Abstract

Novel allosteric modulators of the dopamine transporter (DAT) have been identified. We have shown previously that SRI-9804 [N-(diphenylmethyl)-2-phenyl-4-quinazolinamine], SRI-20040 [N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine], and SRI-20041 [N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine] partially inhibit [¹²⁵I]RTI-55 ([¹²⁵I]3β-(4′-iodophenyl)tropan-2β-carboxylic acid methyl ester) binding and [³H]dopamine ([³H]DA) uptake, slow the dissociation rate of [¹²⁵I]RTI-55 from the DAT, and allosterically modulate d-amphetamine–induced, DAT-mediated DA release. We synthesized and evaluated the activity of >500 analogs of these ligands and report here on 36 selected compounds. Using synaptosomes prepared from rat caudate, we conducted [³H]DA uptake inhibition assays, DAT binding assays with [³H]WIN35428 ([³H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane), and DAT-mediated release assays with either [³H]MPP⁺ ([³H]1-methyl-4-phenylpyridinium) or [³H]DA. We observed three groups of [³H]DA uptake inhibitors: 1) full-efficacy agents with a one-site fit, 2) full-efficacy agents with a two-site fit, and 3) partial-efficacy agents with a one-site fit—the focus of further studies. These agents partially inhibited DA, serotonin, and norepinephrine uptake, yet were much less potent at inhibiting [³H]WIN35428 binding to the DAT. For example, SRI-29574 [N-(2,2-diphenylethyl)-2-(imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine] partially inhibited DAT uptake, with an IC₅₀ = 2.3 ± 0.4 nM, without affecting binding to the DAT. These agents did not alter DAT-mediated release of [³H]MPP⁺ in the absence or presence of 100 nM d-amphetamine. SRI-29574 had no significant effect on the d-amphetamine EC₅₀ or E_max value for DAT-mediated release of [³H]MPP⁺. These studies demonstrate the existence of potent DAT ligands that partially block [³H]DA uptake, without affecting DAT binding or d-amphetamine–induced [³H]MPP⁺ release. These compounds may prove to be useful probes of biogenic amine transporter function as well as novel therapeutics.