Binding Mode of an α‑Amino Acid-Linked Quinoxaline-2,3-dione Analogue at Glutamate Receptor Subtype GluK1

Two α-amino acid-functionalized quinoxalines, <b>1a</b> (CNG-10301) and <b>1b</b> (CNG-10300), of a quinoxaline moiety coupled to an amino acid moiety were designed, synthesized, and characterized pharmacologically. While <b>1a</b> displayed low affinity at native AMPA, KA, and NMDA receptors, and at homomeric GluK1,3 receptors, the affinity for GluK2 was in the midmicromolar range (<i>K</i>_i = 136 μM), <b>1b</b> displayed low to midmicromolar range binding affinity at all the iGluRs (<i>K</i>_i = 9–126 μM). In functional experiments (outside-out patches excised from transfected HEK293T cells), 100 μM <b>1a</b> partially blocked GluK1 (33% peak response), while GluK2 was unaffected (96% peak response). Furthermore, <b>1a</b> was shown not to be an agonist at GluK1 and GluK2 at 100 μM. On the other hand, 100 μM <b>1b</b> fully antagonized GluK1 (8% peak response) but only partially blocked GluK2 (33% peak response). An X-ray structure at 2.3 Å resolution of <b>1b</b> in the GluK1-LBD (ligand-binding domain) disclosed an unexpected binding mode compared to the predictions made during the design phase; the quinoxaline moiety remains to act as an amino acid bioisostere, but the amino acid moiety is oriented into a new area within the GluK1 receptor. The structure of the GluK1-LBD with <b>1b</b> showed a large variation in domain openings of the three molecules from 25° to 49°, demonstrating that the GluK1-LBD is capable of undergoing major domain movements