Ann Marie L. Ogden

Kainate receptors are involved in synaptic plasticity

The ability of synapses to modify their synaptic strength in response to activity is a fundamental property of the nervous system and may be an essential component of learning and memory. There are three classes of ionotropic glutamate receptor, namely NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors; critical roles in synaptic plasticity have been identified for two of these. Thus, at many synapses in the brain, transient activation of NMDA receptors leads to a persistent modification in the strength of synaptic transmission mediated by AMPA receptors. Here, to determine whether kainate receptors are involved in synaptic plasticity, we have used a new antagonist, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid), which antagonizes kainate receptors at concentrations that do not affect AMPA or NMDA receptors. We find that LY382884 is a selective antagonist at neuronal kainate receptors containing the GluR5 subunit. It has no effect on long-term potentiation (LTP) that is dependent on NMDA receptors but prevents the induction of mossy fibre LTP, which is independent of NMDA receptors. Thus, kainate receptors can act as the induction trigger for long-term changes in synaptic transmission.

Pharmacological characterization of glutamatergic agonists and antagonists at recombinant human homomeric and heteromeric kainate receptors in vitro.

An increasing body of evidence suggests that native kainate receptors form ion channels from homomeric and heteromeric combinations of five receptor subunits: GluR5, GluR6, GluR7, KA1 and KA2. We have examined the activity of agonists and antagonists at recombinant human kainate receptors expressed in HEK293 cells, using both whole-cell electrophysiological recording and 96-well plate fluo-3 based calcium microfluorimetry (FLIPR). Both homomeric (GluR5 and GluR6) and heteromeric (GluR5/6, GluR5/KA2 and GluR6/KA2) receptors were examined. Heteromeric receptor assemblies showed electrophysiological and pharmacological profiles which were distinct from homomeric channels. Several agonists, including AMPA, ATPA and (S)-5-iodowillardiine, and antagonists, including gamma-D-glutamylaminomethylsulphonic acid (GAMS) and the decahydroisoquinoline compounds LY293558, LY377770 and LY382884, were found to act at GluR5-containing channels while having no effect at GluR6 homomers. AMPA, ATPA and (S)-5-iodowillardiine did activate GluR6/KA2 heteromers, but only as partial agonists. Additionally, ATPA was shown to act as an antagonist at homomeric GluR6 receptors at high concentrations (IC50 approximately 2 mM). Kynurenic acid was also found to differentiate between GluR6 and GluR6/KA2 receptors, antagonizing glutamate at GluR6 (IC50 = 0.4 mM), while having no effect at GluR6/KA2 channels. The results of the current study provide a broad pharmacological characterization of both homomeric and heteromeric recombinant human kainate receptors, and identify which compounds are likely to be useful tools for studying these various receptor subtypes.

Characterisation of UBP296: A novel, potent and selective kainate receptor antagonist

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

Pharmacological Characterization of the Competitive GLUK5 Receptor Antagonist Decahydroisoquinoline LY466195 in Vitro and in Vivo

The excitatory neurotransmitter glutamate has been implicated in both migraine and persistent pain. The identification of the kainate receptor GLUK5 in dorsal root ganglia, the dorsal horn, and trigeminal ganglia makes it a target of interest for these indications. We examined the in vitro and in vivo pharmacology of the competitive GLUK5-selective kainate receptor antagonist LY466195 [(3S,4aR,6S,8aR)-6-[[(2S)-2-carboxy-4,4-difluoro-1-pyrrolidinyl]-methyl]decahydro-3-isoquinolinecarboxylic acid)], the most potent GLUK5 antagonist described to date. Comparisons were made to the competitive GLUK5/α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-decahydroisoquinoline-3-carboxylic acid], other decahydroisoquinoline GLUK5 receptor antagonists, and the noncompetitive AMPA receptor antagonist LY300168 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodi-azepine]. When characterized electrophysiologically in rat dorsal root ganglion neurons, LY466195 antagonized kainate (30 μM)-induced currents with an IC50 value of 0.045 ± 0.011 μM. In HEK293 cells transfected with GLUK5, GLUK2/GLUK5, or GLUK5/GLUK6 receptors, LY466195 produced IC50 values of 0.08 ± 0.02, 0.34 ± 0.17, and 0.07 ± 0.02 μM, respectively. LY466195 was efficacious in a dural plasma protein extravasation (PPE) model of migraine with an ID100 value of 100 μg/kg i.v. LY466195 was also efficacious in the c-fos migraine model, with a dose of 1 μg/kg i.v. significantly reducing the number of Fos-positive cells in the rat nucleus caudalis after electrical stimulation of the trigeminal ganglion. Furthermore, LY466195 showed no contractile activity in the rabbit saphenous vein in vitro. The diethyl ester prodrug of LY466195 was also efficacious in the same PPE and c-fos models after oral administration at doses of 10 and 100 μg/kg, respectively while having no N-methyl-d-aspartate antagonist-like behavioral effects at oral doses up to 100 mg/kg.

