Regulating dopamine transport through allosteric modulation - Functional and Behavioral Studies - The plasma-membrane monoamine transporters (MATs), including the serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, serve a pivotal role in limiting monoamine-mediated neurotransmission through the reuptake of their respective neurotransmitters. MATs are targets for the treatment of numerous neurological disorders such as depression, anxiety, and attention deficit hyperactivity disorder (ADHD), and they serve as target proteins for major drugs of abuse such as amphetamine and cocaine. The continuing need for therapeutic drugs to treat brain disorders involving aberrant monoamine signaling provides a compelling reason to further our understanding of transporter function and to identify novel ways of targeting them. This project builds on our recent discovery of an allosteric site (A2) within the MATs that can serve as a target site for modulating their activity. Previous experiments in our group targeted the allosteric A2 site in SERT and identified molecules that interact with this site and display remarkable transporter-modulating activities. These compounds have revealed that engaging this site modulate MAT activity in entirely novel ways, including affecting the interaction with transporter ligands such as the selective serotonin reuptake inhibitors (SSRIs) and psychostimulants. In corresponding experiments on DAT, we have identified compounds, KM822 and sydnocarb among others, that similarly modifies DAT function. We find that these compounds interfere with the interaction of DAT with exogenous ligands and attenuates psychostimulant-elicited behaviors in rodents. Computational simulations further support the premise that compounds interacting with the allosteric A2 site can allow transport while interfering with the interaction of the transporter with exogenous ligands like cocaine. The overarching hypothesis of this project is that the specific engagement of the allosteric site in DAT will provide valuable information regarding mechanisms of the dopamine transport process and could provide novel therapeutic avenues for developing DAT-based medications. We propose to pursue this idea by further characterizing the compounds to study allosteric modulation of DAT. We will in Aim 1 elucidate mechanisms of allosteric transporter modulation through computational modelling and molecular simulations coupled with functional and biochemical studies. In aim 2 we will evaluate the in vivo utility of the compounds by examining their effects on psychostimulant-elicited behaviors in rodents. Finally, in Aim 3, we will employ structure-based design to identify A2-specific compounds with improved properties. Consequently, the successful completion of this project will result in the development of novel ligands of DAT that can be employed as experimental tools to provide critical mechanistic information regarding allosteric transporter modulation. Furthermore, the design and development of novel allosteric modulators of DAT will enable a systematic evaluation of the beneficial potential of these compounds to ultimately provide new therapeutic opportunities.
