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Structural and Molecular Determinants of Glutamate Transporter Allosteric Modulators
Journal article   Peer reviewed

Structural and Molecular Determinants of Glutamate Transporter Allosteric Modulators

Katelyn L. Reeb, Satyaki Saha, Xiaowei Bogetti, Adi N.R. Poli, Joseph M. Salvino, Mary Hongying Cheng, Ole V. Mortensen, Ivet Bahar and Andréia C.K. Fontana
Molecular pharmacology, Forthcoming
Apr 2026
DOI: https://doi.org/10.1016/j.molpha.2026.100122
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Abstract

allosteric modulators docking simulations EAATs glutamate transporters Mutagenesis
Excitatory Amino Acid Transporters (EAATs) are critical regulators of synaptic glutamate levels in the central nervous system (CNS). Dysregulated CNS glutamatergic homeostasis is implicated in many neurological diseases, highlighting the key role of EAATs in neurological health. We previously identified a library of small compounds that function as either positive or negative allosteric modulators (PAMs or NAMs) of EAATs, with diverse selectivity for subtypes EAAT1, EAAT2, and EAAT3, including astrocytic EAAT1 and EAAT2, and neuronal EAAT3. In this work, we characterize compounds from our library using molecular modeling, mutagenesis and pharmacological approaches. We focused on three representative compounds: NA-014, an EAAT2-selective PAM, DA-038, an EAAT1-3 PAM; and NA-010, an EAAT2-selective NAM. Binding studies demonstrated that these compounds do not interact with the orthosteric glutamate-binding site, confirming an allosteric action. Docking studies suggested several potential binding poses of NA-014 between the scaffold and transport domains of EAAT2, which we then studied with mutagenesis approaches. We identified potential binding sites of representative compounds in transmembrane (TM) domains 1, 5, 8 and hairpin 2 (HP2) and demonstrated that these are necessary for their activity. Ten key amino acids residues within a subdomain of EAAT2 substituted into EAAT1 conferred EAAT2-selective PAM activity, demonstrating these residues are required and sufficient to enable selective PAM function. Collectively, these studies identified crucial subdomains and key amino acids linked to PAM activity, advancing our understanding of how to modulate EAAT activity. This knowledge can be integrated in future studies to develop EAAT allosteric modulators for neurological disorders.

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