Allosteric Modulator KM822 Attenuates Behavioral Actions of Amphetamine in through Interactions with the Dopamine Transporter DAT-1.

Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions is unproven given the large number of potential off-target sites. Here, we provide and evidence that the allosteric site engaged by KM822 is conserved between human DAT and DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using and genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In , we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal as a new tool to study allosteric modulation of DAT and its behavioral consequences.