In vitro activity of LY393558, an inhibitor of the 5-hydroxytryptamine transporter with 5-HT(1B/1D/2) receptor antagonist properties.

1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-1(2H)-pyridinyl]ethyl]-3-isopropyl-6-(methylsulphonyl)-3,4-dihydro-1H-2,1,3-benzothiadiazine-2,2-dioxide (LY393558) is a potent inhibitor of [3H]5-hydroxytryptamine ([3H]5-HT) uptake into rat cortical synaptosomes (pIC(50)=8.48+/-0.12). It produces a dextral shift of the 5-HT dose-response curves for the binding of GTPgamma[35S] to human 5-HT(1B) (pK(b)=9.05+/-0.14) and 5-HT(1D) (pK(b)=8.98+/-0.07) receptors and inhibits the contractile response of the rabbit saphenous vein to the 5-HT(1B/D) receptor agonist, sumatriptan (pK(b)=8.4+/-0.2). In addition, it is an antagonist at the 5-HT(2A) (pK(i)=7.29+/-0.19) and 5-HT(2B) (pK(i)=7.35+/-0.11) receptors. Presynaptic autoreceptor antagonist activity was demonstrated by its ability to potentiate the K(+)-induced outflow of [3H]5-HT from guinea pig cortical slices (pEC(50)=7.74+/-0.05 nM) in which the 5-HT transporter had been inhibited by a maximally effective concentration of paroxetine. It is concluded that LY393558 should be an effective antidepressant with the potential to produce an earlier onset of efficacy than selective serotonin uptake inhibitors.

Discovery and evaluation of a series of 3-acylindole imidazopyridine platelet-activating factor antagonists.

Studies conducted with the goal of discovering a second-generation platelet-activating factor (PAF) antagonist have identified a novel class of potent and orally active antagonists which have high aqueous solubility and long duration of action in animal models. The compounds arose from the combination of the lipophilic indole portion of Abbott's first-generation PAF antagonist ABT-299 (2) with the methylimidazopyridine heterocycle moiety of British Biotechnology's BB-882 (1) and possess the positive attributes of both of these clinical candidates. Structure-activity relationship (SAR) studies indicated that modification of the indole and benzoyl spacer of lead compound 7b gave analogues that were more potent, longer-lived, and bioavailable and resulted in the identification of 1-(N, N-dimethylcarbamoyl)-4-ethynyl-3-[3-fluoro-4-[(1H-2-methylimidazo[4,5-c] pyrid-1-yl)methyl]benzoyl]indole hydrochloride (ABT-491, 22 m.HCl) which has been evaluated extensively and is currently in clinical development.

Properties of ABT-299, a prodrug of A-85783, a highly potent platelet activating factor receptor antagonist.

ABT-299 is an aqueous soluble prodrug that is converted rapidly in vivo to A-85783, a novel, highly potent, specific platelet activating factor (PAF) antagonist. The K, for inhibiting PAF binding to rabbit platelet membranes is 3.9 and 0.3 nM for human platelets. Inhibition is selective and reversible and is correlated with functional antagonism of PAF-mediated cellular responses (calcium mobilization, priming of superoxide generation, aggregation and degranulation). The in vivo generation of A-85783 from ABT-299 leads to potent inhibition of PAF-induced inflammatory responses (increased vascular permeability, hypotension and edema) and PAF-induced lethality. When administered i.v., the potency (ED50) of ABT-299 for inhibiting PAF responses was between 6 to 10 micrograms/kg in the rat and mouse and 100 micrograms/kg in the guinea pig. A dose of 100 micrograms/kg in the rat provided greater than 60% protection for 8 to 16 hr against cutaneous and systemic PAF challenge. This duration was also evidenced by ex vivo inhibition of platelet aggregation in guinea pig and sheep. In addition to being active parenterally, ABT-299 exhibited p.o. activity in the rat and mouse (ED50 = 100 micrograms/kg in both species). Pharmacokinetic studies in the rat revealed that ABT-299 was converted rapidly to A-85783 and, in turn, metabolized to the corresponding pyridine-N-oxide and sulfoxide metabolites. These metabolites exhibited significant potency in vitro and in vivo and thus may contribute to the activity observed after administration of ABT-299.

