Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist

Abstract

The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.

Characterizing the binding and function of TARP γ8-selective AMPA receptor modulators

“…four TARP molecules per AMPAR tetramer) ( 25 , 49 ). Although this component has previously been reported to be blocked by LY-481 ( 11 , 36 ), the effect of JNJ-118 on resensitization is currently unknown.…”

“…In orientation 4, we replicated a binding mode described for LY-481 based on a claudin-19 homology model ( 36 ), which we could not reproduce (see supplementary discussion) ( Fig. S5 ).…”

“…2 A1 , green ), had a substantial impact on binding, with the (S)-enantiomer being two orders of magnitude more potent than the (R)-enantiomer (pIC 50 = 7.2 versus 5.1) ( 12 ). Because radiolabeled LY-481 derivatives can associate with free γ8 in the absence of the AMPAR ( 36 ), we hypothesized that the oxindole isostere could directly engage with γ8 to mediate the compounds' effects.…”

“…Docking poses from the AMPAR/γ8 complex were more consistent between the three ligands compared with the γ8-only simulations, suggesting that even a subtle dilation of the γ8 M3 T and M4 T helices suffices for ligand binding. As γ8 itself can bind to a radiolabeled LY-481 derivative in HEK293 cells ( 36 ), free γ8 was an appropriate subject for our studies.…”

Resensitizing AMPA receptors

“…Understanding how γ8 resensitizes AMPA receptors is important because a number of drugs can selectively inhibit γ8-associated AMPA receptors and reduce neuronal excitability in epileptic forebrains (3,4). This suggests that γ8/AMPA receptor complexes can adopt unique conformations.…”