A novel fluorescent protein chromophore analogue to simultaneously probe lysosome viscosity and β-amyloid fibrils

Journal Pre-proof A novel Fluorescent Protein Chromophore Analogue to simultaneously probe lysosome viscosity and ␤-Amyloid fibrils Han Sun, Hua-xiang Leng, Jin-sheng Liu, Gaurab Roy, Jin-wu Yan, Lei Zhang

PII:

S0925-4005(19)31708-3

DOI:

https://doi.org/10.1016/j.snb.2019.127509

Reference:

SNB 127509

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

24 August 2019

Revised Date:

22 October 2019

Accepted Date:

29 November 2019

Please cite this article as: Sun H, Leng H-xiang, Liu J-sheng, Roy G, Yan J-wu, Zhang L, A novel Fluorescent Protein Chromophore Analogue to simultaneously probe lysosome viscosity and ␤-Amyloid fibrils, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127509

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A novel Fluorescent Protein Chromophore Analogue to simultaneously probe lysosome viscosity and β‑ Amyloid fibrils

Han Sun,a Hua-xiang Leng,a Jin-sheng Liu,a Gaurab Roy,a Jin-wu Yanabc,* and Lei Zhangabc,*

School of Biology and Biological Engineering, South China University of

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a

Technology, Guangzhou 510006, P. R. China; b

Guangdong Provincial Engineering and Technology Research Center of

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Biopharmaceuticals, South China University of Technology, Guangzhou 510006, P. R. China. c

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Joint International Research Laboratory of Synthetic Biology and Medicine,

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South China University of Technology, Guangzhou 510006, P. R. China.

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Corresponding author at: School of Biology and Biological Engineering, South

China University of Technology, Guangzhou 510006, P.R. China; Guangdong

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Provincial Engineering and Technology Research Center of Biopharmaceuticals, South China University of Technology, Guangzhou 510006, P.R. China; Joint International Research Laboratory of Synthetic Biology and Medicine, South China University of Technology, Guangzhou 510006, P. R. China.

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E-mail address: [email protected] (J. Yan); [email protected] (L. Zhang).

Graphical Abstract

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A GFP Chromophore Analogue C25 could simultaneously probe lysosome viscosity and β‑ Amyloid fibrils with distinguishable turn-on fluorescence signals.

Highlights:

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 A turn-on fluorescent probe for selective and simultaneous detection of Aβ fibrils and lysosomal viscosity was synthesized.

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 The FP chromophore was developed for detection of Aβ fibrils for the first time.

 The probe can detect Aβ fibrils and viscosity with one-excitation and

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dual-emission.

 The fluorescent imaging demonstrated its value of practical application.

Abstract Alzheimer's disease (AD) is an incurable neurodegenerative disease. Better diagnosis of AD is extremely important for therapeutic interventions. The amyloid cascade hypothesis and its revised version identify insoluble β-amyloid deposition as a good diagnostic biomarker for AD. We hypothesize that lysosomal viscosity may also act as an auxiliary biomarker for AD based on literature research. Herein, we have successfully developed a FP chromophore analogue C25 as a dual-functional fluorescent probe which can respond effectively to the Aβ fibrils and enhanced

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lysosomal viscosity with distinguishable turn-on fluorescence signals around 570 nm

and 600 nm, respectively. The probe C25 can also monitor the Aβ degradation in the lysosomes. The probe C25 can serve as a promising tool for developing early

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diagnosis and innovative therapeutics of AD.

Keywords: Alzheimer’s Disease; Aβ fibrils; lysosomal viscosity; dual-functional;

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fluorescent probe; FP chromophore

1. Introduction Alzheimer disease (AD) is a progressive, incurable neurodegenerative disorder that is clinically characterized by the accumulation of β-amyloid (Aβ) in extracellular plaques and tau protein in intracellular neurofibrillary tangles (NFTs).[1-3] While much effort has been devoted to deciphering AD pathogenesis and discovering novel drug treatments, the comorbid nature of this disease hinders the therapeutic effectiveness verification of these methods.[4] Due to the fact that AD patients often are not diagnosed until it is advanced, most therapeutic attempts are rendered

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ineffective. Thus, to improve the diagnostic level of AD in the early stage is essential to early treatment and effectively utilizes disease-modifying drugs. The amyloid

cascade hypothesis and its revised version identify insoluble β-amyloid deposition as

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a good diagnostic biomarker for AD[4-6].

