Despite decades of committed research, no agents are clinically available to combat psychostimulant abuse and addiction. This prodigious void can be linked to sparse information on the 3-D structure and function of the dopamine transporter (DAT), which pharmacological and behavioral studies indicate is the brain receptor chiefly responsible for the reinforcing / reward properties associated with amphetamines and cocaine. There is a fundamental gap in understanding the mechanism of inhibitors and the structural elements and conformational states associated with the DAT. The long-term goal of our research is to develop chemical entities towards understanding how abused and therapeutic compounds interact with the DAT at the amino acid level. The objective of this particular application is to develop non-tropane irreversible ligands for mapping drug-binding pockets within the DAT. The central hypothesis is that non-tropane irreversible ligands can serve as complimentary probes to existing tropane-based (e.g., cocaine, benztropine) irreversible ligands for high- resolution elucidation of DAT 3-D structure. Guided by the literature and strong preliminary data, this central hypothesis will be tested by pursuing two specific aims: 1) Identify the structural elements of pyrovalerone- based irreversible ligands essential for labeling the DAT;and 2) Identify the structural elements of methylphenidate-based irreversible ligands for labeling the DAT. The specific hypotheses for these aims will test whether these non-tropane-based inhibitor scaffolds can be structurally modified to covalently label the DAT via an affinity or photoaffinity labeling experiment. Specifically, pyrovalerone and methylphenidate will be modified to contain an iodine as a tracer tag and either an aryl azide or aryl isothiocyanate as a reactive labeling functional group. The synthesized analogs will then be pharmacologically evaluated at the DAT in order to identify candidate probes for irreversible DAT labeling experiments. Additionally, a systematic design approach towards DAT irreversible ligands will be explored to address noted shortcomings associated with established tropane-based DAT irreversible ligands. The proposed research is significant because the binding sites and conformational preferences for pyrovalerone and methylphenidate as non-tropane therapeutically relevant compounds are unknown relative to tropane-based DAT ligands. A battery of developed probes based on these chemical scaffolds will advance the field towards rational structure-based drug design for a myriad of DAT-implicated disorders. PUBLIC HEALTH RELEVANCE: The proposed studies seek to provide chemical probes for 3-D mapping of drug-binding pockets within the dopamine transporter (DAT). The elucidation of details associated with DAT structure and function in turn will contribute to an understanding of how DAT protein components discriminate between therapeutic (e.g., benztropine, methylphenidate, bupropion) and abused (e.g., cocaine, amphetamine) compounds. Such findings will promote the rational design of safe and effective therapeutics for DAT-implicated conditions such as Parkinson's disease, Alzheimer's disease, schizophrenia, Tourette's syndrome, Lesch-Nyhan disease, attention deficit hyperactivity disorder, depression, obesity, and psychostimulant abuse.