Behavioral and biochemical studies of novel allosteric modulators of the dopamine transporter with therapeutic potential

Cocaine is a highly abused drug used recreationally for social purposes. The intense sensation of euphoria induced by cocaine is the root cause of its high abuse potential. Cocaine inhibits the dopamine transporter (DAT), a membrane bound protein responsible for the clearance of dopamine (DA) neurotransmitter from the synaptic cleft. Cocaine binding to the DAT blocks DA reuptake leading to an increase extracellular DA concentration, resulting in enhanced neurotransmission causing euphoria. Currently, there are no Food and Drug Administration approved treatments for cocaine abuse and addiction. Many attempts have been made to prevent the cocaine-DAT interaction, but have largely failed due to lack of efficacy, leading to an urgent need for clinically efficacious therapeutics to treat cocaine addiction. Previously, our lab found that psychostimulants such as cocaine and amphetamines have low affinity towards the monoamine transporters (SmMATs) of the parasite, Schistosoma mansoni as compared to the human MATs (hMAT) even though both transport their respective neurotransmitters with equal or comparable efficacies. By comparing the structure of SmMATs to the hMATs, secondary allosteric sites were discovered in the human serotonin transporter (hSERT) and subsequently in the human DAT. A database of several molecules was virtually screened for binding to this site, resulting in KM822 as one of the hits which showed promising results in reducing the effects of cocaine in preliminary in vitro experiments. To improve solubility and other drug-like properties, a series of analogs of KM822 were synthesized including NP-1-152. Our current study focuses on the pharmacological characterization of NP-1-152 and to test if this compound has therapeutic potential for the treatment of cocaine addiction. We employed biochemical experiments such as substituted cysteine accessibility method (SCAM) to explore the binding site of KM822 and NP-1-152, and compared the results with two known DAT inhibitors, Modafinil and Sydnocarb. In addition, two behavior models were used to assess NP-1-152's effect on cocaine-induced behaviors like hyperactivity and reward association in Long Evans male rats: locomotor behavior paradigm and conditioned placed preference. We found that NP-1-152 decreased cocaine-induced locomotion which was dose and time dependent. During the active wake-cycle period, NP-1-152 alone decreased locomotion at varying doses suggesting off-target effects. NP-1-152 alone did not promote place preference but did significantly decrease cocaine conditioned place preference. Modeling of this novel allosteric site using NP-1-152 might suggest a viable mechanism for the prevention and treatment of cocaine addiction.