A Fluorogenic Molecule for Probing Islet Amyloid Using Flavonoid as a Scaffold Design

Article abstract of DOI:10.1021/acs.biochem.0c00076

Aggregation of polypeptides and proteins is commonly associated with human and other vertebrate diseases. For example, amyloid plaques consisting of amyloid-β proteins are frequently identified in Alzheimer's disease and islet amyloid formed by islet amyloid polypeptide (IAPP, amylin) can be found in most patients with type 2 diabetes (T2D). Although many fluorescent dyes have been developed to stain amyloid fibrils, very few examples have been designed for IAPP. In this study, a series of environmentally sensitive fluorescent probes using flavonoid as a scaffold design are rationally designed and synthesized. One of these probes, namely 3-HF-ene-4′-OMe, can bind to IAPP fibrils but not nonfibrillar IAPP by exhibiting a much stronger fluorescent enhancement at 535 nm. In addition, this probe shows better detection sensitivity to IAPP fibrils compared with that of conventionally used thioflavin-T. We demonstrate that 3-HF-ene-4′-OMe can be used to monitor the kinetics of IAPP fibril formation in vitro even in the presence an amyloid inhibitor. To test the specificity of the probe, we attempt to incubate this probe with amyloid fibrils formed from other amyloidogenic proteins. Interestingly, this probe shows different responses when mixed with these fibrils, suggesting the mode of binding of this probe on these fibrils could be different. Moreover, we show that this probe is not toxic to pancreatic mouse β-cells. Further structural optimization based on the structure of 3-HF-ene-4′-OMe may yield a specific probe for imaging islet amyloid in the pancreas. That would improve our understanding of the relationship between islet amyloid and T2D.

Full text of DOI:10.1021/acs.biochem.0c00076

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Article
A Fluorogenic Molecule to Probe Islet
Amyloid Using Flavonoid as a Scaffold Design
Wei-Ling Chen, Shih-Ting Ma, Yun-Wen Chen, Yen-
Cheng Chao, Ai-Ci Chan, Ling-Hsien Tu, and Wei-Min Liu
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.0c00076 • Publication Date (Web): 08 Apr 2020
Downloaded from pubs.acs.org on April 9, 2020
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A Fluorogenic Molecule to Probe Islet Amyloid Using Flavonoid as a
Scaffold Design
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Wei-Ling Chen1,†, Shih-Ting Ma2,†, Yun-Wen Chen3, Yen-Cheng Chao2, Ai-Ci Chan1,
Ling-Hsien Tu1* and Wei-Min Liu2*
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1. Department of Chemistry, National Taiwan Normal University, Taipei 116,
Taiwan.
2. Department of Chemistry, Fu Jen Catholic University, New Taipei City 24205,
Taiwan.
3. Department of Pharmacology, College of Medicine, National Cheng Kung
University, Tainan 701, Taiwan.
† These authors contributed equally
*
To whom correspondence should be addressed: Ling-Hsien Tu, E-mail:
litu@ntnu.edu.tw; Wei-Min Liu, E-mail: 133797@mail.fju.edu.tw
Abstract
Aggregation of polypeptides and proteins is commonly associated with human and
other vertebrate diseases. For example, amyloid plaques consist of amyloid-β proteins
(Aβ) are frequently identified in Alzheimer’s disease and islet amyloid formed by islet
amyloid polypeptide (IAPP, Amylin) can be found in most patients with type-2 diabetes
(T2D). Although many fluorescent dyes have been developed to stain amyloid fibrils,
very few examples are designed for IAPP. In this study, a series of environmentally
sensitive fluorescent probes using flavonoid as a scaffold design is rationally designed
and synthesized. One of these probes, namely 3-HF-ene-4’-OMe, can bind to IAPP
fibrils but not non-fibrillar IAPP by exhibiting a much stronger fluorescent
enhancement at 535 nm. In addition, this probe shows better detection sensitivity to
IAPP fibrils compared with conventionally used Thioflavin-T. We demonstrate that 3-
HF-ene-4’-OMe can be used to monitor the kinetics of IAPP fibril formation in vitro
even in the presence amyloid inhibitor. To test the specificity of probe, we attempt to
incubate this probe with amyloid fibrils formed from other amyloidogenic proteins.
Interestingly, this probe shows different responses when mixing with these fibrils
suggesting the binding mode of this probe on these fibrils could be different. Moreover,
we show that this probe is not toxic to pancreatic mouse beta cells. Further structural
optimization based on the structure of 3-HF-ene-4’-OMe may accomplish a specific
probe for imaging islet amyloid in the pancreas. That would improve our understanding
on the relationship between islet amyloid and T2D.
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Introduction
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The self-assembly of amyloidogenic proteins or peptides plays a crucial role in
many human diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and type-
2 diabetes (T2D)1, 2. Although these proteins are different in sequence and length, under
incompletely defined circumstance, they would misfold, associate into typical β-sheet
conformation, and form characteristic aggregated oligomers or fibrils. The mechanism
of amyloid formation has not been fully elucidated, however, many studies have
suggested that the occurrence of fibril formation is frequently associated with the rate
of disease onset. Consequently, tons of efforts were consistently made by scientists to
reveal the mechanism of protein aggregation, to design methods for preventing protein
aggregation, and to develop better probes for detecting protein aggregation.
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Thioflavin-T (ThT) is the most commonly used fluorescent probe for detecting
fibril growth kinetics in vitro3, 4. However, there are several drawbacks and cautions
should be taken when using ThT as an amyloid probe. For example, the non-specific
binding of ThT to other hydrophobic patches sometimes results in a false positive
signal5, 6. The binding affinity of ThT to amyloid fibrils varies significantly and that is
highly dependent on the pH and the ionic strength7 of working solution. An excess of
ThT, relative to amyloid fibrils, is generally required to achieve high fluorescence
enhancement8. As a result, the ThT assay does not always accurately report the presence
of amyloid fibrils. Furthermore, it may not always be reliable using ThT for inhibition
studies, because some potential inhibitory candidates compete with ThT for the same
amyloid binding site or quench ThT fluorescence resulting prejudiced conclusions9-11
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Therefore, the development of efficient and specific fluorescent probes is necessary and
the use of multiple dyes in drug screening is needful. Recently, although many specific
fluorescent probes have been reported for different neurodegenerative amyloidosis 11-
18, there are very few examples designed in particular for islet amyloid polypeptide
(IAPP)19-21. IAPP is a 37-residue hormone polypeptide with a disulfide bridge between
Cys-2 and Cys-7 (Figure 1A) and co-secreted with insulin from pancreatic β-cells. The
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aggregation process of IAPP has been shown to cause the loss of β-cells
.
