Abstract
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 (Ki = 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 Kd and Bmax of [125I]RTI-55 binding to hSERT, we found that TB-1-099 decreased the Bmax in a dose-dependent manner and affected the apparent Kd in a manner well described by a sigmoid dose-response curve. TB-1-099 increased the Kd 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.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.084376.
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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; SERT, 5-HT transporter; RTI-55, 3β-(4′-iodophenyl)tropan-2β-carboxylic acid methyl ester; hNET, cloned human norepinephrine transporter; hDAT, cloned human dopamine transporter; NE, norepinephrine; DA, dopamine; SoRI-6238, ethyl 5-amino-3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-ylcarbamate; SoRI-9804, 4-[(diphenylmethyl)-amino]-2-phenylquinazoline; GBR12909, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine; TB-1-099, 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1-(2-trifluoromethyl-benzyl)-piperidine; hSERT, cloned human 5-hydroxytryptamine transporter; HEK, human embryonic kidney; BB, binding buffer; SA, specific activity; TB-1-101, 4-{2-[bis-(4-fluoro-phenyl)-methoxy]-ethyl}-1-(2-methyl-benzyl)-piperidine oxalate.
- Received February 1, 2005.
- Accepted April 20, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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