| Numerous in vitro and in vivo studies implicate that certain ligands that interact with DAT, nAChRs, and mGlu2 have tremendous potential as anti-addiction therapeutics. However, understanding how these promising anti-addiction compounds interact with their major drug targets at the molecular level is limited because of the absence of human DAT, nAChRs, and mGlu2 x-ray crystal structures. This knowledge gap is important towards rationally designing new therapeutics for psychostimulant abuse and addiction. The objective of this research was to develop irreversible chemical probes based on promising anti-addiction lead compounds (i.e., pyrovalerone, bupropion, BINA, etc) to map their binding sites and poses within the DAT, select nAChR subtypes, or mGlu2. The central hypothesis was that these compounds could be rationally derivatized, without significant alteration in their pharmacological activity, with a photoreactive group capable of forming a covalent bond to their target protein and a tag for application of a "Binding Ensemble Profiling with (f)Photoaffinity Labeling (BEProFL)" experimental approach. BEProFL rationally couples photoaffinity labeling, chemical proteomics, and computational molecular modeling to allow structure-function studies of the target proteins. This central hypothesis was tested by pursuing three specific aims: 1.) Identification of non-tropane photoprobes based on pyrovalerone (PV) suitable for DAT structure-function studies, 2.) Identification of bupropion (BP)-based photoprobes suitable for DAT, and nAChR structure-function studies, and 3.) Identification of irreversible mGlu2 PAM ligands as chemical probes suitable for mGlu2 structure-function studies. In the first aim, PV, a non-tropane DAT inhibitor, was structurally modified to contain a photoreactive group (i.e., an aryl azide) and a tag (i.e., 125I). These photoprobes were then pharmacologically evaluated to identify suitable candidates for DAT structure-function studies. In the second aim, BP was structurally modified to contain an aryl azide and 125I. This probe successfully identified the exact location of the bupropion-binding site within the Torpedo nAChR. Under the third aim, biphenyl-carboxylic acid indanone- and pyridone-based mGlu2 PAMs were structurally modified to contain a photoreactive group (e.g., aryl azide, acetophenone) and a tag (e.g., terminal alkyne, aliphatic azide). These compounds, at present, are being subjected to mGlu2 pharmacological evaluation to identify suitable chemical probe candidates for mGlu2 structure-function studies. |