Accumulation of islet amyloid in the pancreas also has become one of the important
features of T2D23-25. Therefore, it is believed that the development of specific dye for
the detection of islet amyloid would enhance our understanding of this disease and help
us find useful inhibitors against islet amyloid aggregation.
Flavonoids are a class of natural products which are capable of disrupting
aggregation of amyloidogenic proteins26, 27. For instance, epigallocatechin gallate
(EGCG) and silibinin sharing a similar flavonoid like core structure (Figure 1B) were
found to efficiently prevent aggregation of IAPP28. On the other hand, it is also worth
noting that flavonoid-based small molecules, 3-hydroxyflavone (3-HF) and its
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derivatives, are environmentally sensitive (solvatochromic) fluorescent dyes with
characteristic separated dual emission bands29. The dual-band emissions are due to
intramolecular proton transfer (ESIPT) in the excited state and that produces two
excited state forms, namely normal state (N*, excited state intramolecular charge
transfer, ESICT product) and tautomer state (T*, ESIPT product). In comparison with
N*, the T* results in a longer wavelengths emission and that can be easily modulated by
dipolar and H-bonding interaction30. The ratio of the intensities of these two bands
highly depends on the polarity of the environment31. Because of these unique features, 3-
HF and its derivatives have been widely applied in the studies of polymers32, reverse
micelles33, and proteins34-36. Along these lines, flavonoid was considered as an ideal
scaffold for the design of our fluorescent probes. Besides, the conjugated 4’-
methoxybenzene motif of stilbene derivative is another well-known molecular skeleton
with high binding affinity for amyloid fibrils37. In order to optimize the binding affinity
of our probes to IAPP fibrils, different length of conjugated π-electron chain was
employed. Thus, a series of environmentally sensitive fluorescent probes was rationally
designed and synthesized (Figure 1B). Herein, one of these probes, namely 3-HF-ene-
4’-OMe, exhibits better detection properties for IAPP amyloid, acts as an alternative
fluorescent dye in the studies of IAPP aggregation kinetics in vitro, and is also suitable
for IAPP inhibitory studies. 3-HF-ene-4’-OMe displays unique maximum emission
wavelength at 535 nm upon binding to IAPP fibrils but not to non-fibrillar IAPP or
other amyloid fibrils suggesting its potential to be developed as a specific dye. In this
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Figure 1. (A) The amino acid sequences of IAPP. IAPP is amidated at C-terminal and contains a
disulphide bond between residue 2 and 7. (B) Design of environmentally sensitive fluorescent probes for
detecting IAPP amyloid fibrils.
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study, we chose amyloid fibrils formed by amyloid beta (Aβ42) and human calcitonin
(hCT) for comparison with IAPP fibrils. Deposition of amyloid plaques composed of
Aβ in the brain is highly associated with Alzheimer’s disease. hCT is also a member of
amylin protein family. These two peptides are both considered as amyloidogenic
peptides. Additionally, we also demonstrate that non-cytotoxic 3-HF-ene-4’-OMe can
be applied in live cells imaging. However, further structural optimization based on 3-
HF-ene-4’-OMe is needed to improve its nonspecific binding to cell membrane. The
probe holds promising potential for imaging of islet amyloids in the pancreas.
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Materials and Methods
General protocol for the synthesis of a series of flavonoid-based probes.
To a solution of 2’-hydroxyacetophenone (1 equiv.) and corresponding aldehydes
1a-1c (1 equiv.) in MeOH was added 15 M NaOH under ice bath. The reaction mixture
was stirred at room temperature until no aldehyde remained, checking by TLC. Until
the reaction was complete, solution was acidified by concentrated HCl (37%) until pH
1 ~ 2. The acidified reaction solution was filtered through Büchner funnel. The filter
cake was directly used for the next step or was purified by recrystallization using EtOAc
as solvent. The products from previous step were dissolved in MeOH, then NaOH (5
equiv.) and H2O2 (25 equiv.) were added into the reaction mixture at 0 oC. The reaction
mixture was allowed to stir at room temperature for 24 hr. After that, it was diluted with
ice water and 4N HCl aqueous solution was added to adjust the pH until 1. The resulting
precipitates were filtered and washed with distilled water, giving the desired products
or was further purified by crystallization or purified by flash column chromatography,