Lysosomal storage diseases (LSDs) are a group of over 70 diseases, the hallmark

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of which is the aberrant and excessive storage of cellular material in lysosomes.[7, 8] Niemann–Pick disease type C (NPC), a well-studied LSD, leads to cholesterol and

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sphingolipid accumulation since deficiency of NPC1 and the cell biology of NPC could accelerate amyloid precursor protein processing[9, 10]. Meanwhile, dementia is a common symptom of NPC and many histopathological features are associated with

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late-onset AD[10]. We hypothesize that viscosity within the lysosome lumen will increase with the accumulation of cellular materials. If so, the lysosomal viscosity could act as a potential biomarker for NPC as well as an auxiliary biomarker for AD. Mounting evidence has also identified lysosomes to be a major degradative route for

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Aβ and a central hub of Aβ accumulation[11, 12]. Thus, providing a novel method to simultaneously detect lysosomal viscosity and β‑ Amyloid fibrils could be a fertile ground for the early diagnosis of AD thereby facilitating its therapeutic remission. Fluorescence imaging probes, with high sensitivity and low cost are commonly

used in clinical investigations and diagnosis of AD. Fluorescent proteins (FPs) which maintain the chromophore within a restrictive β-barrel have been widely used as genetic tags in molecular and cell biology.[13-16] These chromophores,

4-hydroxybenzylidene-imidazolinone (HBI) derivatives, are non-fluorescent owing to ultrafast intramolecular vibration.[17] Based on the bioluminescence principle of GFP, β‑ Amyloid could be utilized as the protein host to mimic the effects of the β-barrel. In 2018, Zhang et al. reported that the fluorescent protein chromophore modifications could detect α-synuclein aggregation with turn-on fluorescence.[18] Our previous probe Lyso-MC and MC-N-2 emitted at 610 nm and 620 nm in the presence of Aβ aggregates and viscous environment, respectively.[19, 20] The short discrepancy of emission wavelength would be hard to distinguish the viscosity and amyloid beta

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when simultaneously imaging. In this work, we further designed and synthesized the novel FP chromophore analogue C25 to magnify the discrepancy of wavelength when interacting with lysosome viscosity and amyloid beta.

In order to improve the affinity of the probe with β‑ Amyloid fibrils, an electron

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donating dimethylamino group was selected to substitute the phenol group in HBI,

owning to the difficulty to deprotonate phenol inside protein aggregates with low

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PH.[18] Then, a quinolone moiety was introduced, which was reported to bind to β‑ Amyloid species, to improve Aβ targeting effects and extend π conjugation to

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generate red-shift.[21-24] Furthermore, the 3-morpholinopropyl -amine moiety was introduced as a lysosome-targeting group.[25] The FP chromophore derivatives

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synthesized outside their protein cavity become mostly nonfluorescent, largely due to rapid nonradiative decay via twisted-intramolecular charge transfer (TICT). The lysosomal viscous environment and protein host can restrict TICT so that restore fluorescence of the probe C25 (Scheme 1). The emission characteristics of C25 proved sensitive to the polarity of the solvent medium (Fig. S2,16). In particular in

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hydrophobic environments, such as hydrophobic binding pocket of Aβ fibrils, C25 generates a blue shift. As a result, we postulated that C25 might have a role to play as a dual-functional probe capable of imaging Aβ fibrils and lysosomal viscosity simultaneously by monitoring the different emission colors.

2. Experimental

2.1. General information Reagents, solvents, apparatus, detailed experimental procedures can be found in the Supporting Information. 2.2. Chemistry synthesis The target probe C25 was prepared as shown in scheme 2 and was characterized by 1H NMR,

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C NMR, and HRMS spectra as indicated in the Supporting

Information.

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Synthesis of (Z)-4-(4-(dimethyl amino) benzylidene) -2-methyloxazol -5(4H)-one (2). To the acetic anhydride (15 mL) of 4-(dimethyl amino) benzaldehyde (2 g, 13.4mmol), N-acetyl glycine (1.57 g, 13.4 mmol) and sodium acetate (1g,

12mmol) was added. The solution was stirred at 100 °C until the starting material

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disappeared (monitored by TLC plate). After cooling to room temperature, the

solution was concentrated and treated with water and CH2Cl2. The organic phase was

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separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, to give the compound 2 (1.36 g, 44.2%). 1H NMR (400 MHz, CDCl3) δ 8.00

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(d, J = 8.5 Hz, 2H), 7.09 (s, 1H), 6.73 (d, J = 8.4 Hz, 2H), 3.08 (s, 6H), 2.37 (s, 3H). C NMR (101 MHz, CDCl3) δ 168.72, 163.02, 151.92, 134.42, 132.73, 127.82,

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121.67, 111.96, 40.22, 15.58. HRMS (ESI): calcd for (M+H)+ (C13H15N2O2+) 231.1134, found 231.11238. calcd for (M+Na)+ (C13H14N2NaO2+) 253.0953, found 253.09425.

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Synthesis

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(Z)-5-(4-(dimethyl

-(3-morpholinopropyl)

-3,5-dihydro

amino) -4H-

benzylidene)-2-methyl-3

imidazol-4-one

(3).

0.94mL

N-(3-Aminopropyl) morpholine and 10ml ethanol were added to the round bottom flask containing 1g compound 2 and 0.6g potassium carbonate. The solution was stirred at 80 °C overnight. After cooling to room temperature, the solution was concentrated and treated with water and CH2Cl2. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, to

give the compound 3(0.95g,61.7%).1H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.2 Hz, 2H), 7.06 (s, 1H), 6.70 (d, J = 8.3 Hz, 2H), 3.69 (m, 6H), 3.05 (s, 6H), 2.55 – 2.35 (m, 9H), 1.91 – 1.84 (m, 2H).13C NMR (101 MHz, CDCl3) δ 170.82, 158.87, 151.53, 134.59, 134.17, 128.83, 122.24, 111.76, 66.71, 55.60, 53.53, 40.06, 38.57, 25.69, 15.62. HRMS (ESI): calcd for (M+H)+ (C20H29N4O2+) 357.2291, found 357.22794.

Synthesis of 5-((Z)-4-(dimethyl amino) benzylidene) -3-(3-morpholinopropyl) -2-((E)-2-(quinolin-6-yl) vinyl) -3,5-dihydro-4H-imidazol-4-one (C25). To the

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ethanol solution (10 mL) of 3 (0.90 g, 2.5 mmol) and 6-quinolinecarboxaldehyde (0.39 g, 2.5 mmol) was added catalytic amount of piperidine. The solution was stirred at 80 °C till starting material disappeared (monitored by TLC plate). After cooling to

room temperature, the solution was concentrated and treated with water and CH2Cl2.

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The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column

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chromatography to afford C25 (0.25 g, 20.2%) as a dark-red solid. 1H NMR (400 MHz, CDCl3) δ 8.93 (d, J = 3.6 Hz, 1H), 8.26 – 8.12 (m, 5H), 8.01 (d, J = 8.1 Hz, 2H),

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7.46 (dd, J = 8.2, 4.2 Hz, 1H), 7.17 (s, 1H), 7.04 (d, J = 15.7 Hz, 1H), 6.76 (d, J = 8.8 Hz, 2H), 3.89 (t, J = 6.7 Hz, 2H), 3.71 – 3.67 (m, 4H), 3.10 (s, 6H), 2.42 (m, 6H),

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1.94 – 1.88 (m, 2H).13C NMR (101 MHz, CDCl3) δ 170.79, 156.06, 151.70, 151.09, 148.80, 138.09, 136.35, 135.46, 134.71, 133.85, 130.22, 129.47, 128.60,128.49, 127.13, 122.85, 121.96, 114.58, 111.88,

66.94, 55.55, 53.68, 40.09, 38.15,

26.18.HRMS (ESI): calcd for (M+H)+ (C30H34N5O2+) 496.2713, found 496.2711.

