Activity and distribution of binding sites in brain of a nonpeptide substance P (NK1) receptor antagonist.

CP-96,345, a nonpeptide substance P antagonist, is selective for the tachykinin NK1 receptor. The compound binds to a single population of sites in guinea pig brain and potently inhibits substance P-induced excitation of locus ceruleus neurons. CP-96,345 should be a useful tool for studying the action of substance P in the central nervous system.

Functional consequences of posttranslational isomerization of Ser46 in a calcium channel toxin.

The venom of the funnel-web spider Agelenopsis aperta contains several peptides that paralyze prey by blocking voltage-sensitive calcium channels. Two peptides, omega-Aga-IVB (IVB) and omega-Aga-IVC (IVC), have identical amino acid sequences, yet have opposite absolute configurations at serine 46. These toxins had similar selectivities for blocking voltage-sensitive calcium channel subtypes but different potencies for blocking P-type voltage-sensitive calcium channels in rat cerebellar Purkinje cells as well as calcium-45 influx into rat brain synaptosomes. An enzyme purified from venom converts IVC to IVB by isomerizing serine 46, which is present in the carboxyl-terminal tail, from the L to the D configuration. Unlike the carboxyl terminus of IVC, that of IVB was resistant to the major venom protease. These results show enzymatic activities in A. aperta venom being used in an unprecedented strategy for coproduction of necessary neurotoxins that possess enhanced stability and potency.

Functional characterization of CP-465,022, a selective, noncompetitive AMPA receptor antagonist.

The hypothesis that aberrant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity contributes to epileptogenesis and neurodegeneration has prompted the search for AMPA receptor antagonists as potential therapeutics to treat these conditions. We describe the functional characterization of a novel quinazolin-4-one AMPA receptor antagonist, 3-(2-chloro-phenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one (CP-465,022). This compound inhibits AMPA receptor-mediated currents in rat cortical neurons with an IC(50) of 25 nM. Inhibition is noncompetitive with agonist concentration and is not use- or voltage-dependent. CP-465,022 is selective for AMPA over kainate and N-methyl-D-aspartate receptors. However, the compound is found to be equipotent for AMPA receptors composed of different AMPA receptor subunit combinations. This is indicated by the finding that CP-465,022 is equivalently potent for inhibition of AMPA receptor-mediated responses in different types of neurons that express different AMPA receptor subunits. Thus, CP-465,022 provides a new tool to investigate the role of AMPA receptors in physiological and pathophysiological processes.

Thiazole as a carbonyl bioisostere. A novel class of highly potent and selective 5-HT3 receptor antagonists.

A novel structural class of highly potent and selective 5-HT3 receptor antagonists is described. The compounds in this new series contain a thiazole moiety linking an aromatic group and a nitrogen-containing basic region; the thiazole group appears to be acting as a carbonyl bioisostere in this system. An optimized member of this series, 4-(2-methoxyphenyl)-2-[[4(5)-methyl-5(4)-imidazolyl]methyl]thiazole (5), exhibits oral activity in the Bezold-Jarisch reflex paradigm comparable to or better than the standard agents ondansetron (1) and ICS-205-930 (2). Several of the structure-activity relationships are rationalized in terms of a computer pharmacophore model for 5-HT3 receptor binding.

Kynurenic acid inhibits synaptic and acidic amino acid-induced responses in the rat hippocampus and spinal cord.

Kynurenic acid, a tryptophan metabolite, inhibits excitatory synaptic transmission in the rat hippocampal slice and the isolated immature rat spinal cord, but does not affect membrane potential or input resistance of hippocampal CA1 pyramidal cells. Kynurenic acid also antagonizes responses induced in the dentate gyrus by excitatory amino acids, particularly N-methyl-DL-aspartate and the endogenous excitant quinolinic acid. These results indicate that kynurenic acid antagonizes synaptic transmission probably by blocking postsynaptic transmitter receptors at putative amino acid mediated synapses.

Antagonism of lateral olfactory tract synaptic potentials in rat prepyriform cortex slices.