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giving the desired products. 3-HF-4’-OMe (Yield: 73%): H NMR (300 MHz, d6-
DMSO, 294 K): δ = 9.47 (s, OH), 8.30 (d, J = 9.0 Hz, 2H, H2’, H6’), 8.07 (dd, J1 = 7.6
Hz, J2 = 1.0 Hz, 1H, H5), 7.75-7.68 (m, 2H, H7, H8), 7.40 (ddd, J1 = 8.0 Hz, J2 = 5.9
Hz, J3 = 2.1 Hz, 1H, H6), 7.08 (d, J = 9.1 Hz, 2H, H3’, H5’), 3.82 (s, 3H, OMe) ppm; 13C
NMR (75 MHz, d6-DMSO, 298 K): δ = 172.61, 160.43,154.42, 138.13, 133.47, 129.39,
124.71, 124.46, 123.56, 121.33, 118.30, 114.03, 55.35 ppm; IR (KBr): 3198, 3018,
2837, 1633, 1614, 1483, 1308, 1132, 1111, 1025, 830, 757 cm-1; The data agreed with
literature reported38. 3-HF-ene-4’-OMe (Yield: 46%): Rf = 0.2 (n-Hexane: EtOAc 7:1
v/v). 1H NMR (300 MHz, CDCl3, 295 K): δ = 8.22 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H,
H5), 7.71 (ddd, J1 = 8.6 Hz, J2 = 7.1 Hz, J3 = 1.7 Hz, 1H, H7), 7.60-7.49 (m, 4H, H2’,
H6’, H8, Hβ), 7.39 (ddd, J1 = J2 = 7.5 Hz, J3 = 1.0 Hz, 1H, H6), 7.21 (d, J = 16.2 Hz, 1H,
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Hα), 6.95 (d, J = 8.8 Hz, 2H, H3’, H5’), 3.86 (s, 3H, OMe) ppm; C NMR (75 MHz,
CDCl3, 300 K): δ = 172.50, 160.84, 155.23, 146.05, 137.57, 134.36, 133.47, 129.23,
128.87, 125.52, 124.42, 121.56, 118.17, 114.54, 113.60, 55.55 ppm; IR (KBr): 3117,
1636, 1617, 1469, 1304, 1255, 1170, 1114, 1024, 963, 818, 800, 757 cm-1; m.p. = 229-
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230 oC; HR-MS m/z: 295.0970 [M+H]+, calcd. [C18H15O4]: 295.0970.; 3-HF-diene-4’-
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OMe (Yield: 55%): Rf = 0.17 (CHCl3). H NMR (600 MHz, d6-DMSO, 300 K): δ =
9.50 (s, OH), 8.05 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H, H5), 7.76 (ddd, J1 = 8.6 Hz, J2 =
7.1 Hz, J3 = 1.7 Hz, 1H, H7), 7.65 (d, J = 8.4 Hz, 1H, H8), 7.53 (d, J = 8.8 Hz, 2H,
H2’,H6’), 7.43 (ddd, J1 = 7.5 Hz, J2 = 7.5 Hz, J3 = 0.9 Hz, 1H, H6), 7.35 (dd, J1 = 15.1Hz,
J2 = 10.8 Hz, 1H, Hβ), 7.12 (dd, J1 = 15.3 Hz, J2 = 11.0 Hz, 1H, Hγ), 6.86 (d, J = 9.0
Hz, 2H, H3’, H5’), 6.85 (d, J = 15.0 Hz, 1H, Hδ), 6.82 (d, J = 15.0 Hz, 1H, Hα), 3.78
(s, 3H, OMe) ppm; 13C NMR (150 MHz, d6-DMSO, 300 K): δ = 172.00, 159.88, 154.35,
146.93, 138.02, 137.11, 135.07, 133.76, 129.47, 128.69, 126.32, 124.96, 124.58,
122.24, 118.48, 118.21, 114.57, 55.45 ppm; IR (KBr) = 3189, 1614, 1590, 1469, 1302,
1260, 1172, 1153, 1114, 1062, 1027, 985, 837, 760 cm-1; m.p. = 226-229 oC; HR-MS
m/z: 321.1129 [M+H]+, calcd. [C20H17O4]: 321.1127. The details of peak assignments
of 1H NMR spectra are also shown in supporting information.
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Peptide synthesis
IAPP, Aβ42, and hCT were synthesized with standard Fmoc solid phase synthesis
using a microwave-assisted CEM Liberty Lite peptide synthesizer. In order to generate
amidated C-terminus, Rink amide ProTide resin as solid phase support was used for
IAPP and hCT synthesis. Fmoc-protected pseudoproline dipeptides were used to avoid
peptide aggregation during IAPP synthesis. For Aβ42, Cl-MPA ProTide resin was used
and a 1.0 M N, N-diisopropylethylamine (DIEA) and 0.125 M KI solution were
prepared for first amino acid coupling. After synthesis, peptides were cleaved from the
resin by using a cocktail containing 92.5% trifluoroacetic acid (TFA), 2.5% water, 2.5%
3,6-dioxa-1,8-octanedithiol (DODT), and 2.5% triisopropylsilane (TIS). Crude IAPP
and hCT were oxidized with CLEAR-OX resin at room temperature to form an
intramolecular disulfide bond. All peptides were purified via reverse-phase high-
performance liquid chromatography (HPLC) using PROTO C18 semi-preparative
column (4.6 mm x 250 mm). A two solution gradient system was used for IAPP and
hCT purification: solution A consisted of 100% H2O and 0.045% HCl (v/v) and solution
B consisted of 80% acetonitrile (ACN), 20% H2O and 0.045% HCl. For Aβ42
purification, we used another alkalic solution system to increase peptide solubility.
Solution A consisted of 100% H2O, 0.05% NH4OH and 0.005 % diethylamine (DEA,
pH was adjusted to 10 by TFA) and solution B consisted of 90% ACN, 10% H2O,
0.05% NH4OH and 0.005 % DIEA (pH was adjusted to 10 by TFA). The molecular
weight of IAPP was confirmed by ESI mass spectrometry. ESI (positive mode) calcd.
for IAPP: 3903.3, found: m/z 976.7 [M+4H]4+ and 1302.3 [M+3H]3+. MALDI-TOF
mass spectrometry confirmed the molecular weight of Aβ42 (expected, 4514.1 Da;
observed, 4514.5 Da) and hCT (expected, 3417.8 Da; observed, 3418.6 Da).
Sample preparations
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Small molecules, 3-HF, 3-HF-4’-OMe, 3-HF-ene-4’-OMe and 3-HF-diene-4’-
OMe were first prepared in pure dimethyl sulfoxide (DMSO) at 32 mM and diluted
into 10 mM Tris buffer at pH 7.4 to make a concentration of 32 or 64 μM solution.
Absorption spectra were collected using SpectraMax M2 multimode plate reader.