ACET is a highly potent and specific kainate receptor antagonist: Characterisation and effects on hippocampal mossy fibre function

Kainate receptors (KARs) are involved in both NMDA receptor-independent long-term potentiation (LTP) and synaptic facilitation at mossy fibre synapses in the CA3 region of the hippocampus. However, the identity of the KAR subtypes involved remains controversial. Here we used a highly potent and selective GluK1 (formerly GluR5) antagonist (ACET) to elucidate roles of GluK1-containing KARs in these synaptic processes. We confirmed that ACET is an extremely potent GluK1 antagonist, with a Kb value of 1.4+/-0.2 nM. In contrast, ACET was ineffective at GluK2 (formerly GluR6) receptors at all concentrations tested (up to 100 microM) and had no effect at GluK3 (formerly GluR7) when tested at 1 microM. The X-ray crystal structure of ACET bound to the ligand binding core of GluK1 was similar to the UBP310-GluK1 complex. In the CA1 region of hippocampal slices, ACET was effective at blocking the depression of both fEPSPs and monosynaptically evoked GABAergic transmission induced by ATPA, a GluK1 selective agonist. In the CA3 region of the hippocampus, ACET blocked the induction of NMDA receptor-independent mossy fibre LTP. To directly investigate the role of pre-synaptic GluK1-containing KARs we combined patch-clamp electrophysiology and 2-photon microscopy to image Ca2+ dynamics in individual giant mossy fibre boutons. ACET consistently reduced short-term facilitation of pre-synaptic calcium transients induced by 5 action potentials evoked at 20-25Hz. Taken together our data provide further evidence for a physiological role of GluK1-containing KARs in synaptic facilitation and LTP induction at mossy fibre-CA3 synapses.

LY339434, a GluR5 kainate receptor agonist

The activity of a gamma-substituted glutamate analogue, (2S, 4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) and (2S,4R)-4-methylglutamic acid at ionotropic glutamate receptors has been examined. Ligand binding studies were performed using [3H] AMPA binding to membranes expressing either homomeric recombinant GluR1, GluR2, GluR4 receptors, and [3H] kainate binding to GluR5 and GluR6 kainate receptors. LY339434 and (2S,4R)-4-methylglutamic acid showed selectivity in ligand binding studies for kainate receptors over AMPA receptors. Within the kainate class of glutamate receptors, LY339434 showed selectivity for GluR5 over GluR6 whereas (2S,4R)-4-methylglutamic acid showed high affinity for both GluR5 and GluR6 kainate receptors. Examination of the functional activity of LY339434 and (2S,4R)-4-methylglutamic acid showed that both compounds evoked inward currents in dorsal root ganglion neurons (DRG) with estimated EC50 values of 0.8 +/- 0.2 microM and 0.17 +/- 0.04 microM, respectively. In GluR5 expressing HEK 293 cells, LY339434 evoked inward currents with an estimated EC50 value of 2.5 +/- 0.9 microM but had little effect on GluR6 expressing cells at concentrations less than 100 microM. LY339434 was a weak AMPA receptor agonist (EC50 values > 300 microM) as determined by activity in acutely isolated cerebellar Purkinje neurons. LY339434 and (2S,4R)-4-methylglutamic acid had agonist activity at NMDA receptors studied in cultured hippocampal neurons with EC50s of 2.5 microM and 11.7 microM, respectively. These results indicate that both LY339434 and (2S,4R)-4-methyl glutamic acid may be useful pharmacological tools for the examination of kainate receptors.