Dose-response data were collected for the inhibition of the monosynaptic excitatory input onto prepyriform neurons from fibers of the rat lateral olfactory tract, using the potent antagonists of excitatory transmission, L(+)-2-amino-4-phosphonobutyrate (L(+)-AP4), kynurenate, N-(p-chlorobenzoyl)piperazine-2,3-dicarboxylate, and N-(p-bromobenzoyl)piperazine-2,3-dicarboxylate. Kynurenate and the piperazine derivatives blocked up to 80% of the synaptic response at doses of 1000 microM, with single-affinity dose-response curves. L(+)-AP4 blocked only 50% of the synaptic response at a dose of 1000 microM, with a multicomponent dose-response curve.

2-Amino-4-phosphonobutyrate selectively blocks mossy fiber-CA3 responses in guinea pig but not rat hippocampus.

The acidic amino acid antagonist D,L-2-amino-4-phosphonobutyrate (DL-APB) is a potent blocker of synaptic transmission at guinea pig but not rat mossy fiber-CA3 synapses in hippocampal slices. The L-isomer of APB is responsible for the potent inhibition at the guinea pig synapse. The L-APB analogue L-serine-O-phosphate (L-SOP) also is more potent against the guinea pig response. These differences may reflect a difference in a synaptic acidic amino acid receptor in these two species. Other acidic amino acid antagonists are less potent than APB or L-SOP and do not discriminate between the mossy fiber responses in the two species.

Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors.

A series of omega-phosphono-alpha-carboxylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (+/- AP5 and +/- AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response. +/- AP5 and +/- AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpotentiated synaptic response amplitude. +/- AP4, +/- AP6 and +/- AP8 did not block amino acid excitant responses or LTP. These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.

Action of 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP): a new and highly potent antagonist of N-methyl-D-aspartate receptors in the hippocampus.

A new compound, 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces L-[3H]glutamate binding in regions of the hippocampus rich in N-methyl-D-aspartate (NMDA) receptors, but not in regions rich in kainate sites. In isolated membrane fraction preparations, CPP displaces L-[3H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA greater than homocysteate greater than aspartate greater than glutamate greater than kainate greater than or equal to quisqualate. CPP has no effect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synaptic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compound is, therefore, a highly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.

Acidic amino acid antagonists of lateral perforant path synaptic transmission: agonist-antagonist interactions in the dentate gyrus.

Acidic amino acid antagonists were tested for their ability to block depolarizations produced by excitatory amino acids in the outer molecular layer of the dentate gyrus in hippocampal slices. 2-Amino-4-phosphonobutyrate (APB) and serine-O-phosphate (SOP), potent synaptic blockers of the lateral perforant path input to this system, are moderately selective antagonists of kainate and N-methyl-DL-aspartate depolarizations, but not depolarizations produced by L-glutamate, quisqualate, or serine-O-sulfate. N-Methyl-DL-aspartate responses were potently blocked by 2-amino-5-phosphonovalerate, but this antagonist is less potent than APB or SOP against the synaptic response.

Kynurenic acid and quinolinic acid act at N-methyl-D-aspartate receptors in the rat hippocampus.

Responses evoked by several amino acid excitants, including the tryptophan metabolite quinolinic acid, were recorded intracellularly from CA1 pyramidal neurons in rat hippocampal slices. Quinolinate, N-methyl-D-aspartate (NMDA), ibotenate and (+/-)-cis-1-amino-1,3-dicarboxycyclopentane produced excitations characterized by burst firing of action potentials, tetrodotoxin-resistant spiking and apparent increases in input resistance measured with brief hyperpolarizing current pulses. L-Glutamate, kainate, quisqualate and (+/-)-2'-amino-3-hydroxy-5-methyl-4-isoxazole-3'-propionate depolarized CA1 pyramidal neurons and induced apparent decreases in input resistance. Quinolinate-, NMDA-, and ibotenate-induced focal depolarizations, but not L-glutamate, kainate- or quisqualate-induced responses, were strongly antagonized by specific NMDA receptor antagonists. The tryptophan metabolite kynurenic acid, at concentrations that antagonized focal depolarizations produced by NMDA, ibotenate and the endogenous excitant quinolinate, did not antagonize quisqualate or L-glutamate responses. In addition to its NMDA-type antagonist action, kynurenate blocked kainate-induced focal depolarizations.

Hebbian synapses in hippocampus.