Excitation and emission wavelengths for individual compound were determined and
shown in supporting information (Figure S2). The environmental sensitivity of these
probes was studied with a DMSO and water cosolvent system (Figure S1D-F). To
prepare non-fibrillar peptide and peptide fibrils, all peptides were first treated with
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to disassembly possible aggregates and
freeze-dried by lyophilizer. 0.1~0.2 mg dry power peptides were dissolved in 100 μL,
10 mM Tris buffer at pH 7.4 and Pierce BCA protein assay kit (BSA as protein standard,
Thermo Scientific) was used to determine peptide concentration. Then peptide stock
was further diluted to the desired concentrations. Non-fibrillar peptide solution was
always fresh prepared before measurements. To prepare fibril solutions, peptides were
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further incubated at 25 C for 2.5 days with continuous shaking (Eppendorf,
ThermoMixer C). Formation of amyloid fibrils was confirmed by ThT assay and TEM.
Fluoresce spectra of 3-HF and its derivatives with non-fibrillar peptide and
peptide fibrils
Both non-fibrillar peptide solution and fibril solution were prepared at 64 μM
determined by BCA assay. Then individual compound DMSO solution was added into
non-fibrillar peptide or fibril solution to reach a final concentration of 64 μM and
DMSO concentration was fixed in 1%. The well-mixed solution was excited at different
wavelengths according to different maximal absorption of compounds, and emission
spectra were recorded for a wide range.
Circular dichroism (CD)
CD experiments were performed using a JASCO J-715 circular dichroism
spectrometer. In order to avoid the interference of DMSO, we first prepared the probe
in acetonitrile (ACN)39. The final concentration of ACN in IAPP solution is 1%. CD
experiments were conducted after incubation of 64 μM IAPP solution with and without
the probe at 25 oC for 3 days with continuous shaking. Spectra were recorded from 200
to 260 at 1 nm intervals at 25 °C. The data were averaged from ten scans and corrected
with background spectrum.
Determination of fluorescence enhancement of ThT and 3-HF-ene-4’-OMe for
IAPP fibrils
The fluorescence of ThT (λem = 485 nm) and 3-HF-ene-4’-OMe (λem = 535 nm)
are recorded when increasing concentration (0-64 μM) of probes were titrated into fixed
IAPP fibrils (64 μM). The half maximal concentration CM were analyzed by Graph Pad
Prism 5.0 using the equation Y= Bmax*X/(CM+ X) where X is the concentration of
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probes, Y is the change of fluorescence intensity, Bmax is the maximum binding of
probes with the same units as Y, and CM is half maximal concentration with the same
units as X.
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ThT and 3-HF-ene-4’-OMe aggregation assay
Aliquot of freshly prepared IAPP stock was diluted into buffer containing 32 μM
ThT (from a 1 mM ThT solution) or 32 μM 3-HF-ene-4’-OMe to make a concentration
of 32 μM solution. A 384-well nonbinding surface microplate (Corning, USA) was used
for ThT measurement. ThT fluorescence was excited at 430 nm and collected at 485
nm while 3-HF-ene-4’-OMe fluorescence was excited at 380 nm and collected at 535
nm using SpectraMax M2 multimode plate reader under quiescent conditions. The
measurement was conducted every two hours. For resveratrol inhibition studies,
resveratrol was first prepared in DMSO and then added into IAPP with ThT or IAPP
with 3-HF-ene-4’-OMe solution to reach desired concentration. The total
concentration of DMSO was fixed in less than 2%.
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TEM
5 μL of IAPP, Aβ42, hCT samples were deposited onto 300-mesh Formvar carbon-
coated copper grids for 1 min. The grids were blotted, washed with droplets of Milli-Q
water, and stained with 2% uranyl acetate. The samples were examined with Hitachi
H-7100 transmission electron microscopy with an accelerating voltage of 120 kV.
Molecular docking
3-HF-ene-4’-OMe docking studies with IAPP and Aβ42 fibrils were executed by
using Autodock 4.2 software package40. The structure of Aβ42 fibrils (PDB: 2MXU)41
was download from the Protein Data Bank and the model of IAPP fibrils was provided
by Prof. Robert Tycko42 and modified by Prof. Daniel P. Raleigh. The geometry of 3-
HF-ene-4’-OMe in the gas phase was optimized with density functional theory at the
B3LYP/6-31G* level using Gaussian09 program43, 44. The docking simulations were
carried out with a box centered on the IAPP fibrils and a box centered on the Aβ42
fibrils. The sizes of the boxes are 126 × 126 × 126 Å using a 0.375 Å grid step for both
IAPP and Aβ42 fibrils. The docking simulation was performed 100 times and the
Lamarckian Genetic Algorithm was employed for the grid search algorithm.
Confocal fluorescence imaging
For the confocal image observation, IAPP amyloid fibrils were prepared at 32 μM
and mixed with 32 μM 3-HF-ene-4’-OMe in 10 mM Tris buffer (pH 7.4). The mixed
solutions were placed onto a glass slide. Confocal images were taken with a laser
scanning confocal microscope (Leica SP5). 3-HF-ene-4’-OMe was excited with an Ar-
ion laser at 380 nm, and detected in the range of 500-580 nm. Transmitted light images
was also obtained to assess the presence of fibrils in the investigated region.
Cell culture
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Murine insulin-secreting MIN6 cells were grown in Dulbecco’s Modified Eagle’s
Medium (DMEM; Gibco) supplemented with 25ꢀmM glucose, 10% heat-inactivated
fetal bovine serum (FBS; Gibco), 100ꢀU/ml penicillin, 100ꢀμg/ml streptomycin (Gibco),
10ꢀmM HEPEs (Gibco), 1ꢀmM sodium pyruvate (Gibco) and 2ꢀmM GlutaMax (Gibco).
Cells were cultured at 37ꢀ°C in a humidified atmosphere containing 95% air and 5%
CO2. Subculture and maintenance were performed as previously described45. Cultured
cells were passaged every 3–4 days. MIN6 cells presented in this study were at passages
20–30.
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Fluorescence imaging
MIN6 Cells were seeded on the glass-bottom dish (1ꢀ×ꢀ105 cells) for three days.
