Functional characterization of CP-465,022, a selective, noncompetitive AMPA receptor antagonist
Introduction
Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system. Glutamate synaptic transmission is mediated by three subtypes of ionotropic receptors: N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate, as well as by G-protein-coupled metabotropic receptors. This neurotransmitter system is involved in almost every aspect of central nervous system physiological function. In addition, it is widely held that a deregulation of glutamate neurotransmission plays a causal role in a number of pathological conditions. Synaptic levels of glutamate are tightly regulated and the extrasynaptic glutamate concentration is normally very low in the brain parenchyma. However, after traumatic brain injury or stroke, glutamate homeostasis is disrupted and glutamate levels can rise several hundred times higher than in healthy brain tissue (Bullock et al., 1995a, Bullock et al., 1995b). This results in unregulated and prolonged activation of glutamate receptors that ultimately produces neuron death. Glutamate receptor activity is also hypothesized to play a role in the neuron death of the chronic neurodegenerative conditions such as Alzheimer's disease and multiple sclerosis. In these latter conditions, subtle but chronic deregulation in neuronal energy metabolism has been suggested to render neurons susceptible to excitotoxicity from physiological glutamate receptor activity (Albin and Greenamyre, 1992). Finally, there is considerable evidence to implicate glutamate receptor deregulation in epileptogenesis (Chapman, 1998).
The hypothesis that overactivation of glutamate receptors causes neuron death and epilepsy has fueled extensive efforts to identify pharmacological agents that inhibit these receptors, in the hope of developing effective treatments for neurodegeneration and epilepsy. While the initial focus was on discovery of compounds that inhibited the NMDA subtype of glutamate receptor (Albers et al., 1992), there has also been interest in the therapeutic potential of AMPA receptor antagonists (Buchan et al., 1993, Rogawski, 1993). The basis for this interest stems largely from studies with the prototype AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) (Sheardown et al., 1990) and structurally related quinoxalinediones such as 6-(1H-imidazol-1-yl)-7-nitro-2,3(1h,4h)-quinoxalinedione hydrochloride (YM-90K) (Namba et al., 1994). The quinoxalinedione class of competitive AMPA receptor antagonists has been found to be broadly effective at blocking seizures in a number of animal models (Namba et al., 1994, Shimizu-Sasamata et al., 1996). Furthermore, these compounds effectively reduce neuronal loss under experimental conditions including middle cerebral artery occlusion, brief global cerebral ischemia, and traumatic brain and spinal cord injury (Buchan et al., 1991, Gill et al., 1992, Sheardown et al., 1993, Shimizu-Sasamata et al., 1996, Teng Dong and Wrathall, 1996). Clinical development of quinoxalinediones has been hampered by the poor pharmaceutical properties of these agents. Initial prototypes such as NBQX are poorly soluble and precipitate in the kidney at projected therapeutic plasma levels. Attempts have been made to improve the solubility of this class through addition of polar substituents; however, this significantly reduces brain penetrability. Thus, there is a continued need to discover new compounds to test the therapeutic potential of AMPA receptor inhibition.
In the present study, we describe the pharmacology of a novel 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 arose from a medicinal chemistry effort (Welch et al., 2001) to increase the potency and selectivity for AMPA receptor inhibition of the known anticonvulsant piriqualone (Koe et al., 1986; Fig. 1). We show in a series of in vitro functional experiments that CP-465,022 is a potent and selective AMPA receptor antagonist that inhibits the receptor through a noncompetitive mechanism. Parts of this work have been published previously in abstract form (Lazzaro and Ganong, 1998).
Section snippets
Methods
Animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996) and the European Communities Council Directive of November 24, 1986.
