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F 11440 (4-methyl-2-[4-(4-(pyrimidin-2-yl)-piperazino)-butyl]-2H,4H-1,2,4-triazin-3,5-dione) was the outcome of a research effort guided by the hypothesis that the magnitude of the intrinsic activity of agonists at 5-HT1Areceptors determines the magnitude of their antidepressant and anxiolytic-like effects. The affinity of F 11440 for 5-HT1Abinding sites (p Ki, 8.33) was higher than that of buspirone (p Ki, 7.50), and somewhat lower than that of flesinoxan (p Ki, 8.91). In vivo, F 11440 was 4- to 20-fold more potent than flesinoxan, and 30- to 60-fold more potent than buspirone, in exerting 5-HT1Aagonist activity at pre- and postsynaptic receptors in rats (measured by, for example, its ability to decrease hippocampal extracellular serotonin (5-HT) levels and to increase plasma corticosterone levels, respectively). F 11440 did not have detectable antidopaminergic activity (unlike buspirone, which inhibited all of the directly observable behavioral effects of methylphenidate in rats), showed no evidence of antihistaminergic activity (unlike flesinoxan, which protected against the effects of a histamine aerosol in guinea pigs), and had a 70-fold separation between its 5-HT1Aagonist and alpha-1 adrenergic antagonist properties (measured as the ability to inhibit the methoxamineinduced increase in blood pressure in rats), unlike flesinoxan, which showed a <3-fold separation. In HeLa cells expressing human 5-HT1Areceptors, F 11440 decreased the forskolin-induced increase in AMP, and, based on its maximal effect, was found to have an intrinsic activity of 1.0 relative to that of 5-HT, which was significantly higher than that of buspirone (0.49), ipsapirone (0.46) and flesinoxan (0.93). Consistent with the aforementioned hypothesis, F 11440 produced anxiolytic- and antidepressant-like effects in animal models (i.e., increased punished responding in a pigeon conflict procedure and decreased immobility in a rat forced swimming test, respectively) that were more substantial than those of buspirone, ipsapirone and flesinoxan. Thus, F 11440, shown here to be a potent, selective, high efficacy 5-HT1Areceptor agonist, appears to have the potential to exert marked anxiolytic and antidepressant activity in humans.

The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1Band h5-HT1Dreceptor sites using a [35S]GTPγS binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTPγS binding to a membrane preparation containing either h5-HT1Bor h5-HT1Dreceptors. The potencies observed for most of the compounds at the h5-HT1Breceptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTPγS binding assay to C6-glial/h5-HT1Dmembranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1Dmembrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1Dthan h5-HT1Bbinding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTPγS binding response. Hence, the h5-HT1Breceptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1Dreceptor sites [e.g. dihydroergotamine and (2′-methyl-4′-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethylsulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTPγS binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1Band h5-HT1Dreceptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at the h5-HT1Breceptor and only L694247 at the h5-HT1Dreceptor. GR127935 (10 μM) exerted little effect on [35S]GTPγS binding via h5-HT1Breceptors (10% stimulation), but potently (pA2: 9.11) antagonized h5-HT1Breceptor-stimulated [35S]GTPγS binding. Ketanserin and methiothepin inhibited [35S]GTPγS binding (by 13–28%) in the absence of an agonist, but were potent and competitive antagonists in the presence of an agonist via h5-HT1B(methiothepin) and h5-HT1D(methiothepin and ketanserin) receptors. The results document the utility of using [35S]GTPγS binding studies to assess agonist efficacy, and to characterize 5-HT1B/Dreceptor ligands as apparently neutral antagonists and inverse agonists at the G-protein level. © 1997 Elsevier Science Ltd.

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy]-1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).

A series of serotonin dimers of formula 4 in which two serotonin moeities are linked together through their 5-hydroxyl residue has been prepared and evaluated as 5-HT1B/1D receptor agonists. Binding experiments at cloned human 5-HT1B, 5-HT1D, and 5-HT1A receptors show that all of these dimers are very potent ligands at 5-HT1B/1D receptors with increased binding selectivity vs the 5-HT1A receptor when compared to serotonin. Studies of inhibition of the forskolin-stimulated c-AMP formation mediated by the human 5-HT1B receptor (formerly the 5-HT1Dβ receptor) demonstrate that all of these serotonin dimers behave as full agonists. Among them, the piperazide derivatives of bis-serotonin, 4g,j, were also identified as very potent agonists in contracting the New Zealand white rabbit saphenous vein (pD2 = 7.6 in each case compared to 5.8 for sumatriptan). Results analysis supports the hypothesis that the important increase in potency of the serotonin dimers can be attributed to the presence of two serotonin pharmacophores in the same molecule, while the enhanced selectivity for 5-HT1B/1D receptor subtypes may be due to the position of the spacer attachment to serotonin.

Functional activity of 5-hydroxytryptamine (5-HT) receptor ligands and beta adrenergic receptor antagonists was studied at 5-HT1B receptor sites in Chinese hamster lung (CHL) fibroblasts by measuring two cellular responses: inhibition of forskolin-stimulated cyclic AMP formation and potentiation of basic fibroblast growth (BFGF) induced mitogenesis. A good correlation was found between the potency of agonists to inhibit forskolin-induced cyclic AMP formation and their potency to potentiate bFGF-induced thymidine incorporation in CHL fibroblasts. Potent agonist activity was measured with 5-methoxy-3,1,2,3,6-tetrahydro-4-pyidinyl- 1H-indole (RU 24,969), 5-carboxamidotryptamine (5-CT), 3-(1,2,5,6)-tetrahydro-4-pyridyl-5-pyrrolo(3,2-b)pyril-5-one (CP 93,129) and 5-HT, whereas sumatriptan displayed weak agonist activity at concentrations different from its binding affinity for 5-HT1B binding sites. In contrast to the observed 5-HT1B receptor-mediated agonist activity in opossum kidney cells for metergoline and the beta adrenergic receptor antagonists: cyanopindolol, 4-(3-tert-butyl-amino-2-hydroxypropoxy)-indole-2 carbonic acid isopropyl ester (SDZ 21,009), isamoltane, (-)-propranolol and (-)-pindolol, antagonist activity at 5-HT1B receptor sites was yielded in CHL fibroblasts in accordance with the reported observations at rat brain 5-HT1B receptors. Methiothepin was the only compound that antagonized both the opossum kidney cell and CHL fibroblast 5-HT1B receptor-mediated responses although the antagonist effect was more pronounced in CHL fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

The Neurospora crassa catabolic enzyme, arginase (L-arginine amidinohydrolase, EC 3.5.3.1), exists in multiple forms. Multiple forms of arginase are found in many vertebrates, but this is the only reported example in a microbial organism. The two major forms are structurally similar with subunit sizes of 36 and 41 kDa, respectively. The larger form is produced by mycelia growing in arginine-supplemented medium. Both forms are localized in the cytosol. The structural gene for arginase, aga, has been cloned and sequenced; it contains a 358-codon open reading frame with three in-frame ATGs at the amino terminus. Mutagenesis of these ATGs revealed that the first ATG initiates the 41-kDa protein and the third ATG initiates the 36-kDa protein. Mutation of the second ATG has no effect on translation. Northern analysis demonstrated that a 1.4-kilobase (kb) transcript is synthesized in minimal medium and both a 1.4- and 1.7-kb transcript are produced in arginine-supplemented medium. Primer extension identified the 5' ends of each transcript and demonstrated that the first and third ATG of the open reading frame are the initial AUGs of the 1.7- and 1. 4-kb mRNA, respectively. The results suggest that a basal promoter produces the 1.4-kb transcript and an arginine "activated" promoter is responsible for the 1.7-kb transcript. Tandem promoters are rare in eukaryotic organisms, and they often regulate developmental or tissue-specific gene expression. The possibility that arginase has a role in differentiation in N. crassa is being investigated.