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

Two alpha-amino acid-functionalized quinoxalines, 1a (CNG-10301) and 1b (CNG-10300), of a quinoxaline moiety coupled to an amino acid moiety were designed, synthesized, and characterized pharmacologically. While 1a 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 (Ki = 136 mu M), 1b displayed low to midmicromolar range binding affinity at all the iGluRs (K-i = 9-126 mu M). In functional experiments (outside-out patches excised from transfected HEK293T cells), 100 mu M 1a partially blocked GluK1 (33% peak response), while GluK2 was unaffected (96% peak response). Furthermore, 1a was shown not to be an agonist at GluK1 and GluK2 at 100 mu M. On the other hand, 100 mu M 1b fully antagonized GluK1 (8% peak response) but only partially blocked GluK2 (33% peak response). An X-ray structure at 2.3 angstrom resolution of 1b 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 1b showed a large variation in domain openings of the three molecules from 25 degrees G; to 49 degrees, demonstrating that the GluK1-LBD is capable of undergoing major domain movements.