Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA

( RS)-2-Amino-3-(5- tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of ( RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring ( S)-glutamic acid (( S)-Glu) receptors. We report here the separation of (+)- and (−)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and >99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of ( S)-(+)-ATPA and ( R)-(−)-ATPA. ( S)- and ( R)-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6+ KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, ( S)-ATPA showed AMPA receptor agonist effects (EC 50=23 μM) approximately twice as potent as those of ATPA. ( R)-ATPA antagonized depolarizations induced by AMPA ( K i=253 μM) and by ( S)-ATPA ( K i=376 μM), and ( R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid ( K i=301 μM and 1115 μM). At cloned AMPA receptors, ( S)-ATPA showed agonist effects at GluR3 and GluR4 with EC 50 values of approximately 8 μM and at GluR1 (EC 50=22 μM), producing maximal steady state currents only 5.4–33% of those evoked by kainic acid. ( R)-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with K i values of 33–75 μM. ( S)-ATPA produced potent agonist effects at GluR5 (EC 50=0.48 μM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, ( S)-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by ( S)-ATPA and kainic acid were not significantly different. ( R)-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither ( S)- nor ( R)-ATPA showed significant effects at GluR6. ( S)-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6+KA2 receptors, whereas ( R)-ATPA was inactive. Thus, ( S)- and ( R)-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA ( S)-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.