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 (EC50 = 23 microM) approximately twice as potent as those of ATPA. (R)-ATPA antagonized depolarizations induced by AMPA (Ki = 253 microM) and by (S)-ATPA (Ki = 376 microM), and (R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid (Ki = 301 microM and 1115 microM). At cloned AMPA receptors, (S)-ATPA showed agonist effects at GluR3 and GluR4 with EC50 values of approximately 8 microM and at GluR1 (EC50 = 22 microM), 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 Ki values of 33-75 microM. (S)-ATPA produced potent agonist effects at GluR5 (EC50 = 0.48 microM). 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.
Stensbøl
T B
TB
PharmaBiotec NeuroScience Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Copenhagen.
Borre
L
L
Johansen
T N
TN
Egebjerg
J
J
Madsen
U
U
Ebert
B
B
Krogsgaard-Larsen
P
P
D000818:Animals; D001667:Binding, Competitive; D002540:Cerebral Cortex / Q000187:drug effects / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D018690:Excitatory Amino Acid Agonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D007555:Isoxazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008968:Molecular Conformation; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011422:Propionates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011810:Quinoxalines / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D018091:Receptors, AMPA / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics; D013237:Stereoisomerism; D014316:Tritium; D014981:Xenopus
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D007555:Isoxazoles; D011422:Propionates; D011810:Quinoxalines; D018091:Receptors, AMPA; D011993:Recombinant Fusion Proteins; D014316:Tritium; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; C081662:alpha-amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionate