Development of bifunctional stilbene derivatives for targeting and modulating metal-amyloid-β species

Article abstract of DOI:10.1021/ic2012205

Amyloid-β (Aβ) peptides and their metal-associated aggregated states have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the etiology of AD remains uncertain, understanding the role of metal-Aβ species could provide insights int

Full text of DOI:10.1021/ic2012205

ARTICLE
pubs.acs.org/IC
Development of Bifunctional Stilbene Derivatives for Targeting and
Modulating Metal-Amyloid-β Species
Joseph J. Braymer,† Jung-Suk Choi,‡ Alaina S. DeToma,† Chen Wang,† Kisoo Nam,‡ Jeffrey W. Kampf,†
Ayyalusamy Ramamoorthy,†,§ and Mi Hee Lim*,†,‡
†Department of Chemistry, ‡Life Sciences Institute, and §Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States
S Supporting Information
b
ABSTRACT: Amyloid-β (Aβ) peptides and their metal-asso-
ciated aggregated states have been implicated in the pathogen-
esis of Alzheimer’s disease (AD). Although the etiology of AD
remains uncertain, understanding the role of metal-Aβ species
could provide insights into the onset and development of the
disease. To unravel this, bifunctional small molecules that can
specifically target and modulate metal-Aβ species have been
developed, which could serve as suitable chemical tools for
investigating metal-Aβ-associated events in AD. Through a rational structure-based design principle involving the incorporation of a
metal binding site into the structure of an Aβ interacting molecule, we devised stilbene derivatives (L1-a and L1-b) and
demonstrated their reactivity toward metal-Aβ species. In particular, the dual functions of compounds with different structural
features (e.g., with or without a dimethylamino group) were explored by UVꢀvis, X-ray crystallography, high-resolution 2D NMR,
and docking studies. Enhanced bifunctionality of compounds provided greater effects on metal-induced Aβ aggregation and
neurotoxicity in vitro and in living cells. Mechanistic investigations of the reaction of L1-a and L1-b with Zn2+-Aβ species by UVꢀvis
and 2D NMR suggest that metal chelation with ligand and/or metalꢀligand interaction with the Aβ peptide may be driving factors
for the observed modulation of metal-Aβ aggregation pathways. Overall, the studies presented herein demonstrate the importance
of a structure-interaction-reactivity relationship for designing small molecules to target metal-Aβ species allowing for the
modulation of metal-induced Aβ reactivity and neurotoxicity.
’ INTRODUCTION
indicate that metal ions are able to influence pathways of Aβ
aggregation and neurotoxicity, but the molecular mechanisms of
metal-Aβ-associated events are not fully understood.
Alzheimer’s disease (AD) is a serious form of dementia that
causes deterioration of cognitive function in over 5.3 million
people in the United States.1,2 One current hypothesis of the
underlying causes of AD involves the accumulation of plaques
composed of aggregated amyloid-β (Aβ) peptides in the brain.2ꢀ5
Cleavage of the transmembrane amyloid precursor protein (APP)
by β- and γ-secretases results in the production of Aβ, which
contains 38ꢀ43 amino acid residues. Through hydrophobic
interactions, Aβ monomers in solution can self-assemble to form
aggregated states (e.g., oligomers, protofibrils, and fibrils).2,5ꢀ7
While the fibril-containing plaques can be identified in the brain of
AD patients, how these peptides and their possible aggregation
states are connected to AD is not completely understood. Current
evidence suggests that Aβ monomers are relatively nontoxic while
aggregated Aβ species (in particular, soluble low molecular weight
(MW) Aβ species, including dimers) are shown to be toxic to
neuronal cells and therefore may lead to AD neuropathogenesis;
however, their relation to AD development remains obscure.2ꢀ4,6
In Aβ plaques, concentrated areas of metal ions such as Fe, Cu,
and Zn have been observed.5,8ꢀ15 These metal ions are known to
facilitate Aβ aggregation upon binding to the Aβ peptide (via three
histidine residues, H6, H13, and H14).5,10,11,13ꢀ20 Additionally,
reactive oxygen species (ROS) can be generated by the redox
cycling of Cu bound to Aβ, which may be responsible for the
neurotoxicity of the disease.2,10ꢀ17,21ꢀ25 These characteristics
Previous studies have shown that metal chelators such as
2,20,200,2000-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA),
bathocuproine, desferrioxamine, and clioquinol (CQ) are
capable of reducing metal-mediated Aβ aggregation and
neurotoxicity.2,10ꢀ12,15ꢀ17,21,22,24ꢀ32 Use of these metal chelat-
ing agents to understand the details of metal-Aβ chemistry and
biology is limited because they may not be specific for metal-Aβ
species in vivo. This may be due to the lack of Aβ interaction
functionality that could hinder metal chelators from targeting
metal ions associated with Aβ species in heterogeneous environ-
ments. There have been recent efforts to rationally design small
molecules that can target metal-Aβ species and modulate their
aggregation pathways by appending metal chelation sites onto
known Aβ imaging agents.5,15,33ꢀ39 Some of these compounds
have truncated Aβ-interaction moieties and/or nonspecific
metal binding properties, which might hamper their capability to
target and interact specifically with metal-Aβ species in vitro and
in vivo. To improve this, we have constructed bifunctional
small molecules via the direct incorporation of a metal binding
site into the scaffold of previously reported Aβ interacting
Received: June 7, 2011
Published: September 28, 2011
r
2011 American Chemical Society
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The metal ion selectivity of L1-b was determined in EtOH with metal
chloride salts (MgCl2, CaCl2, MnCl2, FeCl2, FeCl3, CoCl2, NiCl2, and
ZnCl2) using UVꢀvis according to the previously published report.38 All
stock solutions of metal salts were prepared in EtOH. Studies employing
FeCl2 were conducted anaerobically. The selectivity values (AM/ACu
,
Supporting Information, Figure S2) were obtained by comparing the
absorbance of the solution at 525 nm before and after the addition of
CuCl2. Absorption values were normalized relative to that of L1-b and
CuCl2 at this wavelength (525 nm) to provide minimal interference
between absorption bands. NMR and IR spectra of small molecules were
recordedona 400 MHz Varian NMRspectrometer and ona Perkin-Elmer
Spectrum BX FT-IR instrument, respectively. The NMR investigations of
the interaction of compounds with Aβ were conducted using a 900 MHz
Bruker Avance spectrometer (Michigan State University). Optical spectra
were collected on an Agilent 8453 UVꢀvis spectrophotometer. Mass
spectrometric measurements were performed using a Micromass LCT
Electrospray Time-of-Flight mass spectrometer. A SpectraMax M5 micro-
plate reader (Molecular Devices) was used for measurements of absor-
bance for the cell viability assay and fluorescence for the H2O2 assay.
[Zn(L1-a)Cl2]. Colorless plate crystals were grown by vapor diffu-
sion of diethyl ether (Et2O) into an acetonitrile (CH3CN) solution of
commercially available L1-a (5.0 mg, 27 μmol) and ZnCl2 (3.7 mg, 27
μmol) at room temperature over 2 days. The crystals were isolated,
washed with Et2O three times, and dried in vacuo (7.1 mg, 22 μmol,
81%). FTIR (KBr, cmꢀ1): 3445 (w, br), 3086 (vw, sh), 3085 (vw), 3064
(w), 3024 (w), 1620 (vw, sh), 1594 (s), 1563 (w), 1494 (s), 1476 (m,
sh), 1456 (m, sh), 1445 (s), 1421 (vw, sh), 1385 (vw, sh), 1368 (m),
1339 (vw), 1303 (w), 1280 (w), 1236 (w), 1195 (w), 1186 (vw, sh),
1158 (w), 1111 (m), 1076 (vw), 1050 (w), 1024 (s), 1000 (vw, sh), 976
(w), 959 (w), 916 (m), 840 (vw), 778 (vs), 740 (s), 684 (s), 650 (m),
616 (vw, sh), 567 (w), 535 (m), 481 (vw), 462 (vw, sh), 413 (w). 1H
NMR (400 MHz, CD3CN): δ = 7.54 (t, J = 8 Hz, 1H), 7.61 (t, J = 8 Hz,
2H), 7.80 (d, J = 8 Hz, 2H), 7.94 (t, J = 8 Hz, 1H), 8.16 (d, J = 8 Hz, 1H),
8.36 (t, J = 8 Hz, 1H), 8.85 (d, J = 8 Hz, 1H), 9.03 (s, 1H). ESI(+)MS
(m/z): [M-Cl]+ calcd for C12H10ClN2Zn, 281.0, found 280.9. Anal.
