To enable visualization of the dopamine transporter (DAT) through fluorescence technologies we have synthesized a novel series of fluorescently tagged analogs of cocaine. Previous structure-activity relationship (SAR) studies have demonstrated that the dopamine transporter (DAT) can tolerate sterically bulky N- or 2-position substituents on the 2-beta-carbomethoxy-3-beta-(3 ,4 -dichlorophenyl)tropane (MFZ 2-12) molecule. Hence, we designed and synthesized compounds in which rhodamine red was extended from either the N- or 2-position of MFZ 2-12, using an ethylamino-linker and amine-reactive rhodamine red derivatives. The resulting N-substituted (JHC 1-64) and 2-substituted (JHC 1-53) ligands showed high affinity binding to DAT expressed in HEK 293 cells (Ki= 6.4 and 29 nM, respectively). Their ability to selectively label the DAT was demonstrated by confocal laser scanning microscopy of HEK 293 cells stably expressing DAT. Incubation with a 10 or 25 nM concentration of either JHC 1-64 or JHC 1-53, respectively, revealed a distinct labeling of the plasma membrane within minutes that was blocked by addition of excess (10 microM) cocaine. Moreover, this labeling was not observed in untransfected control cells. The possibility of using these ligands for direct labeling of the DAT in living cells represents a new and important approach for understanding cellular targeting and trafficking of the DAT. Moreover, these fluorescent ligands might also provide the molecular tools for the development of non-radioactive binding assays for the DAT.
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Society for Neuroscience 34th annual meeting, 2004