The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces, and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. Thus, DAT is a viable target for the treatment of psychostimulant abuse and addiction. We have recently identified a novel allosteric site that lies outside the central substrate and inhibitor binding pocket of DAT. This allosteric site was identified using information derived from comparative modeling of human and parasite Schistosoma mansoni monoamine transporters. Site-directed mutagenesis has validated the functional significance of this allosteric site. In addition, the hybrid structure–based (HSB) method has identified conformation-specific molecules that can potentially stabilize the transporter in certain beneficial conformations that may alter its interaction with cocaine and amphetamine without affecting uptake activity of DA. Interestingly, KM822, one of the ligands identified in a virtual screening experiment, was found to decrease the affinity of cocaine for DAT by 3-folds in an in vitro cell-based assay. In addition, KM822 also reduced the potency of cocaine towards DAT-mediated DA reuptake inhibition in an ex vivo model of striatal synaptosome preparations. The preliminary in vivo effects of KM822 on cocaine potency were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. We have further identified the structural determinants of KM822 allosteric binding site by employing substituted cysteine scanning accessibility methods (SCAM) and biotinylation experiments. Additional studies are being employed to directly determine the point of interaction of KM822 within the allosteric pocket by using affinity-based labeling and biorthogonal click chemistry techniques. In addition, we have also performed a SAR evaluation of several KM822 analogs to find more potent and efficient DAT modulators. Overall, KM822 provides a unique opportunity as a molecular probe to identify novel mechanistic aspects of DAT function and can lead to the development of more unique compounds with promising mechanism of action.

Structure-function studies of a novel allosteric site of the dopamine transporter as a target for alternative therapeutics against cocaine use disorder

2019 Spring National Meeting

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