A combination of current- and voltage-clamp techniques applied to hippocampal brain slices was used to evaluate the role of postsynaptic electrogenesis in the induction of associative synaptic enhancement. In accordance with Hebb's postulate for learning, repetitive postsynaptic spiking enabled enhancement in just those synapses that were eligible to change by virtue of concurrent presynaptic activity. However, the essential postsynaptic electrogenic event that controlled the enhancement was shown to involve biophysical processes that were unknown when Hebb formulated his neurophysiological postulate. The demonstrated spatiotemporal specificity of this pseudo-Hebbian conjunctive mechanism can account qualitatively for the known neurophysiological properties of associative long-term potentiation in these synapses, which in turn can explain the "cooperativity" requirement for long-term potentiation.

Effects of excitatory amino acid antagonists on evoked and spontaneous excitatory potentials in guinea-pig hippocampus.

Evoked and spontaneous excitatory post-synaptic potentials (e.p.s.p.s) at the mossy fibre input to CA3 pyramidal neurones were recorded intracellularly in slices from the guinea-pig hippocampus. The effects of several amino acid antagonists on these responses were examined. L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP), kynurenate, and N-(p-bromobenzoyl)piperazine-2,3-dicarboxylate (pBB-PzDA) reduced the amplitude of evoked mossy fibre e.p.s.p.s without affecting membrane potential or input resistance. Antagonism of mossy fibre spontaneous miniature e.p.s.p.s (m.e.p.s.p.s) by these compounds fell into two groups. L-AP4 and L-SOP applied at concentrations that blocked evoked e.p.s.p.s did not affect amplitude distributions of spontaneous m.e.p.s.p.s. Kynurenate and pBB-PzDA significantly affected the amplitude distributions and reduced the mean amplitude of spontaneous m.e.p.s.p.s. These results are consistent with a presynaptic site of action for L-AP4 and L-SOP and a post-synaptic site of action for kynurenate and pBB-PzDA as antagonists of e.p.s.p.s at the guinea-pig mossy fibre-CA3 pyramidal neurone synapse.

Arylamine spider toxins antagonize NMDA receptor-mediated synaptic transmission in rat hippocampal slices.

The effects of arylamine spider toxins on synaptic transmission in rat hippocampal slices were investigated. Two different responses were monitored: the AMPA receptor-mediated population spike recorded in control buffer (selectively antagonized by DNQX) and the NMDA receptor-mediated EPSP recorded in nominally magnesium-free buffer containing 20 microM DNQX (selectively antagonized by AP5, AP7, and dizocilpine (MK-801)). The synthetic arylamine spider toxins JSTX-3, argiotoxin-636, and argiotoxin-659 were 26 to 73 times more potent at antagonizing the NMDA receptor-mediated EPSP (IC50 values ranging from 12 to 24 microM) than the AMPA receptor-mediated population spike (IC50 values ranging from 612 to 878 microM). These results indicate that arylamine spider toxins are selective antagonists of NMDA receptors in the mammalian CNS.

Parallel antagonism of synaptic transmission and kainate/quisqualate responses in the hippocampus by piperazine-2,3-dicarboxylic acid analogs.

A new series of potent antagonists of excitatory neurotransmission in the rat hippocampus has been identified. These derivatives of piperazine-2,3-dicarboxylate (PzDA) include the most potent acidic amino acid antagonists yet described for Schaffer collateral-commissural EPSPs. These antagonists also effectively block excitatory synaptic responses recorded in the lateral and medial perforant pathways and in the mossy fiber pathway. The PzDA derivatives also block focal depolarizations produced by kainate, quisqualate, and N-methyl-D-aspartate. N-methyl-D-aspartate responses are more susceptible to inhibition by PzDA derivatives, although the spectrum of antagonism of N-methyl-D-aspartate and synaptic responses by PzDA derivatives is not parallel. However, the antagonism of kainate and quisqualate responses by PzDA derivatives shows the same rank order of potency as synaptic responses. These data indicate that synaptic receptors in the hippocampus have a pharmacologic profile similar to that of kainate or quisqualate receptors.

Atropisomeric quinazolin-4-one derivatives are potent noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists.

Piriqualone (1) was found to be an antagonist of AMPA receptors. Structure activity optimization was conducted on each of the three rings in 1 to afford a series of potent and selective antagonists. The sterically crowded environment surrounding the N-3 aryl group provided sufficient thermal stability for atropisomers to be isolated. Separation of these atropisomers resulted in the identification of (+)-38 (CP-465,022), a compound that binds to the AMPA receptor with high affinity (IC50 = 36 nM) and displays potent anticonvulsant activity.