Behavioral, biological, and chemical perspectives on atypical agents targeting the dopamine transporter

Abstract

Background: Treatment of stimulant-use disorders remains a formidable challenge, and the dopamine transporter (DAT) remains a potential target for antagonist or agonist-like substitution therapies.

Methods: This review focuses on DAT ligands, such as benztropine, GBR 12909, modafinil, and DAT substrates derived from phenethylamine or cathinone that have atypical DAT-inhibitor effects, either in vitro or in vivo. The compounds are described from a molecular mechanistic, behavioral, and medicinal-chemical perspective.

Results: Possible mechanisms for atypicality at the molecular level can be deduced from the conformational cycle for substrate translocation. For each conformation, a crystal structure of a bacterial homolog is available, with a possible role of cholesterol, which is also present in the crystal of Drosophila DAT. Although there is a direct relationship between behavioral potencies of most DAT inhibitors and their DAT affinities, a number of compounds bind to the DAT and inhibit dopamine uptake but do not share cocaine-like effects. Such atypical behavior, depending on the compound, may be related to slow DAT association, combined sigma-receptor actions, or bias for cytosol-facing DAT. Some structures are sterically small enough to serve as DAT substrates but large enough to also inhibit transport. Such compounds may display partial DA releasing effects, and may be combined with release or uptake inhibition at other monoamine transporters.

Conclusions: Mechanisms of atypical DAT inhibitors may serve as targets for the development of treatments for stimulant abuse. These mechanisms are novel and their further exploration may produce compounds with unique therapeutic potential as treatments for stimulant abuse.

Keywords: Atypical effects; DA releasers; DAT inhibitors; Dopamine transporter; Molecular mechanisms; Stimulant abuse treatment compounds.

Figure 1

(A) Model of the conformational cycle for substrate translocation by the dopamine transporter (DAT), based upon crystal structures of the bacterial NSS family protein LeuT. In its default ligand-free (apo) configuration, the transporter protein is thought to be in dynamic equilibrium between outward- and inward-facing conformational states (upper left and lower left structures, respectively). Binding of extracellular Na⁺ ions at the S1 site stabilizes an open-to-out conformation with a fully open extracellular gate (upper right structure), allowing substrate molecules maximum access to the core S1 binding domain. Substrate binding at the S1 site induces closure of the extracellular gate, establishing an occluded, closed-to-out conformation (lower right structure). It has been suggested that interaction of a second substrate molecule with a secondary binding domain in the extracellular vestibule (the S2 site, located 11-13 Å above the S1 site) helps facilitate opening of the intracellular gating network, giving rise to a fully inward-facing (open-to-in) conformation capable of releasing the S1-bound substrate and ions into the cytosol (lower middle structure). (B) Structural overlay of homology models of the human DAT protein (hDAT), constructed using either the LeuT (yellow ribbons) or the dDAT (light blue ribbons) crystals as structural templates. Overall, the two structures show a high degree of geometric congruence. The most profound differences are the position of the second extracellular loop region (EL2) and the orientation of TM12, which is kinked in the center of the helix in the dDAT-based structure (at P572). In the dDAT-based model, a molecule of cholesterol (sticks highlighted with a translucent blue molecular surface) is shown bound to the cavity formed by TM1, TM5 and TM7, as reported for the dDAT crystal structure. (C) Zoomed-in superposition view of both the LeuT- and dDAT-based transporter models with DA docked at the S1 site (rendered as sticks with translucent yellow and blue molecular surfaces, respectively). Residues that line the S1 binding site are labeled and rendered as thin colored sticks. (D – E) Two-dimensional molecular interaction diagrams of DA bound at the S1 site of the dDAT-based model (D) and the LeuT-based model (E). For each panel, the interaction map depicts respective DAT residues located within 4.5 Å of the bound DA molecule (hydrophobic residues are colored green and polar residues are purple). The most significant (non van der Waals) DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond (green), main-chain hydrogen bond (blue), cation-π bond ( +) or aromatic π-stacking ( ).