N-(acyloxyalkyl)pyridinium salts as soluble prodrugs of a potent platelet activating factor antagonist.

Pyrrolothiazole 4 is a potent antagonist of platelet activating factor-mediated effects in a variety of in vitro and in vivo assays. Despite its positive activity in models of inflammation and septic shock, 4 lacks the aqueous solubility necessary for intravenous administration. This deficit was overcome by conversion of 4 to water-soluble pyridinium prodrugs. A two-step procedure was used to prepare a series of N-(acyloxyalkyl)pyridinium salts, all of which exhibited aqueous solubility of greater than 20 mg/mL. The rate of conversion of these prodrugs to 4 was faster in human plasma than in pH 7 aqueous buffer. This rate difference was shown to be due to serum enzymes since the conversion in plasma was significantly slower in the presence of a serine esterase inhibitor. A strong correlation between prodrug structure and buffer/plasma half-life was established. The N-(acetyloxymethyl)pyridinium prodrug 11 (ABT-299) is currently undergoing clinical evaluation for the treatment of sepsis.

Synthesis and structure-activity relationships of a series of novel benzopyran-containing platelet activating factor antagonists.

A class of N-substituted tetrahydrobenzopyrano[3,4-c]pyridines, I, have been identified as antagonists of platelet activating factor (PAF). The structural features essential for PAF binding were determined by systematic modification of three sites in the molecule. While O-alkyl analogues had little effect on binding potency, N-alkyl analogues exhibited a wide range of activity. Structural changes in the core ring system generally resulted in a loss of binding activity. Optimization of the N- and O-substituents resulted in the analogues 25-27 which exhibited Ki values ranging between 131 and 167 nM in a [3H]PAF binding assay. Compound 23 was also active in a model of PAF-induced shock in the mouse following intravenous administration.

C5a structural requirements for neutrophil receptor interaction.

A number of C5a modifications were tested to determine effects on receptor binding to polymorphonuclear leukocyte (PMNL) membrane receptors and triggering of PMNL chemokinesis and myeloperoxidase (MPO) release. Site-directed mutagenesis was used to probe relationships of key C-terminal residues, and suggested a role for additional sites, particularly Lys19-20. A synthetic peptide based on C5a 19-30, weakly inhibited C5a binding. Potency of the C-terminal octapeptide, a full agonist, was markedly improved by a single Phe substitution for His67, and a Phe point mutation at this site was shown to enhance activity of the full recombinant protein.

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

C5a is an inflammatory mediator potentially involved in a number of diseases. To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis. Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity. Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor. Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR. Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15. Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency.

Identification and regulation of alpha 2-adrenergic receptors in rabbit ileal mucosa.

The alpha 2-adrenergic receptors in rabbit ileal mucosal membranes can be identified by using [3H]clonidine. [3H]Clonidine bound to a homogeneous population of sites (30-120 fmoles/mg protein) with a KD of 2.2 nM at 25 degrees. Alpha-adrenergic agonists and antagonists competed with [3H] clonidine for the binding sites with an order of potency typical for alpha 2-receptors. Mg2+, Ca2+, or Mn2+ (2.4 mM) markedly increased the binding of [3H]clonidine. At the maximally effective concentration, Mg2+ increased both the binding affinity of [3H]clonidine and the number of receptor sites. Both NaCl and GppNHp, the guanyl nucleotide, inhibited [3H]clonidine binding. NaCl decreased the binding affinity of [3H]clonidine, with no appreciable effect on the number of receptor sites. These findings indicate that ileal mucosal alpha 2-receptors can exist in multiple affinity states, which can be regulated by divalent cations, NaCl, and guanyl nucleotides. It appears that NaCl and GppNHp regulate alpha 2-receptors in ileal mucosa by different mechanisms.

Effects of 2,3-diphosphoglyceric acid on the human erythrocyte membrane phosphorylation system.

