Specific Turn-On Fluorescent Probe with Aggregation-Induced


Specific Turn-On Fluorescent Probe with Aggregation-Induced...

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Letter pubs.acs.org/ac

Specific Turn-On Fluorescent Probe with Aggregation-Induced Emission Characteristics for SIRT1 Modulator Screening and LivingCell Imaging Yi Wang,*,† Yaqi Chen,† Haibo Wang,† Yiyu Cheng,† and Xiaoping Zhao*,‡ †

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang China College of Preclinical Medicine, Zhejiang Chinese Medical University, Hangzhou, Zhejiang China



S Supporting Information *

ABSTRACT: SIRT1 is an important protein that catalyzes the nicotinamide adenine dinucleotide (NAD)+-dependent deacetylation reaction, which is regarded as a novel target to treat metabolic disorders and aging-related diseases. However, there is lack of appropriate approach for SIRT1 modulator screening and bioimaging of SIRT1 in living cells. We designed and synthesized a “turn-on” fluorescent probe by connecting a specifically recognized peptide to tetraphenylethene core. It exhibits excellent selectivity and sensitivity in homogeneous measurement of SIRT1 activity for screening both SIRT1 inhibitors and activators. 20(S)-ginsenoside Rg3 and ophiopogonin D′ were found to activate SIRT1. It was also successfully applied to monitor SIRT1 modulation in the cardiomyocytes as well as in the wild-type and SIRT1−/− mesenchymal stem cells.

S

shaped rotor-like structures are nonemissive in solutions but greatly boost emission efficacies in the aggregate state.17,18 Probes with AIE characteristics exhibit great advantages in biomedical fields for biomacromolecule detection and living cell imaging19−23 because of their high luminosity, excellent biocompatibility, low background interference, and low cytotoxicity.24 Several attempts have been conducted to use AIE-based fluorescent probes to screen inhibitors of acetylcholinesterase (AchE),25 angiotensin converting enzyme (ACE),26 and caspases.27 However, fluorescence “turn-on” assays for detecting enzymes or proteins located in the nucleus of cells such as SIRT1 are rarely reported. In this contribution, a novel fluorescent probe is designed for detecting SIRT1 activity and screening SIRT1 modulators as well as for cell imaging. The probe consists of a hydrophobic tetraphenylethene (TPE) core and a hydrophilic Gly-Lys-TyrAsp-Asp (GKYDD) peptide sequence with a modification of acetyl-Lys (Ac−K). As shown in Scheme 1, the fluorescent probe is nonemissive in aqueous medium. However, the acetyl group of lysine can be deacetylated by SIRT1. With the aid of lysyl endopeptidase, the N-terminal peptide of lysine was removed, and the hydrophobic TPE residues aggregate to induce the light-up fluorescence in aqueous solution. This probe could be utilized for the evaluation of activation and

irtuin type 1 (SIRT1) belongs to a family of evolutionarily conserved intracellular protein deacetylases named Sirtuins (SIRTs), which catalyze nicotinamide adenine dinucleotide (NAD)+-dependent deacetylation reaction in mammals, including humans. Recent studies revealed that SIRT1 serves a critical function in transcriptional network as a cellular sensor in response to the metabolic and redox state.1 Activation or inhibition of SIRT1 leads to various biological effects in distinct disease models, including antiatherosclerosis,2 antidiabetes,3 neuroprotection,4 and anticancer.5 Interestingly, SIRT1 activators such as resveratrol are believed to extend lifespan.6,7 Therefore, the discovery of SIRT1 modulators gains great interest in the development of drugs for treating metabolic disorders and aging-related diseases. Commonly used approaches that measure deacetylation activity of SIRT1 include traditional radioactive assays using radiolabeled acetate or a nicotinamide adenine dinucleotide (NAD),8,9 high-pressure liquid chromatography (HPLC), or mass spectrometry (MS) based method,10,11 fluorescence polarization,12 and immunoblotting.13 The fluorescence resonance energy transfer (FRET) assay of SIRTs has been conducted through linking a quenching group to fluorescencelabeled peptide.14 However, the use of fluorescently tagged substrates has been reported to produce artifacts in screening SIRT1 activators.15 In addition, none of those methods are eligible for real-time detection of SIRT1 activity in living cells. Thus, a simple and specific approach for the biochemical assay of SIRT1 is preferred. Aggregation-induced emission (AIE) is a unique phenomenon found by Tang et al.16 that fluorogens with propeller© XXXX American Chemical Society

