A Pyridazine-Based Fluorescent Probe Targeting Aβ Plaques in Alzheimer's Disease

Accumulation of β-amyloid (Aβ) plaques comprising Aβ40 and Aβ42 in the brain is the most significant factor in the pathogenesis of Alzheimer's disease (AD). Thus, the detection of Aβ plaques has increasingly attracted interest in the context of AD diagnosis. In the present study, a fluorescent pyridazine-based dye that can detect and image Aβ plaques was designed and synthesized, and its optical properties in the presence of Aβ aggregates were evaluated. An approximately 34-fold increase in emission intensity was exhibited by the fluorescent probe after binding with Aβ aggregates, for which it showed high affinity (KD = 0.35 µM). Moreover, the reasonable hydrophobic properties of the probe (log P = 2.94) allow it to penetrate the blood brain barrier (BBB). In addition, the pyridazine-based probe was used in the histological costaining of transgenic mouse (APP/PS1) brain sections to validate the selective binding of the probe to Aβ plaques. The results suggest that the pyridazine-based compound has the potential to serve as a fluorescent probe for the diagnosis of AD.


Introduction
e misfolding and aggregation of proteins cause numerous neurodegenerative diseases, such as Alzheimer's disease (AD), prion disease (PrD), and Parkinson's disease (PD) [1]. AD, one of the most common protein misfolding diseases (PMDs), is characterized by the accumulation of misfolded β-amyloid (Aβ) peptides and neuro brillary tangles (NFTs) containing tau protein in the brain. A recent report revealed that the buildup of Aβ plaques in the brain plays a signi cant role in the pathogenesis of AD [2,3]. erefore, approaches to visualize Aβ deposition might prove useful for diagnosing AD and evaluating the e cacy of AD therapeutics [4][5][6].
Based on these requirements, we developed and reported uorescent pyridazine probes targeting Aβ plaques [24]. ese pyridazine probes can be used for imaging through selective binding but lack the required binding a nity for Aβ plaques. Here, we describe the optimization of pyridazine derivatives based on the conjugation of an electron acceptor with an electron donor.
To optimize these uorescent probes, the electron-donating p-dimethylamino group and electron-accepting cyano group were introduced to construct a compound with a donorπ-acceptor structure ( Figure 1). In this paper, we describe the synthesis and optical and biological properties of a cyanobased probe based on pyridazine. e ex vivo staining of Aβ plaques in APP/PS1 mice brain sections by this uorescent probe is also presented.

General Experimental
Methods. 1 H NMR spectra were recorded in CDCl 3 unless otherwise noted (values in ppm) using TMS as the standard with a JNM-ECA 500 spectrometer. Low resolution mass spectra were recorded using a Varian MAT 212 mass spectrometer. IR spectra (KBr) were measured with a Bruker-Vector 22 instrument (Bruker, Bremen). Flash column chromatography was performed using silica gel (70-230 mesh). All reagent-grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), and synthetic Aβ 42 peptide was purchased from rPeptide (Bogart, GA, USA). (2). A mixture of 1 (300 mg, 0.97 mmol), 3,4-dihydroxybenzaldehyde (147 mg, 1.06 mmol), and K 2 CO 3 (293 mg, 2.12 mmol) was dissolved in DMF (20 ml) and re uxed for 24 h. After evaporating the solvent under reduced pressure, H 2 O (100 ml) and methylene chloride (50 ml) were added. e organic layer was separated and dried over MgSO 4 . e pure product (2) was obtained by column chromatography on silica gel using CH 2

UV/VIS and Fluorescence
Analysis. UV/VIS and uorescence spectra were recorded and analyzed. For the UV/VIS spectra, an In nite M200 Pro Microplate reader (Tecan, Switzerland) equipped with cells with a 1.0 cm path length was used. e scan rate was 120 nm/min. e excitation and emission λ max values of probe 3 (10 μM) were recorded with a detector (slit of 1 mm) and a data interval of 5 nm in DMF.

Preparation of Aβ42
Aggregates and Fluorescence Spectrum Measurement. Aggregated Aβ peptide was prepared by diluting Aβ42 to a nal concentration of 100 μM in PBS (pH 7.4). is solution was incubated at 200 rpm and 37°C for 3 days. e formation of Aβ brils was con rmed by T assay. e excitation and emission λ max values of probe 3 were measured using an In nite M200 Pro Microplate reader (Tecan, Switzerland) equipped with a detector (slit 1 mm) with a data interval of 5 nm. e scan rate was 120 nm/min. Probe 3 (10 μM) was reacted with and without 20 μM Aβ aggregates for 20 min in PBS at 37°C. e emission spectra and uorescence intensity of the samples were measured. e fold increase was calculated by comparing the uorescence intensity with and without 20 μM Aβ aggregates.

Lipophilicity (log P)
. Probe 3 was added to a premixed suspension containing 500 μL of octanol and 500 μL of PBS solution, and the resulting suspension was vortexed vigorously for 10 min and centrifuged at 3000 rpm for 5 min. Two layers separated out, and 100 μL aliquots from octanol and the PBS solution layers were removed and analyzed for their uorescence intensity. e log P value was calculated as the logarithm of the ratio of the uorescence intensity in octanol versus that in PBS solution.