Figure 2

Chemical structures of representative atypical DAT ligands of various types: atypical DA uptake inhibitors (top and middle rows), C₁-substituted cocaine analogs (bottom left) and substrate-like partial DA releasers (bottom right). Despite their structural and mechanistic heterogeneity, atypical DAT ligands all exhibit a similar constellation of behavioral effects when compared to classical DAT inhibitors (e.g. cocaine and methylphenidate), including attenuated locomotor stimulant activity and reduced addictive liability.

Figure 3

Dose-dependent effects of 3α-diphenylmethoxytropane analogs on locomotor activity in mice. Ordinates, horizontal activity counts after drug administration. Abscissae, dose of drug in μmol/kg, log scale. Each point represents the average effect ± SEM determined in eight mice. The data are from the 30-min period during the first 60 min after drug administration, in which the greatest stimulant effects were obtained. Note that the fluoro-substituted compounds (A) were generally more efficacious than the other compounds, and that the bromo-substituted compounds (B) were generally the least efficacious. (Data are from Katz et al., 1999a; Hiranita et al. 2014)

Figure 4

Effects of presession treatment with the N-substituted BZT analogs, JHW 007 and AHN 2-005, or methylphenidate on cocaine self-administration. Each point represents the mean ±S.E.M. (n = 6–10). JHW 007, AHN 2-005, or methylphenidate were administered orally at 300, 240 or 60 min before sessions, respectively. Filled symbols represent the intravenous self- administration of cocaine after a pre-session administration of vehicle. Open symbols represent increasing doses of the pretreatments administered by gavage. Doses of JHW 007 or AHN 2-005 were 10.0 (O), 32.0 (Δ), or 100.0 (∇) mg/kg, p.o. Doses of methylphenidate were 3.2 (O), 10.0 (Δ), or 32.0 (∇)mg/kg, p.o. Abscissae: cocaine self-administration dose in milligrams per kilogram. Each point represents the mean and standard error of the mean (n=6–11). Data are from Hiranita et al. (2014).

Figure 5

Different effects on the cysteine accessibility of DAT T316C/C306A induced by various inhibitors. (A) Hong et al., unpublished results. (B) Adapted from Hiranita et al, 2014. HEK293 cells stably transfected with T316C/C306A human DAT were labeled with maleimide-PEO2-biotin in the presence of DAT inhibitors. Biotinylated DAT proteins were enriched with avidin beads from cell lysates and detected with DAT-specific antibodies. Mean densities of DAT bands were quantified using the NIH ImageJ software and normalized to percent of vehicle (error bar is SEM). Representative blot from n = 3 - 6 experiments. Quantified DAT band densities were analyzed by one way ANOVA with post hoc Dunnett's test or Fisher's least significant difference test. **, P<0.01, *P<0.05 compared with vehicle. See method details in Hong and Amara (2010) and (Hiranita et al., 2014).

Figure 6

Generic pharmacophore for biogenic amine transporter ligands. Note that transportable substrate ligands exhibit size constraints defined by the red circle. Functional groups attached to the nitrogen, α-carbon or phenyl ring that extend beyond the “edge” of the pharmacophore will generate partial substrates, transporter blockers or be inactive.

Figure 7

Effects of DAT releasers on extracellular dopamine (DA) and horizontal locomotor activity (HLA) in male rats. Rats undergoing microdialysis in the n. accumbens received sequential intravenous doses of the fully efficacious releaser PAL 1046, the partial releaser PAL-1045 or saline vehicle. Arrows indicate time of injections (1 or 3 mg/kg i.v.). Dialysate DA concentrations were assayed by HPLC-ECD. Data are mean ± SEM for n=6-7 rats/group.

Figure 8

Effects of mixed DAT/SERT releasers on extracellular dopamine (DA), extracellular serotonin (5-HT) and horizontal locomotor activity (HLA) in male rats. Rats undergoing microdialysis in the n. accumbens received sequential intravenous doses of the DAT-selective releaser PAL-353 or the non-selective DAT/SERT releaser PAL-313. Arrows indicate time of injections (1 or 3 mg/kg i.v.). Dialysate DA and 5-HT concentrations were assayed by HPLC-ECD. Data are mean ± SEM for n=6-7 rats/group.