Dopamine Transporter Dynamics of N-Substituted Benztropine Analogs with Atypical Behavioral Effects

Abstract

Atypical dopamine transporter (DAT) inhibitors, despite high DAT affinity, do not produce the psychomotor stimulant and abuse profile of standard DAT inhibitors such as cocaine. Proposed contributing features for those differences include off-target actions, slow onsets of action, and ligand bias regarding DAT conformation. Several 3α-(4',4''-difluoro-diphenylmethoxy)tropanes were examined, including those with the following substitutions: N-(indole-3''-ethyl)- (GA1-69), N-(R)-2''-amino-3''-methyl-n-butyl- (GA2-50), N-2''aminoethyl- (GA2-99), and N-(cyclopropylmethyl)- (JHW013). These compounds were previously reported to have rapid onset of behavioral effects and were presently evaluated pharmacologically alone or in combination with cocaine. DAT conformational mode was assessed by substituted-cysteine accessibility and molecular dynamics (MD) simulations. As determined by substituted-cysteine alkylation, all BZT analogs except GA2-99 showed bias for a cytoplasmic-facing DAT conformation, whereas cocaine stabilized the extracellular-facing conformation. MD simulations suggested that several analog-DAT complexes formed stable R85-D476 "outer gate" bonds that close the DAT to extracellular space. GA2-99 diverged from this pattern, yet had effects similar to those of other atypical DAT inhibitors. Apparent DAT association rates of the BZT analogs in vivo were slower than that for cocaine. None of the compounds was self-administered or stimulated locomotion, and each blocked those effects of cocaine. The present findings provide more detail on ligand-induced DAT conformations and indicate that aspects of DAT conformation other than "open" versus "closed" may facilitate predictions of the actions of DAT inhibitors and may promote rational design of potential treatments for psychomotor-stimulant abuse.

Fig. 1.

Chemical structures of cocaine and the presently studied BZT analogs.

Fig. 2.

Modulation of hDAT T316C/C306A cysteine accessibility by BZT analogs. (A) Human embryonic kidney 293 cells expressing T316C/C306A hDAT were incubated with drugs at 4°C for 15 minutes, followed by incubation with drugs at 4°C for 45 minutes in the presence of maleimide-PEO₂-biotin, a membrane-impermeant, thiol-selective probe that reacts with accessible cysteine residues in cell surface proteins. After cell lysis, labeled DAT proteins were enriched with NeutrAvidin beads, separated by SDS-PAGE, and detected by immunoblotting (see Methods for details). Summarized results (average ± S.E.M., top panel) are from n = 3 to 4 experiments, with a representative immunoblot showing labeled DAT (∼75 kDa), which is glycosylated on the cell surface (middle panel). Quantified DAT band densities were normalized to vehicle treatment. **P < 0.01, compared with vehicle, one-way ANOVA with post hoc Dunnett’s test. DAT proteins in cell lysates were of approximately the same amount (bottom panel). “Coc” represents cocaine. (B and C) Structure cartoons of inward-facing LeuT_Aa and outward-facing dDAT (PDB codes 3TT3 and 4XP4, respectively), generated using UCSF Chimera 1.12 software. Both show cross-section surface representations with the residue corresponding to T316C in hDAT (G249 in LeuT_Aa; T315 in dDAT) in focus (highlighted in magenta). In the outward-facing conformation, the residue is exposed on the surface of a vestibule accessible from the extracellular side; it is buried inside the protein in the inward-facing conformation. TMs are colored in a rainbow pattern from blue to red, with TM12 (red) aligned to the left.

Fig. 3.

Molecular dynamics (MD) of inhibitor-DAT complexes. Leftmost column: Side view of hDAT computational model featuring the approximate S1 ligand binding pocket (inscribed by orange oval) and R85-D476 salt bridge strut of the outer gate. Remaining columns: Top row: MD traces (n = 2–4) of representative simulations of each of four inhibitors docked to the DAT model. Ordinate indicates average distance between the outer gate R85 terminal nitrogen atoms and D476 carboxylate oxygen atoms as a function of simulation time point. Average distance for an intact salt bridge (∼3 Å) is represented by the red horizontal bar. A vertical line after 300 ns and color-coded by inhibitor ligand indicates the time point at which “snapshot” poses were taken. Bottom row: Side view snapshot poses of DAT MD simulations involving cocaine (purple), GA2-99 (gold), BZT (cyan), or GA1-69 (green). For each pose, nearby surrounding S1 pocket side chains are shown (atomtype color), with the outer gate R85 and D476 residues annotated in red.

Fig. 4.

In vivo displacement of specific [¹²⁵I]RTI-121 accumulation in mouse striatum at various times following intraperitoneal injection of cocaine, GA1-69, GA2-59, GA2-99, and JHW013. Ordinates: specific [¹²⁵I]RTI-121 binding as a percentage of that obtained after vehicle injection. Abscissae: time. For each point, the number of replicates was typically from three to seven. The bottom right panel shows maximal occupancy of [¹²⁵I]RTI-121 binding sites as a function of dose. Ordinates, %DAT occupancy. Abscissae, drug dose in micromoles per kilogram. Note that maximal displacement of [¹²⁵I]RTI-121 was obtained at different time points after injection. Those times were for cocaine, 45 minutes at 40 mg/kg; GA1-69, 30 minutes at 17 mg/kg; GA2-50, 10 minutes at 100 mg/kg; GA2-99, 30 minutes at 56 mg/kg; and JHW013, 30 minutes at 30 mg/kg.

Fig. 5.

Effects of N-substituted BZT analogs on locomotor activity and stereotyped behavior in rats. (A) Effects on horizontal locomotor activity. Abscissae: drug dose in micromoles per kilogram, log scale; Ordinates: horizontal locomotor activity counts per minute over a 30-minute period after drug administration. Each point represents the average effect determined in six subjects. (B) Effects on stereotypy determined as described in the Methods. Abscissae: drug dose in micromoles per kilogram, log scale; Ordinates: stereotypy score determined over a 30-minute period after drug administration. Each point represents the average effect determined in six subjects. (C–G) Effects of combinations of BZT analogs or WIN35,428 with cocaine. Abscissae: dose of BZT analog or WIN35,428 (log scale) alone (filled symbols) or in combination with a 29.4 µmol/kg (10 mg/kg) dose of cocaine (open symbols) that produced intermediate changes in locomotor activity or stereotypy. Ordinates: horizontal locomotor activity counts per minute (circles) or stereotypy score (triangles) determined over a 30-minute period after drug administration. Vertical bars represent the S.E.M. The data are from the 30-minute period immediately after drug administration. Note that none of the BZT analogs produced a stimulation of activity or stereotypy that was equivalent to that of cocaine and that all but GA2-50 blocked the stimulation of activity produced by cocaine, whereas none of the compounds blocked cocaine-induced stereotypy.

Fig. 6.

Dose-dependent interactions of N-substituted BZT analogs with cocaine on locomotor activity in mice. Ordinates: horizontal locomotor activity counts after drug administration. Abscissae: dose of cocaine in milligrams per kilogram, log scale. Each point represents the average effect determined in six mice. Vertical bars represent the S.E.M. Filled points represent the effects of cocaine administered with saline. Disconnected points on the left above V represent the effects of the BZT analogs administered with vehicle. The data are from the 30-minute period immediately after drug administration. Note that each of the BZT analogs [GA1-69 (A), GA2-50 (B), GA2-99 (C), JHW013 (D)] at some doses blocked the stimulation of activity produced by cocaine.

Fig. 7.

Effects of various doses of cocaine and N-substituted BZT analogs in rats trained to discriminate injections of cocaine (10 mg/kg) from saline at various times after injection. Ordinates: Percentage of responses on the cocaine-appropriate key. Abscissae: drug dose in milligrams per kilogram (log scale). Each point represents the effect in six rats. Top row shows effects of each N-substituted BZT analog administered alone compared with effects of cocaine. Filled circles, reproduced in each panel of the figure, represent the effects of cocaine. Open triangles show effects obtained with the BZT analog injected 5 (triangles up) or 60 minutes (triangles down) before testing. Bottom row shows effects of each N-substituted BZT analog administered in combination with cocaine. Filled circles, reproduced in each panel of the figure, represent the effects of cocaine administered alone. Open symbols show effects obtained with combinations of cocaine and each of the BZT analogs at various doses.

Fig. 8.

Substitution of different doses of WIN35,428, BZT analogs, or saline in rats trained to self-administer cocaine [0.09–2.94 µmol/kg/injection]. Ordinates, responses per second. Abscissae, injection dose in milligrams per kilogram. Points above EXT (extinction) represent response rates when responses had no consequences. Each point represents the mean (with bars showing S.E.M., n = 6). (A) WIN35,428 or saline substitution for cocaine. (B) GA1-69, GA2-50, GA2-99, JHW013, or saline substitution for cocaine.

Fig. 9.

Effects of pre-session treatments with WIN35,428 or BZT analogs in rats trained to self-administration cocaine [0.09–2.94 µmol/kg/injection]. Each point represents the mean ± S.E.M. Top row: effects of test compounds [WIN35,428 (A), GA1-69 (B), GA2-50 (C), GA2-99 (D), JHW013 (E)] on self-administration of cocaine (n = 6). Ordinates, responses per second. Abscissae, injection dose of cocaine in micromoles per kilogram. Points above EXT (extinction) represent response rates when responses had no consequences. Bottom row: effects of pre-session treatments with test compounds [WIN35,428 (F), GA1-69 (G), GA2-50 (H), GA2-99 (J), JHW013 (K)] on responding maintained by cocaine injections or food presentations. Ordinates, response rates as percentage of control response rates (sessions before drug tests). Abscissae, micromoles per kilogram of the test compounds (i.p.), log scale. Rates of responding were from the fourth 20-minute component of the session (see Methods). All test compounds were administered 5 minutes before sessions. Rates of responding maintained by food reinforcement averaged 0.549 ± 0.059 responses/s (n = 18), whereas those maintained by cocaine averaged 0.287 ± 0.050 responses/s (n = 12). Significant difference was found in the control rates across the groups (F_1,28 = 9.84; P = 0.04, one-way ANOVA).