H]flunitrazepam from the benzodiazepine receptor. These data suggest that clozapine acts to increase [3H]flunitrazepam binding via a GABAergic mechanism, independent of the chloride channel.

Muscarinic, benzodiazepine, GABA, chloride channel and other binding sites in frontal cortex in hepatic coma in man.

Alterations in several neurotransmitter systems in brain have been implicated in the pathophysiology of hepatic coma (HC). Studies on human autopsy material are few. We investigated 3H-quinuclidinylbenzilate (QNB), 3H-spiperone, 3H-imipramine, 3H-PN-200-110, 3naloxone, 3H-flunitrazepam, 3H-muscimol, 35S-t-butylbicyclophosphothionate and 3H-cyclohexyladenosine binding sites in frontal cortex from seven patients with HC and five controls. The density of 3H-QNB binding sites was significantly decreased and the affinity slightly increased in HC. The functional significance of these selective changes in muscarinic receptor binding sites is unclear. Further studies evaluating cholinergic function in HC are indicated. Acute studies in animals point to an increase in GABA and BZ binding sites in HC. The present results show that the BZ/GABA-receptor-chloride-ionophore complex is unchanged in HC in man. Serotonergic (5HT-2), adenosine (A-1), imipramine (5HT uptake sites), opiate (naloxone) and calcium channel antagonist binding sites are unchanged in HC.

Effects of purine nucleotides on the binding of [3H]cyclopentyladenosine to adenosine A-1 receptors in rat brain membranes.

Adenine nucleotides displace the binding of the selective adenosine A-1 receptor ligand [3H]cyclopentyladenosine (CPA) to rat brain membranes in a concentration-dependent manner, with the rank order of activity being ATP greater than ADP greater than AMP. Binding was also displaced by GTP, ITP, adenylylimidodiphosphate (AppNHp), 2-methylthioATP, and the beta-gamma-methylene isostere of ATP, but was unaffected by the alpha-beta-methylene isosteres of ADP and ATP, and UTP. At ATP concentrations greater than 100 microM, the inhibitory effects on CPA binding were reversed, until at 2 mM ATP, specific binding of CPA was identical to that seen in controls. Concentrations of ATP greater than 10 mM totally inhibited specific binding. Inclusion of the catabolic enzyme adenosine deaminase in the incubation medium abolished the inhibitory effects of ATP, indicating that these were due to adenosine formation, presumably due to ectonucleotidase activity. The inhibitory effects were also attenuated by the alpha-beta-methylene isostere of ATP, an ectonucleotidase inhibitor. Adenosine deaminase, alpha-beta-methylene ATP (100 microM), and beta-gamma-methylene ATP (100 microM) had no effect on the "stimulatory" phase of binding, although GTP (100 microM) slightly attenuated it. Comparison of the binding of [3H]CPA in the absence and presence of 2 mM ATP by saturation analysis showed that the KD and apparent Bmax values were identical. Examination of the pharmacology of the control and "ATP-dependent" CPA binding sites showed slight changes in binding of adenosine agonists and antagonists. The responses observed with high concentrations of ATP were not observed with GTP, AppNHp, the chelating agents EDTA and EGTA, or inorganic phosphate. The divalent cations Mg2+ and Ca2+ at 10 mM attenuated the stimulatory actions of high (2 mM) concentrations of ATP, whereas EGTA and EDTA (10 mM) enhanced the "stimulatory" actions of ATP. EDTA (10 mM) abolished the inhibitory effects of ATP, indicating a specific dependence on Mg2+ for the inhibitory response. The effects of ATP on [3H]CPA binding were reversible for antagonists but not agonists. The mechanism by which ATP reverses its own inhibitory action on adenosine A-1 radioligand binding is unclear, and from the observed actions of the divalent cations and chelating agents probably does not involve a phosphorylation-dependent process.

Evaluation of the binding of the A-1 selective adenosine radioligand, cyclopentyladenosine (CPA), to rat brain tissue.

The binding of [3H]-Cyclopentyladenosine (CPA), an N6-substituted analog of adenosine, was examined in vitro. CPA bound with high affinity (Kd = 0.48 nmol/l) to rat brain membranes. Specific binding, which represented 90-97% of the total counts bound at a ligand concentration of 1 nmol/l, was saturable, reversible and sensitive to protein denaturation. The pharmacology of binding was consistent with the labeling of an A-1 receptor, the R- and S-diasteromers of N6-phenylisopropyladenosine (PIA) showing a sixteenfold difference in their ability to displace CPA. The prototypic A-1 selective adenosine agonist, N6-cyclohexyladenosine (CHA) was twofold less active than CPA in displacing radiolabeled CPA. Comparison of the ability of cold CHA and CPA to displace [3H]-CPA gave rate dissociation constants of 1.88 and 1.80 X 10(4) s-1, respectively suggesting that both CHA and CPA bound to the same recognition site. In contrast however, comparison of the binding of [3H]-CPA with that of [3H]-CHA showed distinct differences. The Kd for CHA was approximately twice that of CPA while the apparent Bmax was 60% greater. In comparing the pharmacology of CPA binding with that of CHA, it was found that CHA, S-PIA and the antagonist, PACPX were more active in displacing CHA than CPA. In general however, CPA has a binding profile very similar to that observed with CHA.