Maestro Images
Analysis. An optical data study was performed using a Maestro 2.0 in vivo imaging system. e images were acquired as described previously [25].

3.
e brain from 12-month-old transgenic APP/PS1 mice was removed and cut into 5 μm sections. e mouse brain sections were stained with probe 3 and anti-Aβ using the following method: rst, the brain sections were equilibrated in PBS solution for 10 min, washed with PBS containing 0.1% Tween 20 (PBS-T) and 5% BSA for 30 min, and washed again with PBS-T supplemented with 1% BSA for 5 min 3 times. Second, the washed sections were incubated with primary antibody (rabbit anti-Aβ, 1 : 100 dilution in PBS-T supplemented with 1% BSA) overnight at 4°C, washed with PBS-T supplemented with 1% BSA 3 times, and stained with secondary antibody (Alexa 555 goat antirabbit IgG, 1 : 100 dilution in PBS-T supplemented with 1% BSA). After washing with PBS, the prestained sections were stained with 10 µM probe 3 for 30 min. e stained section was washed with PBS and analyzed under an FV1000D (Olympus, Tokyo, Japan) confocal laser scanning microscope.

Results and Discussion
e synthesis of probe 3 is outlined in Scheme 1. First, commercially available 3,4-dihydroxybenzaldehyde was converted to the corresponding catechol aldehyde (2) by reacting it with compound 1. The Knövenagel condensation of compound 2 with cyanoacetic acid a orded the nal uorescent probe (3). e optical properties of the synthesized uorescent probe (3) with aggregated Aβ42 peptides in PBS (pH 7.4) were analyzed, and the results are shown at Table 1. Probe 3 exhibited an excitation maximum at 408 nm and an emission maximum at 670 nm (Table 1 and Figure 2).
To operate as a uorescent probe targeting Aβ plaques, a compound must show a signi cant rise in uorescence intensity upon binding with Aβ aggregates compared to the uorescence intensity of free Aβ aggregates in solution [15]. erefore, we compared the uorescence intensity of probe 3 to the uorescence intensity of the probe in the presence of Aβ aggregates (Figure 3(a)). As shown in Table 1, we observed a remarkable increase (35-fold) in the uorescence intensity of probe 3 in the presence of Aβ aggregates. Additionally, the gain in uorescence intensity was visually con rmed using a Maestro uorescence imaging system (Figure 3(c)). is e ect is due to conformational changes: When the probe in solution with Aβ aggregates is in the unbound state, free rotation through a single bond is permitted, whereas upon binding to Aβ aggregates, the probe exhibits a signi cant increase in uorescence intensity due to restricted movement [26]. e binding of probe 3 to Aβ aggregates was also accompanied by a blueshift in the emission spectrum [15]. e emission wavelength of probe 3 exhibited signi cant blueshifts (66 nm, Table 1), indicating that probe 3 likely intercalated into the hydrophobic pocket of the Aβ aggregates. is result suggested that probe 3 could be "turned on" via an increase in uorescence intensity and a blueshift in its emission wavelength upon interacting with Aβ aggregates.
Next, we measured the apparent binding constant (K D ) of uorescent probe 3 to Aβ aggregates. e uorescence intensity of solutions of probe 3 at various concentrations in the presence of Aβ aggregates was measured, revealing that the K D value of probe 3 was 0.35 ± 0.03 μM (Table 1 and Figure 3(b)). is binding constant was signi cantly higher than that of our previously reported uorescence probe, probe 1 (1.83 ± 0.31 μM) [24]. e lipophilicity (log P) of probe 3 was also evaluated to determine whether it could permeate through the blood brain barrier (BBB). The log P value of probe 3 was found to be 2.94 ( Table 1), suggesting that probe 3 has desirable properties regarding BBB permeability [21].  e probe developed in this paper, probe 3, meets the requirements for a uorescence imaging probe for AD: high uorescence receptivity, strong binding a nity, and hydrophobicity. To assess whether uorescent probe 3 could stain Aβ plaques in mouse brain tissue, we further evaluated the histological costaining of Aβ plaques in APP/PS1 mouse brain sections with probe 3 and anti-Aβ. Aβ plaques in the mouse brain section were identi ed by staining with anti-Aβ as a control. As shown in Figure 4, the brain section exposed to probe 3 exhibited signi cant uorescence. Notably, the merged images showed colocalization of the areas stained with probe 3 and anti-Aβ, which demonstrates the selective targeting of Aβ plaques by probe 3.

Conclusions
In summary, we successfully synthesized probe 3 as a novel Aβ plaque-targeting uorescent probe by applying the concept of a donor-π-acceptor structure to the sca old of a previously reported pyridazine dye, probe 1. Probe 3 exhibited a strong uorescence response (F Aβ /F 0 > 34-fold), high a nity for Aβ42 aggregates (K D � 0.35 ± 0.03 µM), and   su cient hydrophobicity to penetrate the BBB (log P � 2.94). Furthermore, probe 3 speci cally stained the Aβ plaques in APP/PS1 mouse brain sections. ese results indicate probe 3 as a novel uorescence imaging agent for the study of AD.

Conflicts of Interest
e authors declare that they have no con icts of interest.