Received: March 20, 2015 Accepted: April 23, 2015

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DOI: 10.1021/acs.analchem.5b01069 Anal. Chem. XXXX, XXX, XXX−XXX

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Letter

RESULTS AND DISCUSSION The design of the probe is based on the following considerations. GK(Ac)Y is a peptide sequence located at the N-domain of LKB1 (amino acids 47−49), which is a protein kinase located predominantly in the nucleus. The acetylated lys48 has been proven to be the key lysine where SIRT1 modulates LKB1 function.28 The connection of hydrophilic peptide GK(Ac)YDD to TPE-core could convert the probe into highly water-soluble species. The TPE-GK(Ac)YDD probe was synthesis in four steps, as shown in Scheme S1 in the Supporting Information. The final product was purified using HPLC and further characterized using 1H NMR and electrospary ionization-mass spectrometry (ESI-MS) (Figures S1−S3 in the Supporting Information). In comparison with the typical AIE behavior of TPE-COOH, the probe is virtually nonfluorescent (Figure S4 in the Supporting Information) and exhibits a good solubility in Tris-HCl buffer. After preincubation, the probe with SIRT1 and site-specific lysyl endopeptidase, deacetylation products of TPE-GK(Ac)YDD can be detected by LC−MS (Figures S5 and S6 in the Supporting Information). The fluorescence intensity of the probe (20 μM) at 37 °C with SIRT1 was measured in the absence and the presence of SIRT1 inhibitor (EX527) or activator (SRT1720). As shown in Figure 1A, the fluorescence intensity of the probe significantly

Scheme 1. Schematic illustration of TPE-GK(Ac)YDD (A) for SIRT1 Modulator Screening and Cell Imaging (B)

inhibition of SIRT1 in its deacetylate capability in vitro and in living cells.



EXPERIMENTAL SECTION Synthesis of TPE-GK(Ac)YDD. Synthesis of TPE-COOH was prepared according to previous report. Detailed experimental procedures can be found in Supporting Information. Through solid phase peptide synthesis, amino group of specific designed peptide was reacted to TPE-COOH to obtain TPEGK(Ac)YDD. General Procedure for Fluorescent Measurement. In order to ensure the deacetylation reaction of SIRT1, SIRT1 (60 μg/mL) was added to TPE-GK(Ac)YDD (20 μM) and NAD+ (Sigma, 3 mM) for 3 h incubation at 37 °C in the presence or absence of the inhibitor and activator. Lysyl endopeptidase was also added. The assay of dose-dependent manners of TPEGK(Ac)YDD with different concentrations (1, 5, 10, 15, 20, 25, and 30 μM). All the PL spectra were measured from 400 to 600 nm (excitation wavelength 320 nm) with a JASCO FP-6500 spectrophotometer. For the enzyme kinetics assay, the fluorescence intensity was measured by a TECAN infinite F200 multifunction microplate (Tecan, Austria) with an excitation wavelength of 320 nm and an emission wavelength of 465 nm. Living Cell Imaging of Neonatal Cardiomyocytes and Mesenchymal Stem Cells Using TPE-GK(Ac)YDD. After being primary cultured for 48 h, neonatal cardiomyocytes and mesenchymal stem cells were exposed to resveratrol (20 μM) and EX527 (20 μM) for a 12 h incubation. The previous culture medium was replaced by fresh medium, followed by 3 h incubation in the incubator with TPE-GK(Ac)YDD (50 μM) at 37 °C. Before fluorescent images were obtained by a Nikon A1R laser scanning confocal microscope equipped with a 405 nm laser, the dishes were washed by PBS. The images were captured with a 60× lens. The cells were also stained with fluorescein diacetate (FDA) (10 μg/mL) for 10 min, and the dual fluorescent images of neonatal cardiomyocytes were captured. Excitation and emission wavelengths, 488 and 525 nm for FDA; 405 and 450 nm for TPE- GK(Ac)YDD. The fluorescence intensity was calculated with the software ImageJ by summing the mean intensity of each cell from six pictures.