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3. Results and Discussion

3.1. Fluorescence Response to Solvent Viscosity With the probe in hand, we firstly evaluated its optical response to viscosity. The

fluorescence intensity of C25 was enhanced with an increase in the proportion of glycerol due to the immobilization of rotation and inhibition of TICT, and the fluorescence peak locates at around 600 nm (Fig.1A). A good linear relationship apparently existed between log I600 and log η in the range from 1.0 CP (100 % PBS)

to 945 CP (100 % Glycerol) (Fig.1A).As is shown in Fig.S4, the fluorescence intensity maxima of C25 gradually increased in glycerol with the gradual reduction of temperature whereas the fluorescence of C25 barely changed in PBS under similar conditions, indicating C25 could be instrumental to report viscosity. 3.2. Fluorescence Response to Solvent Aβ fibrils We studied another function of the probe in recognition of Aβ fibrils which had been characterized by ThT assay and TEM method (Fig. S5-6). The fluorescence

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intensity of C25 exhibited remarkable enhancement at 570nm (13-fold) and significantly increased with the increasing concentration of fibrils in a solution

containing Aβ1–42 fibrils. The fluorescence intensity presented a good linear relationship with Aβ fibrils (Fig.1B, table S1). It is interesting to note that the probe

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C25 mildly responded to Aβ oligomers as can be seen in Fig. S3. Moreover, to

quantitatively evaluate the binding affinity for Aβ, the apparent binding constant Kd

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was measured to be 189.2 nM, indicating that the affinity of C25 was good enough to image Aβ fibrils as it was comparable to that of ThT (Ki = 580 nM) and AOI-987

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(Kd=220 nM)[26]. 3.3. Selectivity and photostability

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To evaluate the selectivity of C25, we tested whether many biomolecules, metal ions (Fig. S3) and pH (Fig. S7) may potentially influence the response signals. As expected, we found that C25 displayed a high specificity toward viscosity and Aβ fibrils (Fig. S3,7). Furthermore, the probe C25 also exhibited good photo stability

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with exposure to different lights in long time (Fig.S8). In addition, the tested log P value was 2.40, suggesting probe C25 has potential ability to effectively traverse the Blood-Brain-Barrier (BBB) (table S1). It is also to be noted that the probe has a large emission shift of about 30 nm between the emission peak of the response of Aβ fibrils and that of viscosity, rendering it favorable for the simultaneous detection of two analytes in complicated biological systems (Fig. S3, table S1). To explain the mechanism of two different emission colors, the photophysical properties of C25 in

solvents with different polarities were investigated. As shown in Fig.S2 and Fig.S16, the fluorescence emission spectra exhibited apparent solvatochromism and the emission maximum of C25 was blue shifted with decreasing solvent polarity. 3.4. Cytotoxicity Tests The standard MTT assay with SH-SY5Y cells was conducted to evaluate biological toxicity of C25. The results indicated that over 90% of cells survived after 24h (32 µM C25 incubation), implying that C25 has no marked cellular cytotoxicity

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(Fig. S9). 3.5. Colocalization test of C25

To characterize the intracellular localization of C25, a colocalization experiment

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was firstly carried out using the commercially available dyes lyso-Tracker Green and

Mito-Tracker Green. Cell staining results confirmed that C25 could easily penetrate

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into the cytoplasm and get localized into the lysosomes. As displayed in Fig.2 A4, the intensity scatter plot of the lysosome-Tracker Green channel and the C25 channel are

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high correlated with a Pearson’s correlation factor of 0.92. On the other hand, the color of distributions of C25 and Mito-Tracker Green are almost variance with a Pearson’s correlation factor of 0.43(Fig.2 B4). The results confirmed that C25

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targeted lysosome as anticipated.

3.6. Cell imaging Studies of C25 as a lysosomal Viscosity Probe As reported, low temperatures and dexamethasone could act as potent stimuli for increasing the lysosomal viscosity. To identify the effectiveness of C25 in lysosomal