Pharmacological effects of AMPA receptor potentiators LY392098 and LY404187 on rat neuronal AMPA receptors in vitro

The present study describes the pharmacological activity of two novel positive allosteric modulators at AMPA receptors in acutely isolated rat cerebellar Purkinje neurons and cultured rat hippocampal neurons. Currents elicited by application of glutamate (100 microM) to isolated cerebellar Purkinje neurons were potentiated by LY392098, LY404187, cyclothiazide, CX516 and aniracetam. The rank order of potency was LY404187> LY392098> cyclothiazide > CX516> aniracetam. LY392098 displayed a higher maximal efficacy than the other compounds examined. AMPA-activated inward currents in cultured rat hippocampal neurons were potentiated by LY392098, LY404187 and cyclothiazide in a reversible and concentration-dependent manner although considerable heterogeneity in the magnitude of response from cell to cell was observed. LY392098 was ineffective in potentiating AMPA receptor responses when dialyzed via the intracellular solution. The selectivity profiles of the two novel AMPA receptor potentiators were examined. LY392098 or LY404187 had minimal activity on NMDA receptor responses, on voltage-gated calcium channel currents in cultured hippocampal neurons and on GluR5 kainate receptor currents in acutely isolated rat dorsal root ganglion neurons.

Antiallodynic and Antihyperalgesic Effects of Selective Competitive GLUK5 (GluR5) Ionotropic Glutamate Receptor Antagonists in the Capsaicin and Carrageenan Models in Rats

GLUK5 kainate receptor subunits are abundant in pain pathways, including dorsal root ganglia and spinothalamic neurons, as well as in the thalamus and brain stem. A growing body of evidence indicates that the GLUK5 kainate receptor subtype plays a prominent role in pain transmission, particularly in persistent pain. In the present studies, compounds from a novel series of amino acid GLUK5 receptor antagonists were evaluated for their effectiveness in reversing capsaicin-induced mechanical allodynia as well as carrageenan-induced thermal hyperalgesia. In vitro, the amino acid compounds were efficacious in blocking glutamate-evoked calcium flux in cells expressing GLUK5 but not GLUK6 or GLUA2, homomeric receptors. Electrophysiologically, the compounds exhibited selectivity for kainate receptors in dorsal root ganglion cells relative to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide and N-methyl-d-aspartate receptors in hippocampal pyramidal neurons. The amino acid compounds were poorly efficacious in the pain tests after s.c. or p.o. administration. However, compounds were highly efficacious after central intracisternal administration, and the rank order of potencies correlated with their rank order of affinities at GLUK5 receptors determined in vitro, indicating that the lack of activity after systemic administration was due to poor oral bioavailability. To increase oral bioavailability, isobutyl or 2-ethylbutyl ester prodrugs of the parent amino acids were prepared. The prodrugs, which produced robust plasma levels of parent amino acids, were highly efficacious in the capsaicin and carrageenan tests. The present studies provide further evidence that selective GluK5 kainate receptor subtype antagonists can reverse allodynia and hyperalgesia, particularly in persistent pain states.

GluK1 antagonists from 6-(tetrazolyl)phenyl decahydroisoquinoline derivatives: in vitro profile and in vivo analgesic efficacy.

We have explored the decahydroisoquinoline scaffold, bearing a phenyl tetrazole, as GluK1 antagonists with potential as oral analgesics. We have established the optimal linker atom between decahydroisoquinoline and phenyl rings and demonstrated an improvement of both the affinity for the GluK1 receptor and the selectivity against the related GluA2 receptor with proper phenyl substitution. In this Letter, we also disclose in vivo data that led to the discovery of LY545694·HCl, a compound with oral efficacy in two persistent pain models.

GluK1 antagonists from 6-(carboxy)phenyl decahydroisoquinoline derivatives. SAR and evaluation of a prodrug strategy for oral efficacy in pain models

The synthesis and structure-activity relationship of decahydroisoquinoline derivatives with various benzoic acid substitutions as GluK1 antagonists are described. Potent and selective antagonists were selected for a tailored prodrug approach in order to facilitate the evaluation of the new compounds in pain models after oral administration. Several diester prodrugs allowed for acceptable amino acid exposure and moderate efficacy in vivo.