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Glutamate excitotoxicity is the result of increased extracellular glutamate levels, which causes overactivation of post synaptic neurons, excessive calcium influx and further downstream signaling resulting in cell death. Excitotoxicity is associated with a number of acute and chronic CNS disorders such as stroke, traumatic brain injury (TBI), epilepsy, Huntington's disease, Parkinson's disease, mental illness, Alzheimer's disease, neuropathic pain and drug addiction. Excitatory amino acid transporter 2 (EAAT2) is responsible for rapid removal of the majority of extracellular glutamate, thus maintaining the glutamate homeostasis and preventing excitotoxicity. Therefore, enhancing the function of this transporter could be a promising approach towards preventing or treating the harmful effects of excitotoxicity. Previous studies have identified a compound from a spider venom that enhanced the function of EAAT2 and displayed neuroprotective properties. Later, the site of interaction of this compound on the transporter was identified, and this information was employed to generate a pharmacophore, that in turn was used to perform a virtual screening of a large library of compounds. This approach identified novel compounds that were characterized as positive allosteric modulators (PAM) of EAAT2, in glutamate uptake assays in transfected COS-7 cells and in glial cultures. Nonetheless, the compounds derived from the virtual screening had poor drug-like properties. To address this issue and progress the studies toward drug development, several analogs were designed and synthesized in collaboration with a medicinal chemistry lab. The purpose of this study was to screen these compounds for efficacy, potency and selectivity in glutamate transport assays. Additionally, a compound derived from Parawixia bistriata venom, Parawixin10 (PWX-10) was also studied. Our goal was to identify a lead compound that is an EAAT2 PAM with good drug-like properties that can progress to in vivo studies. In structure activity relationship (SAR) studies, compounds were first screened in assays in overexpressing COS-7 cells (transfected with EAAT1, EAAT2 and EAAT3 respectively) and in cultured astrocytes. The selectivity of stimulatory compounds were confirmed through dose response assays in other types of neurotransmitter transporters (GABA, Glycine and monoamine transporters) and the mechanism of allosteric modulation was confirmed through kinetic studies by comparing the Vmax and Km values. Selected stimulatory compounds were studied for their neuroprotective properties in in vitro models in neuron/glia cultures subjected to excitotoxic insults by application of glutamate and by deprivation of oxygen and glucose. Using this approach, we have identified three non-selective PAMs of EAAT1-3 transporters: VY-3-285, VY3-286 and NA-005, three PAMs of EAAT1 and EAAT2: DA-050, DA-058, PWX-10, and a few inactive compounds and negative allosteric modulators (NAMs) of glutamate transporters. Moreover, we identified one selective EAAT2 PAM, NA-014, with good efficacy and potency and, importantly, remarkable neuroprotective properties in two in vitro models of excitotoxicity. Future directions include examining the effects compound NA-014 in in vitro and in vivo models, such as TBI, neuropathic pain and epilepsy. The NAMs of EAAT2 and other subtypes of transporters identified can be used in future studies to understand mechanisms of allosteric modulation of glutamate transport. |