Figure 1. (A) PL spectra of TPE-GK(Ac)YDD incubated with/ without SIRT1. In the presence of inhibitor EX527 and activator SRT1720, PL intensity relatively decreased and increased. (B) PL spectra of TPE-GK(Ac)YDD in the presence of dose-dependent concentrations of SIRT1. (C) PL spectra of dose-dependent concentrations of TPE-GK(Ac)YDD when incubated with SIRT1. The incubation time was 3 h. [inhibitor] = EX527 200 nM; [activator] = SRT1720 500 nM; λex = 320 nm.

increased after incubation with SIRT1, whereas the inhibition and activation of SIRT1 could be sensitively detected. After considering the effects of pH (Figure S7 in the Supporting Information), the fluorescence spectral change of the probe incubated with SIRT1 at various concentrations is shown in Figure 1B. The PL intensity steadily increased with the increasing concentration of SIRT1. To optimize the amounts of SIRT1 and probe in the screening assay, the probe with varied concentrations from 0 to 30 μM was incubated with SIRT1. As shown in Figure 1C, the PL intensity increased moderately, when the concentration of TPE-GK(Ac)YDD was above 15 μM. Therefore, 20 μM of TPE-GK(Ac)YDD was chosen for determining the SIRT1 activity and screening SIRT1 modulators. The kinetic activity of the enzymatic reaction was measured through time-course experiments (Figure 2A). Upon the addition of SIRT1, the fluorescence intensity gradually increased in 120 min, whereas the assay system without SIRT1 exhibits a low and stable fluorescence. In the application of cell imaging, shorter incubation time (such as 30 or 60 min) can obtain a fluorescent image. B

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(Figure S11 in the Supporting Information). As shown in Figure 3, overlapping fluorescence images of neonatal

Figure 3. Fluorescence images of neonatal cardiomyocytes by Nikon Laser scanning confocal microscope (60×). Excitation and emission wavelengths, 488 and 525 nm for fluorescein diacetate (FDA); 405 and 450 nm for TPE-GK(Ac)YDD.

Figure 2. (A) Plot of the fluorescence intensity of the assay system at 465 nm versus reaction time in the presence of SIRT1 (60 μg/mL). (B) Dose-dependent PL intensity of TPE-GK(Ac)YDD in the presence of SIRT1. (C) Dose-related inhibition of SIRT1 by EX527. (D) Plot of (I − I0)/I0 versus varied enzyme/proteins. λex = 320 nm. λem = 465 nm.

cardiomyocytes that were obtained through confocal microscopy also suggest that the observed fluorescence (blue) from the TPE-based probe was chiefly localized to the nucleus of cells, whereas green fluorescence that was stained by fluorescein diacetate indicated the whole living cells. To assess the capability of the probe for in situ screening of SIRT1 modulators, two known compounds, resveratrol (SIRT1 activator) and EX527 (SIRT1 inhibitor), were used to treat neonatal cardiomyocytes. The modulatory effects of agents were evaluated by comparing the change of fluorescence intensity with normal cells. As shown in Figure 4, normal

