Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine

Abstract

Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake.

Fig. 1.

Chemical structures of the DAT (A) and A₃AR (B) ligands referred to in the text.

Fig. 2.

Drug-induced enhancement or inhibition of [¹²⁵I]RTI-55 binding and [³H]mazindol binding to HEK-hDAT cell membranes. Assays were conducted as described in Materials and Methods. A total of n = 3–9 independent experiments were conducted in duplicate, except for compounds with no effect, where n = 2 independent experiments were conducted. (A) [¹²⁵I]RTI-55 binding of compounds 2, 6, 8, 9, and 22; (B) [¹²⁵I]RTI-55 binding of compounds 4, 5, 7, 16, 17, and 19; (C) [³H]mazindol binding of compounds 2, 6, 8, 9, and 22; (D) [³H]mazindol binding of compounds 4, 5, 7, 16, 17, and 19 (n = 3–8 independent experiments, except for drugs with no effect, where n = 2).

Fig. 3.

Drug-induced enhancement of [¹²⁵I]RTI-55 binding to HEK-hNET cell membranes is not observed with [³H]mazindol binding to HEK-hNET cell membranes. A total of n = 2–6 independent experiments were conducted in duplicate, except for compounds with no effect, where n = 2 independent experiments were conducted. (A) [¹²⁵I]RTI-55 binding of compounds 2, 6, 8, 9, and 22; (B) [¹²⁵I]RTI-55 binding of compounds 4, 5, 7, 16, 17, and 19; (C) [³H]mazindol binding of compounds 2, 6, 8, 9, and 22; (D) [³H]mazindol binding of compounds 4, 5, 7, 16, 17, and 19 (n = 3–10, except for drugs with no effect, where n = 2).

Fig. 4.

Lack of drug-induced enhancement of [¹²⁵I]RTI-55 binding and [³H]mazindol binding to HEK-hSERT cell membranes. (A) [¹²⁵I]RTI-55 binding of compounds 2, 6, 8, 9, and 22; (B) [¹²⁵I]RTI-55 binding of compounds 4, 5, 7, 16, 17, and 19; (C) [³H]mazindol binding of compounds 2, 6, 8, 9, and 22; (D) [³H]mazindol binding of compounds 4, 5, 7, 16, 17, and 19 (n = 3–7 independent experiments, except for drugs with no effect, where n = 2).

Fig. 5.

Scatchard plots of [¹²⁵I]RTI-55 binding in the presence of compound 9 or 16. Saturation [¹²⁵I]RTI-55 binding experiments were conducted in the absence or presence of varying concentrations of compound 9 (A) or compound 16 (B), as described in Materials and Methods. Data shown are from a representative experiment conducted in triplicate, which was replicated at least two times with similar results. In the Scatchard analysis, the (−) reciprocal of the slope of the line is an estimate of the Kd value for the radioligand. The steeper slope in the presence of higher concentrations of drugs indicates a lower Kd value and increased affinity.

Fig. 6.

Effect of compounds on HEK-hDAT functional assays: [³H]DA uptake and [³H]DA release. (A) Effect of compounds 6, 9, 11, and 16 on [³H]DA uptake into HEK-hDAT cells (n = 3–5). (B) Effect of compound 9 (1 nM to 1 µM) on cocaine-induced inhibition of [³H]DA uptake into HEK-hDAT cells (n = 2–5 for each concentration of 9). (C) Lack of effect of compound 2 (0.3 and 10 µM) and compound 8 (0.3 and 10 µM), compared with methamphetamine (METH) (1 and 10 µM), on preloaded [³H]DA release from HEK-hDAT cells. Data are from a representative experiment that was repeated with similar results.

Fig. 7.

Summary of the SAR of the nucleoside derivatives at DAT and other SLC transporters. The structure of the 3,4-difluorophenyl derivative (compound 6) is shown with substitutions leading to thienyl derivatives (compounds 9 and 16), and when truncated to the 2-chlorophenyl derivative (compound 23). Colored regions correspond to structural features on the most potent modulators, which both enhance the binding of tropane radioligands and inhibit DA uptake. The colors correspond to 5′ (blue), N⁶ (green), and C2-terminal thienyl (yellow) substituents.