Studies of the biogenic amine transporters 15. Identification of novel allosteric dopamine transporter ligands with nanomolar potency

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 [ 125 I]RTI-55 ([ 125 I]3β-(4′-iodophenyl)tropan-2β-carboxylic acid methyl ester) binding and [ 3 H]dopamine ([ 3 H]DA) uptake, slow the dissociation rate of [ 125 I]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 [ 3 H]DA uptake inhibition assays, DAT binding assays with [ 3 H]WIN35428 ([ 3 H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane), and DATmediated release assays with either [ 3 H]MPP 1 ([ 3 H]1-methyl-4-phenylpyridinium) or [ 3 H]DA. We observed three groups of [ 3 H]DA uptake inhibitors: 1) full-efficacy agents with a one-site fit, 2) fullefficacy 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 [ 3 H]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 IC 50 5 2.3 ± 0.4 nM, without affecting binding to the DAT. These agents did not alter DAT-mediated release of [ 3 H]MPP 1 in the absence or presence of 100 nM d-amphetamine. SRI-29574 had no significant effect on the d-amphetamine EC 50 or E max value for DATmediated release of [ 3 H]MPP +. These studies demonstrate the existence of potent DAT ligands that partially block [ 3 H]DA uptake, without affecting DAT binding or d-amphetamine-induced [ 3 H]MPP 1 release. These compounds may prove to be useful probes of biogenic amine transporter function as well as novel therapeutics.