Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors

Abstract

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propionic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC₅₀ value of 150 μM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC₅₀ value of 170 μM and a relative efficacy of 0.17±0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED₅₀ >300 μM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED₅₀=14±2 μM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 μM) or DNQX (25 μM). A structure–activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.

Introduction

(S)-Glutamic acid (Glu), which is the main excitatory amino acid (EAA) neurotransmitter in the central nervous system (CNS), acts through four different classes of receptors. In addition to three heterogeneous classes of ionotropic EAA receptors (iGluRs), named N-methyl-d-aspartic acid (NMDA), (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) acetic acid (AMPA), and kainic acid receptors (Collingridge and Watkins, 1994, Wheal and Thomson, 1995, Monaghan and Wenthold, 1997), a heterogeneous class of metabotropic EAA receptors (mGluRs) has been shown to have important functions in central excitatory neurotransmission processes (Conn and Patel, 1994). Accumulating evidence suggests that iGluRs as well as mGluRs play important roles in the healthy as well as the diseased CNS, and that all subtypes of these receptors are potential targets for therapeutic intervention in a number of diseases (Lodge, 1988, Danysz et al., 1995, Knöpfel et al., 1995, Herrling, 1997).

Using the nonselective EAA receptor agonist, ibotenic acid, as a lead structure a number of structurally related 3-isoxazolol amino acids have been synthesized and characterized as selective EAA receptor ligands (Krogsgaard-Larsen et al., 1996, Sløk et al., 1997). Thus, (S)-AMPA selectively activates AMPA receptors (Krogsgaard-Larsen et al., 1980, Hansen et al., 1983), whereas the (R)-form of the lower homologue of AMPA, (RS)-2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA) is a selective NMDA agonist (Madsen et al., 1996b), and (S)-Homo-AMPA is a specific mGluR6 agonist (Ahmadian et al., 1997). As part of the structure–activity analyses of AMPA analogues, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (Fig. 1) was synthesized and shown to be a moderately potent AMPA receptor agonist (Lauridsen et al., 1985), showing some ability to penetrate the blood-brain barrier (BBB) (Turski et al., 1992, Arnt et al., 1995). ATPA has recently been shown to bind with similar and relatively weak affinities (K_i values of 6–14 μM) to recombinant AMPA receptors (GluR1–4) (Clarke et al., 1997). In this study, ATPA was however, shown to partially discriminate between subtypes of kainic acid receptors, showing relatively weak activity at GluR7 and KA-2 receptors, no activity at GluR6 receptors, but very high affinity for GluR5 receptors, with an IC₅₀ value for inhibition of [³H]kainic acid binding in the low nanomolar range (Clarke et al., 1997).

The 3-carboxymethoxy analogue of AMPA, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl)propionic acid (AMOA) has been described as a relatively selective AMPA receptor antagonist showing neuroprotective effects in vivo and in vitro (Frandsen et al., 1990, Krogsgaard-Larsen et al., 1991). Subsequently, the tert-butyl analogues of AMOA, (RS)-2-amino-3-[3-(carbomethoxy)-5-tert-butyl-4-isoxazolyl)propionic acid (ATOA) and (RS)-2-amino-3-[3-(phosphonomethoxy)-5-tert-butyl-4-isoxazolyl)propionic acid (ATPO) (Fig. 1) were synthesized and shown to be more potent than AMOA as AMPA receptor antagonists (Madsen et al., 1996a). However, whereas AMOA is a neuroprotectant (Frandsen et al., 1990), preliminary studies disclosed neurotoxic effects of ATOA using cultured cerebral cortical neurones and assay conditions similar to those used for studies of AMOA.

Using Xenopus oocytes expressing homo- or heterooligomeric EAA receptors, ATPO has recently been shown to be a dual AMPA/kainic acid ligand with GluR1–4 antagonist effects (K_i values of 4–26 μM) and low-efficacious partial agonism at GluR5 and GluR5/KA-2 receptors (Wahl et al., 1998). Under these conditions, AMOA showed antagonist effects at AMPA-preferring receptors an order of magnitude weaker that those of ATPO but did not show agonist activity at recombinant kainic acid receptors (Wahl et al., 1998). These observations prompted us to study the effects of ATOA at recombinant GluR5 receptors and the pharmacology of the cell damaging effects of ATPA and ATOA using cultured cerebral cortical neurones and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage.