Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Abstract

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC(50) values of HD-205 were 4.1, 14 and 280nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC(50) values of 191, 230 and 840nM at DAT, SERT and NET, respectively. Saturation binding studies with [(125)I]RTI-55 in membranes pretreated with 100nM HD-205 showed that HD-205 significantly decreased the B(max) but not K(D) of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC(50) of HD-244 at DAT was 20nM. [(125)I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [(125)I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.

Fig. 1

Chemical structures of tropane analogs.

Fig. 2

Wash-resistant inhibition of radioligand binding at DAT, SERT and NET by HD-205 and HD-206. Rat striatal or whole brain membranes were preincubated for 1 hr at 25° with various concentrations of tropane analogs, then subjected to five washes before determination of radioligand binding with [¹²⁵I]RTI-55 (DAT), [³H]citalopram (SERT) and [³H]nisoxetine (NET) as described in Methods. For preincubation with HD-206 (600 nM), results averaged for all the three transporters are shown. The individual data for HD-206 at the three transporters (expressed as % control) are: 84% ± 10%, 99% ± 15% and 102% ± 11% at DAT, SERT and NET respectively.

Fig. 3

Wash-resistant effects of HD-205 on Scatchard plots of DAT binding (top) and SERT binding (bottom). Membranes were preincubated with 100 nM HD-205 for 1 hr at 25°, followed by five washes prior to radioligand binding. Data are typical plots from experiments repeated at least three times. Lines represent best-fit parameters for single-site analysis.

Fig. 4

Purification and pharmacological characterization of radiolabeled [¹²⁵I]HD-244 by HPLC. After reaction of HD-243 with 600 μCi of Na[¹²⁵I] with iodo-beads, samples were eluted on a C18 HPLC column using a linear gradient of 20 mM HEPES, pH 7.0 and acetonitrile. Arrow indicates the elution position of non-radioactive HD-244. Radioactivity (closed triangles) was determined in 10 μl aliquots from each fraction; expressed as cpm × 10⁻³. Equal amounts of [¹²⁵I] (60,000 cpm) from fractions 3–11 were used in DAT binding assays with rat striatal membranes in the presence (closed circles) and absence (open circles) of WF-23 to distinguish total and non-specific binding.

Fig. 5

Covalent labeling of striatal membrane proteins with [¹²⁵I]HD-244. Rat striatal membranes were incubated with [¹²⁵I]HD-244 for 1 hr at 25° in the presence and absence of 1 μM WF-23, solubilized in SDS buffer, and proteins separated by SDS-PAGE. Left, autoradiography of SDS-PAGE gel. TOT: total binding (without WF-23); NSB: non-specific binding (with WF-23). Right, Western blot of unlabeled membranes from rat striatum (STR) and cerebellum (CER) indicating the position of immunoreactive DAT protein in striatal membranes, using primary antibody directed against rat DAT (Abcam).