Effects of CP-465,022 on AMPA receptor-mediated currents in cortical neurons
The effect of CP-465,022 was examined on kainate-induced whole-cell currents in primary cultures of rat cortical neurons. That these kainate-induced responses are mediated by AMPA receptors is indicated by the pharmacology of this response. Specifically, (1) the EC50 of kainate (approximately 145 μM, see below) is consistent with kainate acting as a low-affinity AMPA receptor agonist (Partin et al., 1993), (2) the kainate-induced response is blocked (84% at 32 μM) by the AMPA receptor-specific
Discussion
The studies presented herein demonstrate that CP-465,022 is a potent antagonist for native AMPA receptors. In cultured rat cortical neurons, CP-465,022 inhibits kainate-induced, AMPA receptor-mediated whole-cell currents with an IC50 of 25 nM. Similar potency for inhibition of AMPA receptor-mediated currents was observed in the human neuronal hNT cell line (IC50 of 15 nM) and, qualitatively, in cultured rat hippocampal neurons. The inhibitory effect of CP-465,022 was observed to develop
Acknowledgements
J.L.T., B.L.C., F.E.E., J.H., W.M.W., A.H.G. and F.S.M. are supported by Pfizer Global Research and Development. J.L. is supported by a grant from the Spanish Ministry of Science and Technology (PM99-0106). A.V.P. holds a postdoctoral fellowship from the Community of Madrid. We wish to thank Dr Christine Strick for helpful discussions on the sequence similarities between AMPA and kainate receptors. Some of these data were presented previously in abstract form : Lazzaro, J.L., Ganong, A.H.,
References (37)
- et al.
Activity of 2,3-benzodiazepines at native rat and recombinant human glutamate receptors in vitro: stereospecificity and selectivity profiles
Neuropharmacology
(1996) Glutamate receptors in epilepsy
Progress in Brain Research
(1998)- et al.
The distribution of glutamate receptors in cultured rat hippocampal neurons: postsynaptic clustering of AMPA- selective subunits
Neuron
(1993) - et al.
The neuroprotective actions of 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo(f)quinoxaline (NBQX) in a rat focal ischaemia model
Brain Research
(1992) - et al.
Localization of AMPA receptors in the hippocampus and cerebellum of the rat using an anti-receptor monoclonal antibody
Neuroscience
(1992) Spermine regulates N-methyl-d-aspartate receptor desensitization
Neuron
(1992)- et al.
The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function
Neuron
(1995) - et al.
Antiepileptogenic and anticonvulsant effects of NBQX, a selective AMPA receptor antagonist, in the rat kindling model of epilepsy
Brain Research
(1994) - et al.
Modulation of N-methyl-d-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells
Brain Research
(1991) - et al.
Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin-A
Neuron
(1993)
Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons
Neuron
Therapeutic potential of excitatory amino acid antagonists: channel blockers and 2,3-benzodiazepines
Trends in Pharmacological Science
AMPA, but not NMDA, receptor antagonism is neuroprotective in gerbil global ischaemia, even when delayed 24-h
European Journal of Pharmacology
Atropisomeric quinazolin-4-one derivatives are potent non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists
Bioorganic and Medicinal Chemistry Letters
Do NMDA antagonists prevent neuronal injury? Yes
Archives of Neurology
Alternative excitotoxic hypothesis
Neurology
AMPA antagonists: do they hold more promise for clinical stroke trials than NMDA antagonists?
Stroke
Delayed AMPA receptor blockade reduces cerebral infarction induced by focal ischemia
NeuroReport
Cited by (63)
Structural Bases of Noncompetitive Inhibition of AMPA-Subtype Ionotropic Glutamate Receptors by Antiepileptic Drugs
2016, NeuronCitation Excerpt :Fitting of concentration dependencies with the logistic equation yielded the following values of the half-maximal inhibition concentration (IC50) and the Hill coefficient (nHill) for PMP: IC50 = 0.88 ± 0.06 μM and nHill = 1.08 ± 0.04 for GluA2Del (n = 6) and IC50 = 0.89 ± 0.07 μM and nHill = 1.08 ± 0.05 for GluA2WT (n = 5); for GYKI: IC50 = 18.9 ± 1.8 μM and nHill = 1.29 ± 0.08 for GluA2Del (n = 5) and IC50 = 14.5 ± 1.6 μM and nHill = 1.29 ± 0.08 for GluA2WT (n = 6); and for CP: IC50 = 0.76 ± 0.14 μM and nHill = 1.73 ± 0.21 for GluA2Del (n = 5) and IC50 = 0.76 ± 0.03 μM and nHill = 1.72 ± 0.08 for GluA2WT (n = 6). Finally, we tested the noncompetitive character of the GluA2WT and GluA2Del receptor-mediated current inhibition (Balannik et al., 2005; Chen et al., 2014a; Donevan and Rogawski, 1993; Lazzaro et al., 2002). Typical of noncompetitive antagonists, inhibition of currents by GYKI, PMP, and CP was voltage independent (Figures 1C and 1D).