Calcd for C12H10Cl2N2Zn: C, 45.25; H, 3.16; N, 8.80. Found: C, 45.24;
H, 3.17; N, 8.70. UVꢀvis [CH3CN; λ/nm (ε ꢁ 104, Mꢀ1 cmꢀ1)]: 235
(1.1), 241 (1.2), 328 (1.9), 341 (1.8).
Figure 1. Rational structure-based design strategy for bifunctional small
molecules. Incorporation of a metal binding site into an Aβ interacting
molecule is the design basis for L1-a and L1-b. L1-a = N-(pyridin-2-
ylmethylene)aniline; L1-b = N1,N1-dimethyl-N4-(pyridin-2-ylmethylene)-
benzene-1,4-diamine; S0 = (E)-1,2-diphenylethene; S1 = N,N-dimethyl-
4-(2-phenylethenyl)benzamine. *I = 123I or 125I.
molecules without major structural modification (the rational
structure-based design).33,34,37ꢀ39 Our first-generation com-
pound (L1-b) was devised by applying this synthetic design
strategy employing the structure of an Aβ imaging agent,
p-123/125I-stilbene (Figure 1).37 Our small molecule showed
bifunctionality (metal chelation and Aβ interaction) as well as
control of Cu2+-induced Aβ aggregation processes and neuro-
toxicity in vitro and in living cells.37 In addition, our recent
studies using other bifunctional small molecules suggest that
chemical structural moieties such as the dimethylamino func-
tionality are important to tune their reactivity toward metal-
induced Aβ aggregation and neurotoxicity.38 To understand a
structure-interaction-reactivity relationship of the stilbene frame-
work, we report herein the studies of metal binding and Aβ
interaction of L1-a (the compound that does not contain a
dimethylamino group, Figure 1) and L1-b, the effects on the
modulation of metal (Cu2+ or Zn2+)-mediated Aβ aggregation
and Cu-Aβ-induced ROS production in vitro, as well as the
influence on metal-Aβ neurotoxicity in living cells. Furthermore,
Aβ interaction of L1-a and L1-b was compared to that of S0 and
S1 that have the stilbene scaffold without a metal coordination
site (a structure-interaction relationship). Our current findings
demonstrate that slight difference in chemical structures of small
molecules is able to influence their Aβ interaction, which can
improve reactivity toward metal-Aβ species.