The linear range of the assay system for SIRT1 activity was measured using a fluorescence microplate reader. As shown in Figure 2B, the PL intensity exhibits a good linear curve versus with the increase of SIRT1 concentration from 0.5 to 100 μg/ mL (R2 = 0.9862). Moreover, the modulation of SIRT1 with EX527 and SRT1720 was evaluated quantitatively using the proposed assay system. The IC50 value of EX527 was 0.548 μM (Figure 2C), which is close to the literature report.29 Dosedependent activation of SIRT1 by SRT1720 at submicromolar concentrations was also observed (Figure S8 in the Supporting Information). These results suggest the utility of the probe for screening both inhibitors and activators of SIRT1. It was also utilized to screen SIRT1 modulators from Shengmai Fang, a botanical drug for treating ischemic heart disease. Several active compounds were found, in which 20(S)-ginsenoside Rg3 is a recently reported SIRT1 activator30 and ophiopogonin D′ was found to dose-dependently activate SIRT1 for the first time (Figure S9 in the Supporting Information). Thus, the proposed TPE-GK(Ac)YDD can be applied in screening SIRT1 modulators both in vitro and in living cells. Before the application for living cell imaging, the specificity of TPE-GK(Ac)YDD toward other histone deacetylases (HDACs) and proteins existed in cell culture medium was verified. The fluorescence spectra of TPE-GK(Ac)YDD were measured in the presence of HDAC1, HADC3, human serum albumin (HSA), bovine serum albumin (BSA), collagenase I/II, cytochrome C (CYC), lysozyme, thrombin, and typsin. Figure 2D showed that other histone deacetylases and proteins presented relatively weaker changes in (I − I0)/I0 compared with SIRT1. The cytotoxicity of TPE-GK(Ac)YDD was also evaluated. Incubation of the probe (50 μM) with cardiomyocytes for 24 h showed no cytotoxicity (Figure S10in the Supporting Information), which proved that the proposed probe is emerging as an ideal probe for noninvasive tracking of the SIRT1 activity in the cell. The usability of the probe for living cell imaging of SIRT1 was validated in cardiomyocytes and mesenchymal stem cells (MSCs). Among the SIRTs, only SIRT1 contains both nuclear localization and nuclear export sequence and shuttles between the nucleus and the cytoplasm.31 After incubation with 50 μM TPE-GK(Ac)YDD in DMEM for 3 h at 37 °C, the nucleus region of H9c2 cardiomyocytes were specifically stained

Figure 4. Bright-field (BF), fluorescence (FL), and overlay images of neonatal cardiomyocytes, neonatal cardiomyocytes that are treated with resveratrol (Res) and EX527(EX). The images were captured using a laser scanning confocal microscope (Nikon) equipped with a 405 nm laser and 60× lens.

neonatal cardiomyocytes and EX527 treated cells exhibited relative low fluorescent signals, which indicated lower SIRT1 activity in cell populations. An approximate 1.4-fold increase of fluorescence intensity is observed for resveratrol-treated cells (Figure S12 in the Supporting Information). These results are consistent with previous reports that resveratrol can induce the expression of SIRT1 in cardiomyocytes.32 SIRT1 plays pivotal roles in modulating cardiomyocyte survival33,34 and regulating the differentiation of MSCs.35,36 Compared with the fluorescent images of wild-type and C

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Analytical Chemistry SIRT1−/− MSC also demonstrated that inhibition of SIRT1 using lentivirus-mediated shRNA can be quantitatively monitored by the TPE-GK(Ac)YDD probe (Figure S13 in the Supporting Information).

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CONCLUSION In conclusion, a novel TPE-based fluorescent probe with AIE characteristics was designed and synthesized for the measurement of SIRT1 activity and in situ detection of intracellular SIRT1. This probe can be used to screen SIRT1 inhibitors and activators and track the expression of nucleus SIRT1 and its translocation. Compared with previous immunofluorescence approaches for cell imaging of SIRT1 that requires tedious fixing and washing steps, the TPE- GK(Ac)YDD probe offers an alternative approach to tracking intracellular SIRT1 in living cells. This probe may provide novel insight for the discovery of SIRT1 modulators.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01069.



AUTHOR INFORMATION

Corresponding Authors

*Fax/phone: +86-571-88208426. E-mail: [email protected] *Fax/phone: +86-571-86633138. E-mail: [email protected] edu.cn. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was supported by the National Key Scientific and Technological Project of China (Grant 2012ZX09304007) and the Fundamental Research Funds for the Central Universities (Grant 2015FZA7017).



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DOI: 10.1021/acs.analchem.5b01069 Anal. Chem. XXXX, XXX, XXX−XXX