A computational approach yields selective inhibitors of human excitatory amino acid transporter 2 (EAAT2)

Abstract

Excitatory amino acid transporters (EAATs) represent a protein family that is an emerging drug target with great therapeutic potential for managing central nervous system disorders characterized by dysregulation of glutamatergic neurotransmission. As such, it is of significant interest to discover selective modulators of EAAT2 function. Here, we applied computational methods to identify specific EAAT2 inhibitors. Utilizing a homology model of human EAAT2, we identified a binding pocket at the interface of the transport and trimerization domain. We next conducted a high-throughput virtual screen against this site and identified a selective class of EAAT2 inhibitors that were tested in glutamate uptake and whole-cell electrophysiology assays. These compounds represent potentially useful pharmacological tools suitable for further exploration of the therapeutic potential of EAAT2 and may provide molecular insights into mechanisms of allosteric modulation for glutamate transporters.

Keywords: SLC1A; allosteric regulation; drug discovery; excitatory amino acid transporters; glutamate; glutamate transporter; high throughput virtual screening; homology modeling; neurological disease; neurotransmitter.

Figure 1.

Predicted binding sites on hEAAT2 homology models. Yellow, transport domain. Blue, trimerization domain. Top panel, site A, inward-facing conformation (shown in spherical representation). Bottom panel, site B, outward-facing conformation (shown in spherical representation) located on the back side of hEAAT2 as compared with known glutamate- and UCPh101-binding sites. Green, TBOA. Cyan, UCPH101.

Figure 2.

A, uptake of 10 μm [¹⁴C]glutamic acid in HEK293TRex-TO/hEAAT2 cells in the presence of either control (DMSO) or 25 μm compounds. B, compound 1 dose-dependently inhibits the uptake of 10 μm [¹⁴C]glutamic acid in hEAAT2-expressing cells (6.6 ± 0.6 μm). C, compound 1v decreases the maximal transport capacity of the EAAT2 transporter without affecting glutamate K_d, suggesting an allosteric noncompetitive mode of action. D, compound 1 dose-dependently inhibits EAAT2-meduated currents in hEAAT2-expressing cells. The inhibition is reversible, as demonstrated in the inset.

Figure 3.

Top panel, predicted binding modes of compound 1 (orange and yellow) in site B of the hEAAT2 outward facing conformation model (green). Bottom panel, relevant residue differences between hEAAT2 (green) and hEAAT1 (purple) in site B, shown in stick representation.