Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro. Herein, we report on the structure-activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Glutamate transporters, or excitatory amino acid transporters (EAATs), are key proteins that regulate the excitatory tone in the central nervous system (CNS) by clearing synaptic glutamate, maintaining extracellular glutamate concentrations low enough to prevent receptor desensitization and/or glutamate-mediated excitotoxicity. Dysregulation of the function and/or expression of the EAATs is implicated in several diseases, including epilepsy, stroke, traumatic brain injury, drug abuse disorders, neurodegenerative disorders, and neuropathic pain, among others. In this chapter, we will discuss the regulatory mechanisms of EAATs in health and disease states. We will discuss post-translational modifications, trafficking deficits, reverse transport, and other regulatory processes. We will also discuss current approaches on potential therapeutic strategies targeting these transporters for many neuropsychiatric diseases.

Abstract ID 95456Poster Board 324Excitatory amino acid transporters (EAATs) are critical proteins in the CNS that regulate synaptic glutamate levels, crucially preventing excitotoxicity. The astrocytic transporter EAAT2 is responsible for the majority of glutamate clearance in the CNS. Aberrant EAAT2 activity and glutamatergic signaling occurs in many neuropsychiatric disorders. Our work focused on ischemic stroke, a condition that urgently needs new treatments. In ischemic stroke, excessive levels of released glutamate cause excitotoxicity, leading to secondary damage which ultimately results in cognitive deficits. We have developed novel allosteric modulators (AMs) of EAATs, including selective-EAAT2 positive allosteric modulators (PAMs), and non-specific and broad-acting analog AMs. We hypothesize the pharmacological activity of AMs is determined by differential interactions with critical amino acid residues located between the transporter’s scaffold and transport domains. We have two main goals: to further understand the mechanism of these AMs, and to study their effects in an in vitro stroke model. Computational modeling predictions suggest some amino acid residues on EAAT2 that are critical to mediating the action of NA-014, an EAAT2-specific PAM. Dose-response assays evaluating the effect of NA-014 and other AMs offered further insights on which residues are important for their action, and what chemical moieties confer EAAT subtype selectivity and pharmacological action. Additionally, we evaluated potential translatability of EAATs PAMs in a model of ischemic stroke. We hypothesize that these compounds can restore glutamatergic homeostasis by augmenting glutamate clearance. We evaluated NA-014 in an in vitro model of ischemic stroke, oxygen glucose deprivation, in primary neuron-glia cultures, and found that it demonstrated neuroprotective properties. Collectively, these studies expand our mechanistic understanding of the EAAT AMs and demonstrate their clinical utility for ischemic stroke.

The glutamatergic system, located throughout the brain including the prefrontal cortex and nucleus accumbens, plays a critical role in reward and reinforcement processing, and mediates the psychotropic effects of addictive drugs such as cocaine. Glutamate transporters, including EAAT2/GLT-1, are responsible for removing glutamate from the synaptic cleft. Reduced expression of GLT-1 following chronic cocaine use and abstinence has been reported. Here, we demonstrate that targeting GLT-1 with a novel positive allosteric modulator (PAM), NA-014, results in reduction of cocaine-associated behaviors in rats. Pharmacokinetic analysis demonstrated that NA-014 is brain-penetrant and suitable for in vivo studies.We found that 15 and 30 mg/kg NA-014 significantly reduced cocaine-induced locomotion in males. Only the 15 mg/kg dose was effective in females and 60 mg/kg was ineffective in both sexes. Furthermore, 30 and 60 mg/kg NA-014 reduced expression of cocaine conditioned place preference (CPP) in males. 30 mg/kg NA-014 reduced expression of cocaine CPP in females and 15 mg/kg did not affect cocaine CPP in either sex, suggesting GLT-1 influences cocaine-associated behaviors in a sex-dependent manner. NA-014 did not elicit rewarding behavior, nor alter baseline locomotion. Twice daily/7-day administration of 100 mg/kg of NA-014 did not alter GLT-1 or GLAST expression in either sex in the prefrontal cortex (PFC). Collectively, these studies demonstrated that NA-014 reduced the locomotor stimulant and rewarding effects of cocaine in male and female rats. In the context of psychostimulant use disorders, our study suggests studying GLT-1 PAMs as alternatives to β-lactam compounds that increase GLT-1 protein levels.