To examine the binding ability of 3-HF-ene-4’-OMe for IAPP fibrils in live cells,
MIN6 cells were incubated with 8 μM IAPP fibrils for 1 hr, and then incubated with 3-
HF-ene-4’-OMe for 2 hr in the growth medium. After that, cells were washed with
PBS buffer for three times prior to fluorescence imaging. The images were taken by
Fluorescence microscopy.
Cell viability assay
MIN6 Cells were seeded in 96 well plates (8ꢀ×ꢀ104 cells/well) and cultured for three
days before exposure to IAPP probe (24 hr, 48 hr, 72ꢀhr) and then the 3-(4,5-
diethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess
the viability of cells after IAPP probe treatment.
Results and Discussion
Before introduction of our probes in the test, the preparation of IAPP fibrils was
further confirmed by traditional ThT assay and transmission electron microscopy (TEM,
Figure 2A and Figure S1). It was shown that ThT with IAPP fibrils exhibited stronger
fluorescence in comparison with freshly prepared IAPP. TEM also showed the typical
fibrillar structure of amyloid. In this study, 3-HF was initially used to evaluate the
ability of flavonoid scaffold for the detection of amyloid fibrils. As shown in Figure
2B, the fluorescence intensity of 3-HF was dramatically increased upon addition to
IAPP fibrils, although a similar result was observed when 3-HF was mixed with non-
Scheme 1. The synthesis of 3-HF-4’-OMe, 3-HF-ene-4’-OMe, and 3-HF-diene-4’-OMe.
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fibrillar IAPP which is considered a very hydrophobic peptide. Encouraged by this
preliminary outcome, the B ring of 3-HF was functionalized with a methoxy group at
para-position to mimic the structure of stilbene derivatives. Different length of
conjugated π-electron chains was created between ring C and ring B of 3-HF and to
improve the fibril selectivity. The synthesis of a series of flavonoid-based probes is
shown in Scheme 1. Aldehyde 1c was prepared from 1b according to the reported
procedure (supporting information)46. When the appropriate aldehydes were prepared,
they were condensed with 2’-hydroxyacetophenone to produce the conjugated diphenyl
intermediates. Subsequently, Algar-Flynn-Oyamada reaction was applied to make
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desired fluorescent probes38, 47
.
First, the UV and fluorescence spectrum for synthetic flavonoid-based probes
were measured. The excitation/maximum emission wavelength of 3-HF-4’-OMe, 3-
HF-ene-4’-OMe, and 3-HF-diene-4’-OMe in dimethyl sulfoxide (DMSO) were
355/555, 390/475, and 415/545 nm, respectively. Next, the environmental sensitivity
of these probes was evaluated to confirm that these molecules are solvatochromic
(Figure S2). As anticipated, dual fluorescence emissions of 3-HF-4’-OMe were found
and the ratio of these two fluorescence peak intensities was strongly depended on the
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Figure 2. 3-HF-ene-4’-OMe is the most sensitive dye to distinguishing non-fibillar IAPP and IAPP
fibrils. Fluorescence emission spectra of (A) ThT, (B) 3-HF, (C) 3-HF-4’-OMe, (D) 3-HF-ene-4’-OMe,
and (E) 3-HF-diene-4’-OMe at 64 μM are measured. Black curve represents probe alone. Red dot curve
represents probe with non-fibillar IAPP. Blue dot curve represents probe with IAPP fibrils. All
measuremnets were performed in 10 mM Tris buffer at pH 7.4 in duplicate. The IAPP fibrils were
prepared by incubation of samples in microtubes for 2.5 days with continuous shaking. (F) The ratio of
fluorescence increase of flavonoid-based probes and ThT mixed with non-fibillar IAPP or fibrils relative
to the fluorescence intensity of free probes in 10 mM Tris buffer at pH 7.4. (Wavelenths were used for
claculation. ThT: 485 nm; 3-HF: 535 nm; 3-HF-4’-OMe: 540 nm; 3-HF-ene-4’-OMe: 535 nm; 3-HF-
diene-4’-OMe: 555 nm)
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water concentration in DMSO (Figure S2D). Instead of dual emission bands, 3-HF-
ene-4’-OMe and 3-HF-diene-4’-OMe display a single broad emission band. The
broadening emission band could result from the increase of the flexibility of conjugated
double bonds (Figure S2E and F). Despite they both exhibit a single broad emission
band, the fluorescence intensity and maximal emission wavelength of these two probes
still strongly depend on the environmental polarity and viscosity. 3-HF-ene-4’-OMe
and 3-HF-diene-4’-OMe exhibit a significant red-shift about 70 nm and 40 nm,
respectively, when the percentage of water in solution increases from 0% to 40%
(Figure S2E and F). Fluorescence of these two probes enhance along with increasing
viscosity (Figure S2G and H). Meanwhile, all of these flavonoid-based probes display
negligible fluorescence emission in aqueous solution (Figure S2C). After understanding
the optical properties of these probes, we next examined their ability in detecting
preformed IAPP fibrils. As shown in Figure 2, in all cases, the fluorescence intensities
of these probes significantly increased when they were incubated with IAPP fibrils. In
particular, 3-HF-ene-4’-OMe was found to be the most sensitive dye in differentiating
non-fibrillar or aggregated IAPP. Basically, this dye exhibits negligible fluorescence in
aqueous solution and mixed with non-fibrillar IAPP, yet its fluorescent property was
turned on upon mixing with IAPP fibrils. The intensity enhancement ratio of fibril to
non-fibrillar species is significantly different (p<0.001, Figure 2F, intensity from dye
alone was used as individual background). The ratio of dual emission bands (T*/N*) of
3-HF-4’-OMe was also significantly different when it was mixed with IAPP fibrils
compared with non-fibrillar IAPP and free probe in solution. This change indicates that
3-HF-4’-OMe binds to the hydrophobic region of IAPP fibrils. As a result, the
fluorescent spectra were majorly dominated by ESIPT product of 3-HF-4’-OMe.
Because of the sensitivity of 3-HF-4’-OMe is still less than 3-HF-ene-4’-OMe, we
chose to use 3-HF-ene-4’-OMe in the following studies. On the other hand, we also
evaluate the specificity of 3-HF-ene-4’-OMe for detection of amyloid fibrils. Bovine
serum albumin (BSA), the relative larger protein with major helix structure was also
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incubated with this probe. Only a slight enhancement of fluorescence intensity was
observed when 3-HF-ene-4’-OMe was mixed with BSA, indicating its specificity for
fibril detection (Figure S3).
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The comparison of fluorescence enhancement from ThT and 3-HF-ene-4’-OMe
with IAPP fibrils was also determined here by in vitro saturation binding assay. The
fluorescent intensity was recorded for each probe at different concentration with pre-
aggregated IAPP fibrils in Tris buffer. The half maximal concentration (CM) of ThT
and 3-HF-ene-4’-OMe were about 48 μM and 12 μM, respectively (Figure 3). The
result indicates that the rational designed 3-HF-ene-4’-OMe exhibits stronger signal
change to IAPP fibrils than the generally used ThT although it seems to have minor
self-quenching effect when higher concentration of 3-HF-ene-4’-OMe was applied. As
described previously, ThT is a commonly used probe to monitor the kinetics of amyloid
formation. Several factors including binding site competition and quenching effect
cause this probe not to always report without bias in amyloid inhibition study.
Resveratrol, a reported IAPP inhibitor 48-50, is one of the examples 51. Previous studies
have reported that there was no detectable ThT fluorescence intensity when an excess
of resveratrol was used for IAPP inhibition studies, even though large amounts of IAPP
fibrils were observed by TEM. This phenomena indicates that resveratrol possibly
competes with ThT for binding to amyloid fibrils. In this context, 3-HF-ene-4’-OMe
was also examined whether it can be used for both kinetic and inhibition studies. As a
useful probe for monitoring the kinetics of fibril formation, the probe itself should not
interfere with the fibril formation. To prove that, 3-HF-ene-4’-OMe along with ThT
were incubated with IAPP and the fluorescence of ThT was monitored during the period
of IAPP fibril formation. As shown in Figure S4, similar kinetic curves were observed
in the presence and absence of 3-HF-ene-4’-OMe revealing that our probe did not
disturb the fibril formation and this probe might have different binding site to IAPP
fibrils compared with ThT. We also used CD to show the β-sheet structure of IAPP
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Figure 3. 3-HF-ene-4’-OMe showed a higher fluorescence signal to islet amyloid than ThT. Fluoresence
spectum were recored when (A) ThT and (B) 3-HF-ene-4’-OMe were titrated into IAPP fibrils. (C) A
plot of the fluorescence intensity at λem 485 nm for ThT and at λem 535 nm for 3-HF-ene-4’-OMe
versus the concentration of probes in the presence of IAPP fibrils. Intensity was recored from
independent duplicate trials and averaged.
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fibrils and this probe did not change the conformation of fibrils (Figure S5). 3-HF-ene-
4’-OMe shares similar core structure with amyloid inhibitors, EGCG and silibinin, but
do not inhibit IAPP aggregation. Although the inhibitory mechanism remains unclear
for numerous flavonoids, several studies have suggested that the phenolic hydroxyl
groups on B ring are important for the effectiveness of these inhibitors. The structure
of 3-HF-ene-4’-OMe is lack of phenolic hydroxyl groups, so this finding may indicate
that the core structure of flavonoid could lead the probe interact with IAPP but not block
IAPP assembly. In addition, a typical sigmoidal fibril growth curve with three
characteristic nucleation (lag phase), elongation, and equilibrium stages (steady phase)
was observed when we use 3-HF-ene-4’-OMe as a probe and it is similar to the one
measured by conventional ThT assay (Figure 4a). Even in the condition with performed
fibrils as seeds, probe signal still agrees with ThT signal showing the accelerated
aggregation (Figure 4b). Our data clearly showed that 3-HF-ene-4’-OMe could be used
as an alternative probe for IAPP aggregation kinetic studies.
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We next aim to investigate whether 3-HF-ene-4’-OMe can be utilized as a dye for
resveratrol inhibition studies. Resveratrol is one of the naturally occurring polyphenols
mostly present in red wine and grapes. According to previous studies, it was considered
as a potential therapeutic drug for several amyloidosis52. To IAPP, resveratrol is a
moderate inhibitor. It reduces the rate of amyloid formation by IAPP. Studies by using
atomic discrete molecular dynamic simulation also suggested that resveratrol binds to
IAPP monomers and stabilizes IAPP oligomers to retard IAPP self-association53.
However, this compound also was shown to interfere with ThT flurosence40, 51. In this
study, 5 molar equivalents of resveratrol were incubated with IAPP and the kinetics of
fibril formation profile was monitored by either ThT or 3-HF-ene-4’-OMe. As shown
in Figure 5A, ThT fluorescence intensity is greatly reduced when resveratrol is present.
Figure 4. (A) Kinetics of 32 μM IAPP aggregation and (B) kinetics of 32 μM IAPP aggregation with
20% seeds were monitored respectively by 32 μM 3-HF-ene-4’-OMe (blue) and 32 μM ThT (red)
assays. In order to conveniently compare the sigmoidal curves observed from these two assays, each
curve was normalized to its individual maximum fluorescence intensity. Reactions in triplicates were
performed in 10 mM Tris buffer (pH 7.4) at 25 oC. Seeds were prepared from preformed IAPP fibrils.