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viscosity imaging, confocal laser fluorescence images of SH-SY5Y cells stained with C25 were acquired after the treatment of 37℃ ， 4℃ ， DMSO(10 µL) and dexamethasone(5 µM) for 30min.As is shown in fig.2D, the fluorescence signals produced by the C25 probe in the SH-SY5Y cells pre-treated with 4℃ and dexamethasone exhibited obvious enhancement compared to that of 37℃.The fluorescence intensity of cells incubated with DMSO barely changed，which showed

that polarity fluctuation would not interfere with cell imaging of probe C25.Then, we performed spectra analysis of the red spot in lysosomal viscosity imaging experiment (Fig.2 C4). The maximum emission wavelength of the viscosity imaging experiment was the same as that found with binding assay in solution, which confirmed the probe responded to lysosomal viscosity with 570nm emission wavelength (Fig.2 E). Finally, we monitored the real-time changes of red fluorescence intensity using dexamethasone as stimulation regent. The fluorescence intensity showed obvious enhancement after the treatment of dexamethasone for 30 minutes (Fig.S10). All the

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above results highlight its great potential for mapping the lysosomal viscosity in live cells. 3.7. fluorescence staining of Aβ fibrils

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We further examined the feasibility of C25 to probe Aβ aggregates at the cellular level using Aβ overexpressing PC12 cells (PC12 -Aβ cells) and normal PC12 cells.

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The cells were co-stained with C25 and Alexa flour®405 and observed under a confocal fluorescence microscope. We observed that the normal PC12 cells produced

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a weak fluorescence when stimulated with C25 and the secondary antibody Alexa Fluor®405 (Fig.S13). In contrast, C25 could detect Aβ species in cytoplasm of the transfected PC12 cells, which was consistent with an immunofluorescence group

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incubated with the secondary antibody DylightTM594 (Fig.S11).The transfected PC12 cells were co-stained with C25 and Alexa Fluor®405 and the merged image indicated that the staining of C25 fits well with that of the immunofluorescence group with a Pearson’s correlation factor of 0.77 (Fig.3 A-C, Fig. S12). Furthermore, we

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investigated the practical application of C25 for labelling Aβ fibrils in Tg mouse brain tissue. When the brain slices of

the cerebrum and cortex regions of the Tg mouse

were co-stained with C25 and Aβ-fibril tracker thioflavin-S (ThS), the two channels could almost overlap (Fig.3 D-F,Fig.S15,Fig.S13) and C25 could stain the species outside the nucleus of Aβ that may be Aβ oligomer as is shown in Fig3 G-I. No fluorescent spots were observed in the brain slices of the cerebrum and cortex regions obtained from the wild-type mouse (Fig.S15), which also suggested that C25 could

specifically target Aβ species. These data indicated that the C25 reporter is capable of detecting Aβ fibrils at the cellular and tissue level.

3.8. Application of C25 to monitor the process of digesting Aβ in the lysosomes Lysosomes are a major degradative route for Aβ breakdown in APP-transgenic mouse models of AD，so can the probe C25 monitor the process of digesting Aβ in the lysosomes? We tested this process using SH-SY5Y cells and 5-FAM-labelled Aβ

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(Spectral properties of 5-FAM: Abs/Em,491/517 ±5 nm). After incubated with 5-FAM-labelled Aβ for different times and stained with C25, the SH-SY5Y cells were exposed to 488nm channel and confocal images were obtained. Green represents Aβ peptides and red represents lysosomes in Fig.4. As shown in Fig.4 A-C, Aβ peptides

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are combined in the cell membrane and two channels didn’t overlap with 1h FAM-Aβ treatment. After 16h incubation, there were several yellow spots, indicating that

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FAM-Aβ had been phagocytosed in lysosomes (Fig.4 D-F). 24 hours after treatment, more yellow spots were observed (Fig.4 G-I). We performed spectra analysis of the

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merged yellow spot, which confirmed the probe were bound to Aβ with 530 nm emission wavelength of 5-FAM and 570 nm emission wavelength of C25-Aβ complex

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(Fig.4 I). The results showed that C25 could be successfully applied to monitor the lysosomal degradation of Aβ peptides in AD model. 4. Conclusion

In summary, β‑Amyloid have been utilized as the protein host to mimic the

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effects of the β-barrel based on the bioluminescence principle of GFP in this work. we developed the FP chromophore analogue C25 ，which possesses an extended ring for significant red emission shift as well as strengthened interactions with Aβ fibrils. The probe C25 afforded the highly sensitive detection of Aβ fibrils and viscosity in lysosome, owing to its low background fluorescence, with distinguishable turn-on fluorescence signals around 570nm and 600nm, respectively. We confirmed the practical application of C25 by using it not only to detect the Aβ fibrils in solution,

cellular and tissue levels but also the viscosity in solution and cellular level. The probe exhibited obvious distinguishable fluorescence enhancement available for Aβ fibrils and viscosity detection and could serve as a powerful tool for developing the early diagnosis and innovative therapeutics of AD. The designed strategy also provides motivation for FP chromophore to be considered for sensing. Acknowledgements This work was financially supported by the supported by the Fundamental

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Research Funds for the Central Universities (D2191260) and the Medical Scientific Research Foundation of Guangdong Province, China (A2018080 and A2019029). References

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Figure Captions:

Fig. 1. (A) Fluorescence emission spectra of C25 (10 µM) with an increase in viscosity values in a water–glycerol system and the linear relationship between logI600 and log η of C25 (R2=0.96) (B) The fluorescence spectroscopic titration of C25 (1.0 µM) with addition of Aβ1-42 from 0 µM to 10 µM and the linear relationship between fluorescence intensity and concentration of C25 (R2=0.99). Fig. 2. (A-B) Colocalization test of C25.SH-SY5Y cells were stained with 5.0 µM

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C25 and 1.0 µM corresponding commercial probe (A1-A3 and B1-B3). Pearson’s colocalization coefficient of C25 and the commercial probes (A4 and B4). (C)

Confocal laser fluorescence images of C25. (C1) the cells were incubated with C25 (5 µM) only at 37℃ (B2) the cells were incubated with 10 µL DMSO for 30min and then

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treated with 5 µM C25. (C3) the cells were incubated with C25 (5 µM) only at 4℃ (C4) the cells were incubated with 5 µM dexamethasone for 30 min and then treated

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with 5 µM C25.(D)fluorescence intensity of images of C1-C4,which were analysed by Image-J. (E) Fluorescence emission spectra of region of interest in image C4

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marked by arrow. Scale bar=10 µm.

Fig. 3. (A-C) In vivo mapping of Aβ fibrils in AD transfected PC12 cells. (A)

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Immunofluorescence image from Alexa Fluor®405. (B)the fluorescence image from C25(5 µM). (C)the merged image of A-B. Scale bar=10 µm. (D-I) In vitro fluorescent staining of brain slices of cerebrum region from Tg mice (C57BL6, APP/PS1, 12 months old, male) using ThS and C25, respectively. Scale bar=50 µm. Fig. 4. Fluorescent images of SH-SY5Y cells after treatment of 2 µM FAM-Aβ for 1

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h (A-C), 16h(C-E), 24 h (F-H), followed by 5 µM C25 for 30 min. (H) Fluorescence emission spectra of region of interest marked by arrow. Scale bar=5000 nm.

Scheme. 1. Fluorescent protein chromophores can be modified to serve as fluorescent probes to simultaneously detect lysosome viscosity and β-Amyloid fibrils.

Scheme. 2. Synthetic route of C25 using the following reagents and conditions: a) N-Acetyl glycine, sodium acetate, acetic anhydride, 120℃, 4 h; b) N-(3-Aminopropyl) morpholine, K2CO3, EtOH, 80℃, 4 h; c) piperidine, EtOH, piperidine, 80℃,

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Author biogrophies Han sun is a graduate student in South China University of Technology under the supervise of Prof. Yan. His current research interests include the design and synthesis of bio-sensors. Huaxiang Leng is a graduate student in South China University of Technology under the supervise of Prof. Yan. Her current research interests include the design and synthesis of fluorescent probes for bio-imaging. Jinsheng Liu is a graduate student in South China University of Technology under the supervise of Prof. Yan. His current research interests include the design and synthesis of theranostic agents.

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Jinwu Yan received his Ph.D. degree in medicinal chemistry in Sun Yat-sen University in 2013. He is currently an associate professor in the School of Biology and Biological Engineering at South China University of Technology. His research interests currently focus on the design and development of fluorescent sensors for bio-imaging.

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Lei Zhang received his Ph.D. degree in Nankai University in 2000. He is currently the professor in the School of Biology and Biological Engineering at South China University of Technology. His research interests include drug design and development.