The effect of 2,3-diphosphoglycerate (2,3-P2-glycerate) on the phosphorylation of spectrin in solution by purified membrane cyclic AMP-independent protein kinase and in membrane preparations by the endogenous kinase was investigated. 2,3-P2-Glycerate inhibited spectrin phosphorylation both in solution and in the intact membrane. Kinetic analyses showed that 2,3-P2-glycerate had no effect on the Km for ATP but appeared to lower the Vmax of the reaction. When the effect of 2,3-P2-glycerate was examined in the presence of varying concentrations of spectrin, competitive inhibition kinetics were obtained. Interestingly, low concentrations of 2,3-P2-glycerate were found to effect the release of the membrane kinase from erythrocyte membranes. This release reaction may be related to the ability of 2,3-P2-glycerate to interfere with the interaction between the kinase and spectrin. The data suggest the possibility that the kinase may be bound to spectrin in the erythrocyte membrane. 2,3-P2-glycerate also caused the solubilization of 3-phosphoglyceraldehyde dehydrogenase, but not of cyclic AMP-dependent protein kinase. Taken together, our data indicate that 2,3-P2-glycerate may have a regulatory role in membrane protein phosphorylation and also may regulate the extent of association of the kinase with the membrane.

Human brain protein phosphorylation in vitro: cyclic AMP stimulation of electrophoretically-separated substrates.

In vitro phosphorylation of electrophoretically-separated brain proteins was studied in human frontal cortex obtained 3-16 h post-mortem from 13 patients ages 3 days-82 years with extensive, mild or no neuropathological involvement. In 12 of the 13 cases, cyclic AMP increased incorporation of phosphate into acid-precipitable protein. Analysis of the autoradiographic profiles of separate proteins indicated that phosphorylation of the doublet of molecular weight 86-80,000 was stimulated by cyclic AMP in certain samples. This doublet corresponded to the cyclic AMP stimulated doublet from rat frontal cortex we have termed band D-1,2 (proteins Ia and Ib of Ueda and Greengard). Of special interest was the fact that, while co-migration was observed in the other phosphoprotein bands studied, band D-1,2 of humans consistently migrated slightly less than rat protein band D-1,2. This difference was not a function of post-mortem time, subcellular fraction or buffer used in the reaction phosphorylation assay. The use of post-mortem tissue was not a contributing factor as the retardation in band D-1,2 migration was still observed when post-mortem rat brain was used for comparison. In two human post-mortem samples, there was no measureable band D-1,2 phosphorylation even in the presence of cyclic AMP. This was the case in both homogenate and crude synaptosome/mitochondrial preparations. Band F-1 (mol. wt. = 47,000) was not observed in any of the human samples studied. This is consistent with prior studies in rat which show that band F-1 phosphorylation is not detected in post-mortem brain, Band F-2 (mol. wt. 41,000) recently identified as pyruvate dehydrogenase, was lightly phosphorylated under the reaction conditions used in this study.

Endogenous phosphorylation in vitro: differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins.

In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [32P]Labelled phosphoproteins were separated by SDS-slab gel electrophoresis, and the autoradiographs were analyzed by densitometry. We report here that Band F of our previous reports can be separated into two components, F1 and F2, using an improved gel system. This separation is particularly relevant in this report since these components appear to be differentially sensitive to the manipulations used. Specifically, we found that while F1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F2 was relatively stable. While phosphorylation of F2 was also stable 24 h post-mortem, Band F1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F2 somewhat, it eliminated the in vitro phosphorylation of Band F1. We found that under light barbiturate anesthesia, just at the time when the animals lost the righting reflex, the in vitro phosphorylation of Bands D (MR 78,000--80,000 daltons), F1 (MR 47,000--49,000) and F2 (MR 40,000--45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F1 was depressed in tissue prepared from animals that were deeply comatose. These effects of pentobarbital were more pronounced when animals were sacrificed by liquid nitrogen immersion, rather than by decapitation. Cyclic AMP-dependent phosphorylation of Band D exhibited remarkable stability 24 h post-morten (7 days in one case), even when brain tissue was left at room temperature (21--23 degrees C). Phosphorylation of Band F1, however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Band F1 is: (3) increased in phosphorylation in liquid nitrogen P2 preparations, and may be (4) cAMP-independent, (5) rapidly turning over its phosphate in vivo, and (6) altered by a training experience. Other evidence suggests that: (7) Band F1 phosphorylation may be Ca2+-dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. The results suggest an important and perhaps unique role for Band F1 in neuronal function.