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Conversely, a clear sigmodal fibril growth curve with an elongated lag phase was
observed from 3-HF-ene-4’-OMe fluorescence suggesting resveratrol did completely
inhibit IAPP aggregation, although this final intensity is also reduced. Similar to the
previously research51, large amounts of IAPP fibrils in both samples are observed after
incubation for 125 hr (Figure 5B and 5C). The decrease of 3-HF-ene-4’-OMe
fluorescence intensity while mixing with resveratrol may be caused by the interference
of resveratrol with 3-HF-ene-4’-OMe fluorescence, or by the competition between 3-
HF-ene-4’-OMe and resveratrol to the same binding site on IAPP fibrils. To explain
the decline of 3-HF-ene-4’-OMe fluorescence intensity in inhibition study, 3-HF-ene-
4’-OMe in 40% DMSO was first incubated with different amount of resveratrol. The
intensity of 3-HF-ene-4’-OMe retains in the presence of resveratrol indicating that
resveratrol does not quench 3-HF-ene-4’-OMe fluorescence (data not shown). Next,
again we monitored IAPP aggregation by 3-HF-ene-4’-OMe fluorescence. After 96 hr,
resveratrol was added into the aggregated sample when fibril formation has achieved a
steady phase. The 3-HF-ene-4’-OMe fluorescence intensity dramatically decreased
after the addition of resveratrol, suggesting that the binding of 3-HF-ene-4’-OMe on
IAPP fibrils might be replaced by resveratrol (Figure S6). Although the binding of both
ThT and 3-HF-ene-4’-OMe to IAPP fibrils are interfered by resveratrol, 3-HF-ene-4’-
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Figure 5. 3-HF-ene-4’-OMe is suitable to be used in IAPP-Resveratrol inhibition study. (A) The
resveratrol inhibitory effect on 32 μM IAPP aggregation was monitored by 32 μM ThT (no inhibitor,
blue; with inhibitor, light blue) and 32 μM 3-HF-ene-4’-OMe (no inhibitor, red; with inhibitor, pink).
(B and C) TEM images of IAPP with 5 molar equivalents of resveratrol from ThT assay or 3-HF-ene-
4’-OMe assay after incubation of 125 hr. The scale bar represents 200 nm.
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OMe is still more suitable for resveratrol inhibition studies, because a clear sigmoidal
curve with elongated lag time was observed when using 3-HF-ene-4’-OMe as a probe.
To test whether 3-HF-ene-4’-OMe is specific for detection of IAPP fibrils, other
two protein amyloid aggregates formed by Aβ42 and hCT were also prepared in vitro
and the formation of amyloid fibrils was confirmed again by TEM (Figure S7). hCT
and IAPP are both belong to amylin protein family. Calcitonin is a 32 amino acid
polypeptide, like IAPP, there are two conserved cysteines in the N-terminal and the C-
terminal of calcitonin is also amidated. We chose these proteins in our examination
because they are similar to IAPP in peptide length and easy to form amyloid fibrils in
aqueous solution. Strikingly, the incubation of 3-HF-ene-4’-OMe with hCT fibrils did
not lead to obvious fluorescence enhancement (Figure 6). The fluorescence of 3-HF-
ene-4’-OMe was also lighted on upon binding to Aβ42 fibrils, but probe exhibited a
weaker signal and a maximum emission wavelength (λmax) which is about 70 nm
different to the one detected from IAPP fibrils. Owing to the solubility issue of Aβ42 in
Tris buffer, the pH value of Tris buffer for Aβ preparation was adjusted to 8.0, which
is different from the IAPP sample solution. We speculated that the observed different
emission profile could due to the acid-base interactions between amyloid and the probe.
To clarify this possibility, 3-HF-ene-4’-OMe was incubated with IAPP fibrils prepared
at the same pH values as Aβ42 (Figure S8). Similar emission profile was observed as
seen at pH 7.4 and the maximum emission wavelength still falls at 535 nm. The
selectivity of 3-HF-ene-4’-OMe fluorescence emission with IAPP fibrils is not affected
by this minor pH change. Different emission profile of 3-HF-ene-4’-OMe when mixing
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with aggregated IAPP and Aβ42 may due to the difference of the local environment of
Figure 6. 3-HF-ene-4’-OMe shows different spectra for three different kinds of amyloid fibrils.
Fluorescence emission spectra of 3-HF-ene-4’-OMe alone (black) and the spectra measured in the
presence of IAPP fibrils (orange), Aβ42 fibrils (blue), calcitonin fibrils (green), non-fibrillar Aβ42 (light
blue), and non-fibrillar hCT (yellow). The exiciation wavelength was set at 380 nm. All peptide fibrils
were prepared by incubation of peptide samples at 64 μM in microtubes with continuous shaking for 2.5
days at 25 oC. The fibril formation was then further confirmed by ThT and TEM.
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this probe in the amyloid binding pocket. 3-HF-ene-4’-OMe exhibits a shorter
emission wavelength when it is in a less polar environment (Figure S2E). Consequently,
we deduced that the fluorescence discrimination was due to environmental polarity and
the 3-HF-ene-4’-OMe binding site on Aβ42 fibrils is possibly relatively less polar than
IAPP fibrils.
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In order to assess the binding mode of 3-HF-ene-4’-OMe, a molecular docking
approach was carried out using the most commonly studied IAPP and Aβ42 fibril
structure model41, 42. The results showed that only a single binding site was found when
3-HF-ene-4’-OMe binds to Aβ42 fibrils (Figure 7A and Figure S9). 3-HF-ene-4’-OMe
is clamped inside Aβ42 fibrils mainly through π-π stacking interactions with the
imidazole ring of His14 and through hydrophobic interaction with Leu17, Leu34, and,
Ile32. We speculated this is why 3-HF-ene-4’-OMe gave a fluorescent emission at
lower wavelength, 475 nm, which represents a less polar environment. By contrast,
there are multiple binding sites found for 3-HF-ene-4’-OMe on IAPP fibrils. The
binding site with the lowest binding energy is identified inside the IAPP fibrils and the
probe would interact with several residues including Gln10, Ala12, Val32, and Ser34
(Figure 7B and Figure S9). However, the most favorable binding site was determined
on the IAPP surface around Val17 and Phe15. The water molecules surrounding on the
surface of IAPP fibrils can interact with 3-HF-ene-4’-OMe. This docking result
apparently agrees with the observed fluoresce spectrum (Figure 6).
Next, we further examined the potential of 3-HF-ene-4’-OMe in detection of
IAPP fibrils in vitro and in live cells. Due to a significant difference in the fluorescence
intensity of the free dye and fibrils bound dye, we were able to obtain confocal
microscopy images of the 3-HF-ene-4’-OMe stained fibrillar structures (Figure 8A).
From previous studies, it is known that the fluorescent dye containing hydroxyflavone
scaffold might be able to bind membrane and increase its quantum yield54. Therefore,
Figure 7. Docking results of 3-HF-ene-4’-OMe with (A) Aβ42 fibrils and (B) IAPP fibrils. The fibrils
are shown in cartoon mode and the binding site residues are shown in line with CPK color. 3-HF-ene-
4’-OMe is shown in stick mode and carbon atoms from 3-HF-ene-4’-OMe are colored in green, oxygen
atoms in red and hydrogen atoms from the hydroxyl group in white.
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we also used mouse pancreatic beta (MIN6) cells for fluoresce imaging. Mouse
pancreatic beta cell did not produce human IAPP, therefore we added preformed IAPP
fibrils to cells to mimic the conditions that IAPP formed amyloid outside the beta cells.
Before that, MIN6 cells were firstly treated with different concentrations of 3-HF-
ene-4’-OMe for 24 hr, 48 hr, and 72 hr to investigate whether 3-HF-ene-4’-OMe is
cytotoxic. The results showed that there was no significant effect of 3-HF-ene-4’-OMe
on cell viability of MIN6 cells in various concentrations and various incubated times
(Figure S10). Thereafter, MIN6 cells were incubated with IAPP fibrils for 1 hr and then
stained with 3-HF-ene-4’-OMe. The fluorescence images revealed that the intensity of
green fluorescence with IAPP fibrils was much greater than cells in accompanying with
3-HF-ene-4’-OMe, suggesting that 3-HF-ene-4’-OMe could detect IAPP fibrils in live
cells (Figure 8B and 8C). Although the probe still has a chance to bind cell membrane
then fluoresces, this molecule has shown this potential in detection of IAPP amyloid
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fibrils. We will continue to modify the probe structure using flavonoid scaffold.
Figure 8. 3-HF-ene-4’-OMe could detect IAPP fibrils. (A) Confocal microscopy images of 3-HF-ene-
4’-OMe stained IAPP amyloid fibrils. The transmitted light image (right panel) show the presence of
IAPP fibrils in targeted area. Fluorescence only can be detected in the area that fibrils are present. The
scale bar represents 10 μm (B) Fluorescence images of MIN6 Cells treated with IAPP fibrils (top left
panel), with 3-HF-ene-4’-OMe (top middle panel) and with both (top right panel). Transmitted light
images were shown in bottom panels. The scale bar represents 50 μm. (C) The graph shows a quantitative
analysis of fluorescence intensity collected from (B).
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In the past, several groups were also attempting to detect islet amyloid using
fluorescent dye. SYPRO orange was shown to bind IAPP amyloid fibrils with excitation
and emission maxima of 490 nm and 594 nm21. Our probe can absorb light around 380
nm and fluoresce at about 535 nm upon binding to IAPP fibrils. Besides ThT, these two
probes can become useful alternative to monitor IAPP aggregation when a putative
inhibitor may interfere ThT spectrum. Stevens and Bourgault groups both have utilized
the biarsenical dye, FlAsH, to detect the early stage of IAPP aggregation because FlAsH
can recognize tetracysteine motifs from IAPP N-terminal assembly19, 20. It would be
useful to detect IAPP oligomers using FlAsH. However, its application in cell imaging
have not been tested yet. In this study, the flavonoid scaffold was shown to be
successfully employed in the design of environmentally sensitive fluorescent probes for
the detection of IAPP amyloid fibrils. 3-HF-ene-4’-OMe gave the largest enhancement
of fluorescence intensity upon binding to IAPP fibrils. The conjugated double bonds
system seem to make the behavior of the probes as molecular rotor dyes which are
sensitive to the environment, as well as to viscosity. We speculate that the
conformational flexibility of 3-HF-ene-4’-OMe is restricted when it binds to the IAPP
fibrils and that could significantly contribute to the emission enhancement. In addition,
3-HF-ene-4’-OMe was more suitable to be used in resveratrol inhibition assay
compared with conventionally used ThT. We also demonstrated that non-cytotoxic 3-
HF-ene-4’-OMe was able to detect IAPP aggregates in live cells, but further
modification is needed to reduce nonspecific binding to cell membrane. Most
importantly, 3-HF-ene-4’-OMe showed selectivity for detection of islet amyloid as it
did not give significant fluorescent signal upon mixing with hCT fibrils or displayed a
weaker fluorescent emission with a large blue-shift (70 nm) upon binding to Aβ42 fibrils.
The different emission profiles, as revealed by molecular docking calculation, might
due to the polarity of the binding environment of 3-HF-ene-4’-OMe. Although it is not
able to completely distinguish IAPP and Aβ42 fibrils in practical, we believe that use of
flavonoid as a molecule scaffold holds potential to develop a specific dye for detection
of islet amyloid in T2D.
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Supporting Information
Characterization data of all flavonoid-based small molecules and all 1H and 13C NMR
data.
Additional experimental details, fluorescence spectra, CD spectra, TEM images,
docking analysis and data for cytotoxicity tests.
Author contribution
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W. Chen, S. Ma, Y.Chen, Y. Chao, and A. Chan conducted the experiments. L. Tu and
W. Liu designed the concept, analyzed the data, and wrote the manucript.
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Acknowledgments
This work is financially supported by the Ministry of Science and Technology, Taiwan
(project numbers: MOST105-2113-M-030-010-MY2, MOST105-2113-M-003-014-
MY2, and MOST106-2320-B-006-058-MY3), the Department of Chemistry, Fu Jen
Catholic University, the Department of Chemistry, National Taiwan Normal University
and the Department of Pharmacology, College of Medicine, National Cheng Kung
University. Special thanks to Prof. Robert Tycko and Prof. Daniel P. Raleigh who
kindly provide us the model of IAPP fibrils. We also thank Mr. Yu-Jen Chang for
providing technical support in taking confocal fluorescence images.
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For Table of Contents Use Only
An environmentally sensitive non-cytotoxic probe with flavonoid structural feature is
used for detection of IAPP fibrils.
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