[Zn(L1-b)Cl2]. Orange needle crystals were grown by vapor diffu-
sion of Et2O into a CH3CN solution of L1-b (5.0 mg, 22 μmol) and
ZnCl2 (3.0 mg, 22 μmol) at room temperature overnight. The crystals
were isolated, washed with Et2O three times, and dried in vacuo (6.8 mg,
19 μmol, 86%). FTIR (KBr, cmꢀ1): 3440 (w, br), 3077 (vw), 3027 (vw),
3008 (vw), 2974 (vw), 2902 (w), 2863 (vw, sh), 2822 (vw), 1622 (s),
1599 (vs), 1583 (s, sh), 1546 (vs), 1471 (s), 1447 (s), 1415 (vw, sh),
1371 (s), 1328 (vw), 1321 (vw), 1300 (w), 1284 (w), 1250 (w), 1231
(w), 1215 (vw, sh), 1188 (s), 1173 (s), 1159 (s, sh), 1123 (vw, sh), 1105
(vw), 1067 (w), 1050 (vw), 1024 (vw), 1000 (vw), 946 (w), 923 (vw,
sh), 842 (vw, sh), 810 (s), 771 (m), 744 (w), 644 (vw, sh), 642 (w), 551
(vw), 529 (m), 430 (vw), 411 (w). 1H NMR (400 MHz, CD3CN): δ =
3.06 (s, 6H), 6.86 (d, J = 8 Hz, 2H), 7.71 (d, J = 8 Hz, 2H), 7.80 (t, J = 4
Hz, 1H), 8.00 (d, J = 8 Hz, 1H), 8.27 (t, J = 8 Hz, 1H), 8.75 (d, J = 4 Hz,
1H), 8.86 (s, 1H). ESI(+)MS (m/z): [M+H]+ Calcd for
C14H16Cl2N3Zn, 360.0, found 360.0. Anal. Calcd for C14H15Cl2N3Zn:
C, 46.50; H, 4.18; N, 11.62. Found: C, 46.44; H, 4.14; N, 11.57. UVꢀvis
[CH3CN; λ/nm (ε ꢁ 104, Mꢀ1 cmꢀ1)]: 277 (2.0), 477 (3.3).
’ EXPERIMENTAL SECTION
Materials and Procedures. All reagents were purchased from
commercial suppliers and used as received unless stated otherwise. The
compound L1-b was synthesized by the previous method.37 Aβ1ꢀ40
peptide (free acid terminal) was purchased from EZBiolab
(>95% purity, Carmel, IN) or rPeptide (>97% purity, Athens, GA).
The amino acid sequence for the Aβ1ꢀ40 peptide is DAEFRHDS-
GYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV. The hydrogen
peroxide (H2O2) assay using horseradish peroxidase (HRP, Sigma,
St. Louis, MO) and Amplex Red (AnaSpec, Inc., Fremont, CA)
was performed following previously published procedures.23,32,37,38,40
X-ray Crystallographic Study. Single crystals suitable for data
collection were mounted on a Bruker SMART APEX CCD-based X-ray
diffractometer equipped with a low temperature device and fine focus
Mo-target X-ray tube (λ = 0.71073 Å) operated at 1500 W power
(50 kV, 30 mA). The X-ray intensities were measured at 85(2) K.
Empirical absorption corrections were calculated with the SADABS or
TWINABS program.41 Structures were solved and refined with the
SAINTPLUS and SHELXTL software packages.42,43 All non-hydrogen
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ARTICLE
atoms were refined anisotropically. Hydrogen atoms were assigned in
idealized positions, and each was given a thermal parameter equivalent to
1.2 times the thermal parameter of the atom to which it was attached. The
structure solution was checked for higher symmetry with PLATON.44
The highest electron density in the final difference Fourier maps for
[Zn(L1-a)Cl2] and [Zn(L1-b)Cl2] were 1.181 and 1.112 e/Å3, respec-
tively, in the vicinity of the Zn center. In the structure of [Zn(L1-b)Cl2],
indexing performed using the CELL_NOW program45 indicated that the
crystal was a two-component, nonmerohedral twin. The data were
processed with TWINABS and corrected for absorption.41 The domains
are related by a rotation of 179.8 degrees about the direct and reciprocal
[0 1 0] axis. For this refinement, single reflections from component one
as well as composite reflections containing a contribution from this
component were used. Merging of the data was performed in TWINABS,
and an HKLF 5 format file was used for refinement.
E: 118 ꢁ 88 ꢁ 54: 10.202, 9.338, ꢀ4.058. In AutoDock4, each
compound was evaluated with 256 Lamarckian genetic algorithm local
searches (GALS) by a population size of 150 and 2.5 million energy
evaluations. Additional default parameters remained unchanged unless
otherwise specified. Conformations of compounds were analyzed by
clusters in AutoDockTools4. The highest frequency and lowest energy
conformation was chosen and portrayed docked to Aβ using Pymol.
