Development of fluorescence imaging probes for nicotinic acetylcholine α4β2∗ receptors

Abstract

Nicotinic acetylcholine α4β2^∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2^∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [¹⁸F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2^∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4β2^∗ nAChRs in [³H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4β2^∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4β2^∗ nAChRs, labeled with antibodies specific for a β2 subunit extracellular epitope indicating that nifrofam labels α4β2^∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4β2^∗ nAChRs in brain regions.

Keywords: Fluorescence imaging; Nicotine; Nifene; Nifrodansyl; Nifrofam; Nifrolidine.

Figure 1. Lead PET α4β2 nAChRs probes for design of fluorescent probes

1. Chemical structure of [¹⁸F]nifene; 2. Chemical structure of [¹⁸F]nifrolidine showing (blue arrow) the possibility of replacing fluorine-18 (in red circle) with a fluorophore; A. Rat brain slice showing different brain regions (FC: frontal cortex; ST: striata; TH: thalamus; HP: hippocampus; SB: subiculum; CB: cerebellum); B. [¹⁸F]Nifene binding to the brain regions shown in brain slice A; C. [¹⁸F]Nifrolidine binding to the brain regions shown in brain slice A.

Figure 2. Potential Fluorescence Imaging Agents for α4β2 nAChRs

A. Minimized energy structure of nifrolidine, 3 with reported high affinity for α4β2* receptors; B. Energy minimized structure of nifrodansyl, 4, in which the fluorine atom in 3 is replaced with the fluorescent moiety, dansylamide (marked in green bracket in 4). C. Energy minimized structure of nifrofam, 5, in which the fluorine atom in 3 is replaced with the fluorescent moiety, carboxyfluorescein (FAM, marked in green bracket in 5).

Figure 3. Synthesis of Nifrodansyl

A. Sodium azide, DMF, trace water, reflux 24hrs. B. LiBH₄, THF, rt. 24 hrs. C. Dansyl chloride, CH₂Cl₂, Pyridine, rt, 24 hrs. D. Trifluoroacetic acid (TFA), CH₂Cl₂, rt, 24 hrs. Thin layer chromatography (9:1 CH₂Cl₂: CH₃OH) showing dansylamide (Rf= 0.8) on the left lane and the more polar nifrodansyl (Rf=0.2) on the right lane.

Figure 4. Synthesis of Nifrofam

A. FAM 5,6-NHS ester 10, DMSO, rt, 24 hrs. D. Trifluoroacetic acid (TFA), CH₂Cl₂, rt, 24 hrs. Thin layer chromatography (1:1 CH₂Cl₂: CH₃OH) showing carboxyfluorescien NHS ester 10 on the left lane (Rf= 0.95) and the product mixture containing nifrofam 5 in the right lane (Rf=0.2).

Figure 5. Binding affinity curves for Nifrodansyl

(A) Scan of rat brain slice; (B) total binding of [³H]cytisine in different brain regions (AC, anterior cingulate; TH, thalamus; SB, subiculum; FC, frontal cortex; ST, striatum; CB, cerebellum); (C) binding of [³H]cytisine in the presence of 10 nM nifrodansyl; (D) Binding of [³H]cytisine in the presence of different concentrations of nifrodansyl; (E) Competition binding curves of nifrodansyl with [³H]cytisine binding in rat brain regions shown in (B).

Figure 6. Binding affinity curves for Nifrofam

(A). Binding of [³H]cytisine in the presence of nifrofam (AC, Anterior cingulate; TH, thalamus; SB, subiculum; FC, frontal cortex; ST, striatum; CB, cerebellum); (B). Competition binding curves of nifrofam with [³H]cytisine binding in rat brain regions.

Figure 7. α4β2_HA stable cells - live labeling and live cell imaging

HEK cells stably expressing α4β2_HA nicotinic receptor subtypes were untreated (top panel, A–C) or treated with nicotine (10 μM for 17 hr; bottom panel, D–F). Nicotine was washed off prior to labeling the live cells with antibody or ligand. Cell surface receptors were labeled with anti-HA (β2 subunit has an HA tag at the extracellular C-terminus tail) followed by Nifrofam (100 nM) and Alexa Fluor 568 anti-rabbit secondary antibody. Images were taken on Marianas Yokogawa type spinning disk confocal microscope using 100X objective. Images are z-projects of 25 slices taken with 0.2 μm step size. Nifrofam (B, E) labeling showed high degree of co-localization with surface expressed β2 subunits (A, D). Labeling with anti-HA antibodies and nifrofam was performed for 30 minutes at 37°C.

Figure 8. Mouse brain slice fluorescence images of Nifrofam

(A). Binding of nifrofam (0.5 μM) to distinct cell bodies (shown by arrows; 20x) in mouse brain slices auditory cortex region. (B). Adjacent slices treated with ACSF alone (in the absence of nifrofam) did not reveal any distinct fluorescent cell bodies.