Identification and characterization of a novel allosteric modulator (SoRI‐6238) of the serotonin transporter

Abstract

In the present study we describe a novel agent, SoRI-6238 (ethyl 5-amino-3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-ylcarbamate) that partially inhibits 5-HT transporter (SERT) binding and allosterically modulates SERT function. Membranes were prepared from rat brain. SoRI-6238 partially inhibited SERT binding to brain membranes with a plateau at about 40% of control. SoRI-6238 fully inhibited norepinephrine transporter (NET) and dopamine transporter (DAT) binding with IC(50) values of 12.1 microM and 5.8 microM, respectively. The apparent K(d) of [(125)I]RTI-55 binding to SERT increased, then reached a plateau with increasing concentrations of SoRI-6238. SoRI-6238 fully inhibited [(3)H]5-HT uptake, acting to decrease the V(max) (noncompetitive inhibition). In kinetic experiments, SoRI-6238 slowed the dissociation of [(125)I]RTI-55 from SERT and slowed the initial association rate. We conclude that SoRI-6238 partially inhibits SERT binding and function, most likely via an allosteric mechanism.

Studies of the Biogenic Amine Transporters. XI. Identification of a 1-[2-[Bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) Analog That Allosterically Modulates the Serotonin Transporter

“…The transporter binding assays used membranes prepared from frozen rat brains (Nandi et al, 2004). Briefly, frozen rat brains (Pel Freeze, Rogers, AZ) were each thawed in 20 ml of ice-cold binding buffer (BB; 55.2 mM sodium phosphate buffer, pH 7.4) for 15 min and then homogenized with a Brinkmann Polytron (Brinkmann Instruments, Westbury, NY; setting 6 for 20 s).…”

“…Kinetic experiments were conducted with minor modifications of published methods (Nandi et al, 2004). For SERT dissociation experiments, […”

“…Binding surfaces were fit to one-and two-site binding models using MLAB-PC as described elsewhere (Rothman et al, 1991). Dissociation experiments were fit to one-or two-component dissociation models (Nandi et al, 2004 (Boston, MA). Thomas Prizansano kindly provided the highly selective DA uptake inhibitor 4-(2-benzhydryloxyethyl)-1-(4-nitro-benzyl)piperidine oxalate, synthesized as described (Greiner et al, 2003).…”

Therapeutic Potential of Monoamine Transporter Substrates

“…Because substrate-type releasing agents must be transported into nerve terminals to promote transmitter release, reuptake inhibitors can block the effects of releasers. In some cases, drugs can act as allosteric modulators of transporter function [16][17][18][19][20].…”

The promiscuity of the dopamine transporter: Implications for the kinetic analysis of [3H]serotonin uptake in rat hippocampal and striatal synaptosomes

“…Although there is dense serotonergic innervation to rat hippocampus originating in the raphe nuclei, this region is essentially devoid of dopaminergic innervation (Fuxe et al, 1985, Donnan et al, 1989, Mennicken et al, 1992. Although numerous studies have investigated the kinetics of 5-HT re-uptake under varying environmental and pharmacological conditions (O'Reilly and Reith, 1988, Asano et al, 1997, Kokoshka et al, 1998, Wells et al, 1999, Martin et al, 2000, Pollier et al, 2000, Nandi et al, 2004, Nightingale et al, 2005 and in varying rat strains (Martin et al, 2000, Fernandez et al, 2001, Fernandez et al, 2003, few studies have considered the impact of the expression of both DAT and SERT in brain regions such as striatum and the potential contribution of DAT to the analysis of 5-HT uptake kinetics. The heterogeneous expression of DAT and SERT in specific brain regions is also an important factor with regard to selecting specific transporter inhibitors and concentrations to define nonspecific uptake.…”