Amyloid-β (Aβ) Peptide Experiments. Aβ1ꢀ40 peptide (1 mg,
EZBiolab) was purchased, dissolved with ammonium hydroxide (1% v/v,
aq), aliquoted to 10 samples, lyophilized, and stored at ꢀ80 °C. The
stock solution (ca. 200 μM) for the reactions was made by redissolving
Aβ with NH4OH (1% v/v, aq, 10 μL) followed by dilution with ddH2O
or a buffer solution. Preparation of all Aβ samples followed the
previously reported procedures.37ꢀ39,55ꢀ58 The ddH2O and buffer
solutions used for Aβ experiments were treated with Chelex overnight
to remove traces of metal ions. For the inhibition study, solutions of
samples contained Aβ (25 μM), a metal chloride salt (CuCl2 or ZnCl2,
25 μM), and a compound (50 μM, stock solutions in DMSO, 1% v/v
final DMSO concentration). The solutions were incubated for 24 h at
37 °C with constant agitation and analyzed by transmission electron
microscopy (TEM) and Western blotting using an anti-Aβ antibody
(6E10). For the disaggregation study, Aβ (25 μM) and a metal chloride
salt (CuCl2 or ZnCl2, 25 μM) were incubated for 24 h at 37 °C with
constant agitation. Afterward, a compound (50 μM, stock solutions in
DMSO, 1% v/v final DMSO concentration) was added and incubated
for 24 h at 37 °C with constant agitation. Buffered solutions (20 μM
HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid),
pH 6.6 (for CuCl2) or pH 7.4 (for ZnCl2), 150 μM NaCl) were used
for both studies.
Transmission Electron Microscopy (TEM). Samples for TEM
were prepared by following the previously reported methods.37ꢀ39,55ꢀ58
Glow-discharged grids (Formar/Carbon 300-mesh, Electron Micro-
scopy Sciences) were treated with Aβ aggregated samples (5 μL, 25 μM)
for 2 min at room temperature. Excess sample was removed using filter
paper followed by washing twice with ddH2O. Each grid incubated
with uranyl acetate (1% w/v, ddH2O, 5 μL, 1 min) was blotted and dried
for 15 min at room temperature. Images from each sample were taken by
a Philips CM-100 transmission electron microscope (80 kV, 25,000ꢁ
magnification).
Native Gel Electrophoresis and Western Blotting. The
reactions described above (Aβ Peptide Experiments) were visualized
by native gel electrophoresis followed by Western blotting using an
anti-Aβ antibody (6E10).37ꢀ39,57,58 Each sample (10 μL, 25 μM) was
separated on a 10ꢀ20% gradient Tris-tricine gel (Invitrogen). The gel
was transferred onto a nitrocellulose membrane, blocked with bovine
serum albumin (BSA, 3% w/v) in Tris-buffered saline (TBS) containing
0.1% Tween-20 (TBS-T) for 3 h at ambient temperature, and then
incubated with an anti-Aβ antibody (6E10, 1:2,000) in 2% BSA in TBS-
T for 3 h at ambient temperature. The membrane was probed with the
horseradish peroxidase-conjugated goat antimouse antibody (1:10,000)
in 2% BSA in TBS-T for 1 h at ambient temperature. The Thermo
Scientific Supersignal West Pico Chemiluminescent Substrate was used
to visualize protein bands.
Two-Dimensional (2D) 1H-15N Transverse Relaxation Op-
timized Spectroscopy (TROSY)-Heteronuclear Single Quan-
tum Correlation (HSQC) NMR Measurements. The 15N-labeled
Aβ1ꢀ40 was purchased from rPeptide and stored at ꢀ80 °C. Aβ (ca. 0.25
mg) was dissolved into a buffered solution containing sodium dodecyl-
d25 sulfate (SDS-d25, 200 mM), sodium phosphate buffer (20 mM, pH
7.3), and D2O (7%, v/v) and was briefly vortexed.37ꢀ39 The peptide
solution was transferred to a Shigemi NMR tube. After acquiring spectra
of the peptide, about 10 equiv (stock solutions: 5 mM in above buffer
solution) of either L1-a or L1-b were added to the NMR samples of Aβ
(ca. 190 μM Aβ). Control samples were conducted under similar
conditions with the addition of 2.28 μL of either neat dimethyl
sulfoxide-d6 (DMSO-d6) or a 50 mM DMSO-d6 stock solution of S0
or S1 to a 300 μL solution containing 190 μM Aβ.46 For NMR studies
with Zn2+, about 1.0 equiv of ZnCl2 (0.57 μL of 100 mM stock solution
in D2O) was added into the peptide solution followed by treatment with
10 equiv of L1-a or L1-b. For the [Aβ + ZnCl2 + L1-a or L1-b] solution,
no further spectral changes were observed after 1 h of incubation. For
1
NMR studies, the TROSY version of 2D H-15N HSQC spectra were
recorded on the 900 MHz Bruker Avance NMR spectrometer (equipped
with a TCIcryoprobeaccessory) with 14.3kHz and 1.7 kHz spectralwidth
1
and 2048 and 128 complex data points in the H and 15N dimension,
respectively, 4 scans for each t1 experiment and 1.5 s recycle delay; each
spectrum took 10 min for completion. The water 1H peak was referenced
to 4.78 ppm at 25 °C. 1H-15N HSQC peaks were assigned by comparison
oftheobservedchemical shift valueswiththose reportedin the literature.47
1
Combined H and 15N 2D chemicals shifts (ΔδNꢀH) were calculated
from eq 1.48ꢀ50 The 2D data were processed using Topspin software
(version 2.1 from Bruker) and analyzed with Sparky (version 3.112).
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
2
ðΔδHÞ þ ð0:2ðΔδNÞÞ
ΔδN-H
¼
ð1Þ
2
Docking Studies of Compounds with AutoDock4. Docking
of L1-a, L1-b, S0, and S1 with the Aβ monomer was determined using
the computational package AutoDock4 and AutoDockTools4 following
previous reports.38,51ꢀ53 The optimized structures of compounds were
generated for AutoDockTools4 by the MMFF94 energy minimization in
ChemBio3D Ultra 12.0. Ten peptide conformations from a previously
determined NMR structure of Aβ1ꢀ40 monomer in SDS micelles (PDB
1BA4) were employed.54 Of these conformations, five were in agree-
ment with the NMR data and are depicted in Figure 4 and Supporting
Information, Figure S4. The structural files of compounds and peptides
were transferred to AutoDockTools4 and prepared for AutoGrid4 using
a grid volume encompassing the entire peptide with 0.375 Å spacing:
[Conformation: box size (Å): center (x,y,z)] A: 126 ꢁ 84 ꢁ 56: 7.663,
7.889, ꢀ8.706; B: 126 ꢁ 72 ꢁ 66: 0.194, ꢀ6.01, ꢀ7.711; C: 126 ꢁ 72 ꢁ
58: 5.665, ꢀ5.79, ꢀ8.124; D: 126 ꢁ 72 ꢁ 76: 5.432, ꢀ4.614, ꢀ12.983;
Cytotoxicity (MTT Assay). Two human neuroblastoma SK-N-
BE(2)-M17 (M17) and SK-N-AS (AS) cell lines were purchased from
the American Type Culture Collection (ATCC). The M17 and AS cell
lines were maintained in [1:1 Minimum Essential Media (MEM) and
Ham’s F12K Kaighn’s Modification Media (F12K)] and [Dulbecco’s
Modified Eagle Medium (DMEM)] (GIBCO), respectively, containing
10% (v/v) fetal bovine serum (FBS, Atlanta Biologicals), 100 U/mL
penicillin, and 100 mg/mL streptomycin (Invitrogen). The cells were
grown at 37 °C in a humidified atmosphere of 5% CO2. For the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
Sigma-Aldrich) assay, M17/AS cells were seeded in a 96 well plate
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(for cytotoxicity studies of compounds without and with metal ions,
16,000/8,000 and 10,000/5,000 cells (in 100 μL per well) for 24 and
72 h experiments, respectively; for the investigations of regulating metal-
Aβ neurotoxicity by compounds, 16,000 M17 cells (in 100 μL per
well)).37,38,58 After 24 h, cells were treated with [compounds with or
without metal ions] and [Aβ, CuCl2 or ZnCl2, and/or compounds]. The
final concentration of DMSO in each well was fixed to 1%. Then, the
cells were incubated for 24 or 72 h at 37 °C. After the incubation, the
cells were treated with 25 μL MTT (5 mg/mL in PBS) for 4 h at 37 °C
and then were lysed in a buffered solution containing N,N-dimethylfor-
mamide (pH 4.5, 50% (aq, v/v)) and SDS (20% (w/v)) overnight at
room temperature in the dark. The absorbance (A600) was measured
using a microplate reader.
