F 11356, a novel 5-hydroxytryptamine (5-HT) derivative with potent, selective, and unique high intrinsic activity at 5-HT1B/1D receptors in models relevant to migraine.

F 11356 (4-[4-[2-(2-aminoethyl)-1H-indol-5-yloxyl]acetyl]piperazinyl-1-yl] ben zonitrile) was designed to take advantage of the superior potency and efficacy characteristics of 5-hydroxytryptamine (5-HT) compared with tryptamine at 5-HT1B/1D receptors. F 11356 has subnanomolar affinity for cloned human and nonhuman 5-HT1B and 5-HT1D receptors, and its affinity for 5-HT1A and other 5-HT receptors, including the 5-ht1F subtype, is 50-fold lower and micromolar, respectively. In C6 cells expressing human 5-HT1B or human 5-HT1D receptors, F 11356 was the most potent compound in inhibiting forskolin-induced cyclic AMP formation (pD2 = 8.9 and 9.6), and in contrast to tryptamine and derivatives, it produced maximal enhancement of [35S]guanosine-5'-O-(3-thio)triphosphate-specific binding equivalent to 5-HT. F 11356 was equipotent to 5-HT (pD2 = 7.1 versus 7.2) and more potent than tryptamine derivatives in contracting rabbit isolated saphenous vein. In isolated guinea pig trigeminal ganglion neurons, F 11356 was more potent (pD2 = 7.3 versus 6.7) and induced greater increases in outward hyperpolarizing Ca2+-dependent K+ current than sumatriptan. In anesthetized pigs, F 11356 elicited highly cranioselective, more potent (from 0.16 microgram/kg i.v.) and greater carotid vasoconstriction than tryptamine derivatives. Decreases in carotid blood flow were observed in conscious dogs from 0.63 mg/kg oral F 11356 in the absence of changes in heart rate or behavior. Oral activity was confirmed when hypothermic responses were elicited in guinea pigs (ED50 = 1.6 mg/kg), suggesting that F 11356 also accesses the brain. F 11356 thus is a selective, high-potency agonist at 5-HT1B/1D receptors, which distinguishes itself from tryptamine and derivatives in exerting high intrinsic activity at these receptors in vascular and neuronal models relevant to migraine.

Magnitude of 5-HT1B and 5-HT1A receptor activation in guinea-pig and rat brain: evidence from sumatriptan dimer-mediated [35S]GTPgammaS binding responses.

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT1A and 5-HT1B receptors in guinea-pig and rat brain using agonist-stimulated [35S]GTPgammaS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT1B (pKi: 9.22 vs. 7.79 for sumatriptan), h 5-HT1D (9.07 vs. 8.08) and also h 5-HT1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht1E (6.67 vs. 6.19) and h 5-ht1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 microM) stimulated [35S]GTPgammaS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT1B/1D receptor antagonist [3H] GR 125743. The observed [35S]GTPgammaS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT1B receptor subtype, since they were antagonized by the 5-HT1B inverse agonist SB 224289 (10 microM), and not by the 5-HT2A/1D antagonist ketanserin (10 microM). Quantitative assessment of the [35S]GTPgammaS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51+/-10% and 35+/-13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT1B receptors besides 5-HT1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

Dimers of 5HT1 ligands preferentially bind to 5HT1B/1D receptor subtypes.

New dimers of known 5HT1 ligands (5HT, 1-NP or 8-OH-DPAT) have been prepared and evaluated at human cloned 5HT1B, 5HT1D and 5HT1A receptors. Binding experiments show that all these dimers have better affinities at 5HT1B/1D receptors than their corresponding monomeric ligands. Studies of inhibition of the forskolin-stimulated c-AMP formation mediated by the human 5HT1B receptor show that hetero-bivalent ligands [combining an agonist (5HT) with an antagonist (1-NP)] behave as partial agonists while the intrinsic activity of bivalent antagonists (combining two 1-NP residues) was found to be spacer dependent. Surprisingly enough, the dimer of 8-OH-DPAT 6 binds to 5HT1A, 5HT1B and 5HT1D receptors with similar high affinity.

[Inhibitors of Ras farnesylation: tomorrow's anticancer agents?]. / Inhibiteurs de farnésylation de Ras: agents anticancéreux de demain?

Increasing evidence implicates a critical role for aberrant Ras function in promoting the development of human tumours and has provided the impetus for identifying anti-Ras drugs for therapy. In order to be active the protooncogene ras must be associated with the plasma membrane. This feature depends crucially upon its farnesylation (addition of a 15 carbon moiety) by the enzyme farnesyl protein transferase. In the search for new anticancer strategies and agents, potent and selective inhibitors of this enzyme have been designed. The more recent of these compounds have produced impressive results in vivo against human tumours xenografted onto nude mice, without notable toxicity, making them promising candidates for clinical evaluation.

Enzymatic phosphorylation and pyrophosphorylation of 2',3'-dideoxyadenosine-5'-monophosphate, a key metabolite in the pathway for activation of the anti-HIV (human immunodeficiency virus) agent 2',3'-dideoxyinosine.

2',3'-Dideoxyadenosine-5'-monophosphate (ddAMP) is a key intermediate in the metabolic pathway involved in the activation of the anti-retroviral agent 2',3'-dideoxyinosine (ddI) to 2',3'-dideoxyadenosine-5'-triphosphate (ddATP). The potential phosphorylation of ddAMP by adenylate kinase (myokinase) and pyrophosphorylation by the reverse reaction of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase were investigated. Using ATP as phosphate donor, ddAMP was phosphorylated by adenylate kinase with an efficiency of 8.8% of that for AMP, as estimated from the Vmax/Km ratios. In the presence of PRPP, Escherichia coli and rat PRPP synthetases catalysed the pyrophosphorylation of ddAMP with efficiencies of 52 and 35% of that determined for AMP, respectively. Two carbocyclic phosphonate analogues of ddAMP were not substrates of adenylate kinase. Yet, they were pyrophosphorylated by both PRPP synthetases, albeit less efficiently than ddAMP. In vivo, the usual function of PRPP synthetase is to synthesize PRPP from ribose-5-phosphate and ATP. In the forward reaction ddATP proved to be a substrate as efficient as ATP for rat PRPP synthetase. ddATP was also studied as a potential phosphate donor in the reaction catalysed by adenylate kinase with AMP as phosphate acceptor and found to be as efficient as ATP. The relevance of these in vitro results to the in vivo situation is discussed.

9-(Phosphonoalkyl)guanine derivatives as substrates or inhibitors of guanylate kinase.

Several 9-(phosphonoalkyl)guanines (Gua(CH2)nCH2-PO3H2; n = 4-6) and 9-(difluorophosphonoalkyl)guanines (Gua(CH2)nCF2PO3H2; n = 3-7) were studied as potential substrates and inhibitors of guanylate kinase. These compounds are inhibitors of the enzyme except 9-(5-phosphonopentyl)guanine (n = 4) which is a substrate with an efficiency of phosphorylation of about 0.3% that of GMP, as estimated from the Vmax/Km ratios. The phosphonate and difluorophosphonate derivatives with n = 5 produce optimal inhibition. These two compounds have similar affinity, both being competitive inhibitors with respect to GMP and noncompetitive inhibitors with respect to ATP. pH-dependence studies indicate that the dianionic rather than the monoanionic form of these compounds bind to the enzyme. The lack of phosphorylation of 9-(5,5-difluoro-5-phosphonopentyl)guanine by guanylate kinase is explained by the decreased nucleophilic character of the oxygen atoms of the phosphonate group rather than by inadequate binding to the GMP-binding site.