OBJECTIVE:Excitotoxicity is a common hallmark of epilepsy and other neurological diseases associated with elevated extracellular glutamate levels. Thus, here, we studied the protective effects of (R)-AS-1, a positive allosteric modulator (PAM) of glutamate uptake in epilepsy models. METHODS:(R)-AS-1 was evaluated in a range of acute and chronic seizure models, while its adverse effect profile was assessed in a panel of standard tests in rodents. The effect of (R)-AS-1 on glutamate uptake was assessed in COS-7 cells expressing the transporter. WAY 213613, a selective competitive EAAT2 inhibitor, was used to probe the reversal of the enhanced glutamate uptake in the same transporter expression system. Confocal microscopy and Western blotting analyses were used to study a potential influence of (R)-AS-1 on GLT-1 expression in mice. RESULTS:(R)-AS-1 showed robust protection in a panel of animal models of seizures and epilepsy, including the maximal electroshock- and 6 Hz-induced seizures, corneal kindling, mesial temporal lobe epilepsy, lamotrigine-resistant amygdala kindling, as well as seizures induced by pilocarpine or Theiler's murine encephalomyelitis virus. Importantly, (R)-AS-1 displayed a favorable adverse effect profile in the rotarod, the minimal motor impairment, and the Irwin tests. (R)-AS-1 enhanced glutamate uptake in vitro and this effect was abolished by WAY 213613, while no influence on GLT-1 expression in vivo was observed after repeated treatment. INTERPRETATION:Collectively, our results show that (R)-AS-1 has favorable tolerability and provides robust preclinical efficacy against seizures. Thus, allosteric enhancement of EAAT2 function could offer a novel therapeutic strategy for treatment of epilepsy and potentially other neurological disorders associated with glutamate excitotoxicity. ANN NEUROL 2025;97:344-357.

Excitatory amino acid transporters (EAATs) are key proteins in the CNS that regulate glutamate levels and, therefore, control excitatory neurotransmission and limit excitotoxicity. EAATs are trimers, with each protomer consisting of a scaffold domain, and a transport domain that slides along the scaffold domain in an “elevator‐like motion” to move synaptic glutamate into astrocytes and neurons. Our lab has discovered novel positive allosteric modulators (PAMs) of EAAT2, the main EAAT subtype responsible for glutamate clearance in the CNS. We hypothesize that these compounds act by altering the interactions between the scaffold and the transport domain. To test this, we used computational modeling, followed by site‐directed mutagenesis and radioligand uptake assays. This allowed us to identify mutations in specific amino acid residues on astrocytic transporters EAAT1 and EAAT2 that result in increased activity without changes in expression. This suggests that interactions between the domains are responsible for enhanced efficiency of these transporters. These gain‐of‐function mutated EAAT1 and EAAT2 are currently being studied in single molecule Förster Resonance Energy Transfer (smFRET) approaches to further understand the kinetics and dynamics of EAATs. In our work, we also evaluated the neuroprotective properties of our EAAT2 PAMs in models of excitotoxicity. Previously, our lab has demonstrated that EAAT2 PAMs are protective in an in vitro stroke model. We expanded our studies to investigate potential neuroprotective properties an in vitro epilepsy model, using live calcium signaling measured in primary neuron/glia cultures. Lastly, we explored the ability of these compounds to attenuate cocaine seeking behavior in conditioned place preference behavioral experiments. Collectively, these studies expand our understanding of the mechanisms of these PAMs and broaden their potential therapeutic indications.

(R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders.