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

Previous studies identified partial inhibitors of serotonin (5-HT) transporter and dopamine transporter binding. We report here on a partial inhibitor of 5-HT transporter (SERT) binding identified among a group of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine analogs (4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-1-(2-trifluoromethyl-benzyl) -piperidine; TB-1-099). Membranes were prepared from rat brains or human embryonic kidney cells expressing the cloned human dopamine (hDAT), serotonin (hSERT), and norepinephrine (hNET) transporters. β-(4′- ¹²⁵Iodophenyl)tropan-2β-carboxylic acid methyl ester ([ ¹²⁵I]RTI-55) binding and other assays followed published procedures. Using rat brain membranes, TB-1-099 weakly inhibited DAT binding (K_i = 439 nM), was inactive at NET binding ([³H]nisoxetine), and partially inhibited SERT binding with an extrapolated plateau ("A" value) of 20%. Similarly, TB-1-099 partially inhibited [¹²⁵I]RTI-55 binding to hSERT with an extrapolated plateau (A value) of 14%. Upon examining the effect of increasing concentrations of TB-1-099 on the apparent K _d and B_max of [¹²⁵I]RTI-55 binding to hSERT, we found that TB-1-099 decreased the B_max in a dose-dependent manner and affected the apparent K_d in a manner well described by a sigmoid dose-response curve. TB-1-099 increased the K_d but not to the magnitude expected for a competitive inhibitor. In rat brain synaptosomes, TB-1-099 noncompetitively inhibited [³H]5-HT, but not [ ³H]dopamine, uptake. Dissociation experiments indicated that TB-1-099 promoted the rapid dissociation of a small component of [¹²⁵I]RTI-55 binding to hSERT. Association experiments demonstrated that TB-1-099 slowed [¹²⁵I]RTI-55 binding to hSERT in a manner unlike that of the competitive inhibitor indatraline. Viewed collectively, these results support the hypothesis that TB-1-099 allosterically modulates hSERT binding and function.