Fluorescence imaging probes for nicotinic acetylcholine α4β2[asterisk]receptors

1175 Objectives: Nicotinic acetylcholine α4β2[asterisk] receptors (nAChR) have been associated with tobacco dependence and smoking cessation. PET studies have been directed towards understanding the role of this receptor subtype in smokers. In order to further support PET findings using fluorescence imaging at the cellular and subcellular level, we have developed several new fluorescent probes by modifying our PET probe [18F]Nifrolidine. We report here synthesis, binding affinity, cellular and subcellular localization of fluorescent probes suitable for imaging at different wavelengths of the visible spectrum. Methods: The synthesis of rhodamine and biotin analogs of nifrofam, containing fluorescein and nifrodansyl, containing dansyl (Samra et al., BMCL, 2018) was carried out by reaction of 5-(3’-aminopropyl)-3-(N-BOC-2-(S)-pyrrolidinylmethoxy)pyridine with carboxytetramethylrhodamine NHS ester and biotin NHS ester, followed by deprotection of the N-BOC using trifluoroacetic acid. Figure 1 shows the similar chemical structures of nifrorhodamine, nifrobiotin, nifrofam, and nifrodansyl with an R group attached. Binding affinity studies of the compounds were carried out in rat brain slices as well as rat brain homogenates using [3H]cytisine. HEK cells stably expressing α4β2 nAChR were used to test the fluorescent probes. Live cells were also pretreated with nicotine (10 μM), which was washed off prior to labeling, to evaluate nicotine-induced upregulation. Cell surface receptors were labeled with antibody recognizing the β2 subunit tagged with hemagglutinin to confirm co-localization of cell-surface receptors (for nifrofam studies) and Triton-X100 was used for cell permeabilzation and with Hoechst stain to confirm subcellular receptors (for nifrorhodamine studies). Results: The large fluorescent substituent groups were not found to be detrimental to the binding affinity of nifrolidine. Biotin substituent had the weakest affinity which may be due to the additional 4-carbon linker on biotin, making the carbon chain on nifrobiotin too long and thus lowering the affinity. Nifrodansyl, nifrofam and nifrorhodhamine exhibited nanomolar affinities (1 to 17 nM) for the α4β2[asterisk] receptor subtypes measured using [3H]cytisine in different brain regions. Nifrofam and nifrorhodhamine studies with HEK cells expressing α4β2 nAChR confirmed specific binding to the receptors located both on the cell surface as well as subcellular organelles. The upregulation of the receptors post-nicotine treatment was visualized by both nifrofam and nifrorhodhamine. Conclusion: We have successfully developed fluorescent probes for the α4β2[asterisk] nAChRs by modifying our reported PET probes. Our findings suggest that the replacing the fluorine atom with larger fluorophores moieties is tolerable at the receptor binding site with minimal loss in affinity. Our preliminary findings show nifrofam (green fluorescent) and nifrorhodamine (red fluorescent) bind to the α4β2[asterisk] nAChR expressed on HEK cells and in subcellular organelles. Further studies are underway to establish selectivity of binding to nAChR subtypes. These fluorescent probes will be useful in evaluating cellular and subcellular distribution of PET probes such as [18F]Nifene and others. Research Support: NIH R01 DA044760