Table 1. Summary of Optical Resultsa
spectral properties (nm)
unboundb
280, 323
CuCl2
297
ZnCl2
crystalsc
b
b
compound
L1-a
283, 325
[Zn(L1-a)Cl2]
235, 241, 328, 341
[Zn(L1-b)Cl2]
277, 475
L1-b
316, 396
318, 445
403, 484
408,d 473d
a The optical spectra are depicted in Supporting Information, Figure S1.
b Measurements were performed in EtOH at room temperature
following 10 min incubation. Ratio of metal to ligand was 1:1
unless otherwise noted. c Optical data were obtained in CH3CN.
d [ZnCl2]/[L1-b] = 10:1.
’ RESULTS AND DISCUSSION
Design Consideration and Preparation of Bifunctional
Small Molecules for Targeting and Modulating Metal-Aβ
Species. To elucidate the mechanisms of metal-induced Aβ
aggregation and neurotoxicity in AD, chemical tools that can
specifically target and regulate metal-Aβ species are needed. We
have designed bifunctional chemical reagents that contain a
metal binding site in the structure of Aβ interaction
molecules.33,34,37ꢀ39 In particular, we have focused on the
incorporation of a metal chelation moiety into the stilbene
framework (Aβ imaging agent) for bifunctionality (metal chela-
tion and Aβ interaction) (Figure 1).
Recently, we reported that L1-b, in which two nitrogen donor
atoms were introduced into the stilbene scaffold for metal
binding (Figure 1), exhibited bifunctionality and an ability to
modulate Cu2+-induced Aβ events such as aggregation and
neurotoxicity in vitro and in living cells.37 Moreover, amine
derivatives of L1-a and L1-b have been prepared for this purpose
indicating promising applications in biological systems.38 From
this and other studies, the dimethylamino functionality in the
small molecules has been suggested to be an important structural
moiety for Aβ interaction.38,59,60 To understand the structure-
interaction-reactivity relationship, L1-a (that does not have a
dimethylamino group) was studied along with L1-b (vide infra).
The compounds L1-a (commercially available) and L1-b can be
synthesized in high yield (>90%) through Schiff base condensa-
tion of an aldehyde and primary amine following previously
established methods.37,61
Metal Binding Properties of L1-a and L1-b. Following our
rational structure-based design principle, two nitrogen atom
donors were introduced into the stilbene framework to generate
a metal chelation site (Figure 1). To elucidate metal binding
properties of our compounds, the reactions of the compounds
with metal chloride salts in solution were evaluated using
UVꢀvis and the metal complexes [Zn(L1-a)Cl2] and [Zn(L1-
b)Cl2] were isolated and characterized by X-ray crystallography.
First, metal coordination was observed by the shifts in the optical
bands of our compounds upon addition of CuCl2 or ZnCl2 in
EtOH (Table 1 and Supporting Information, Figure S1). Modest
changes in the absorption spectra were shown for L1-a with 1
equiv of CuCl2 or ZnCl2 whereas both metal chloride salts
caused noticeable changes in the spectra of L1-b. The ligand L1-
b interacted with Cu2+ based on the new absorption bands at
about 450 nm. The observed peaks from 470ꢀ480 nm that grew
in upon the addition of ZnCl2 indicated that L1-b could bind
to Zn2+ as well. Furthermore, L1-b having the dimethylamino
group presented more apparent changes in the optical spectra
Figure 2. ORTEP diagrams of [Zn(L1-a)Cl2] (left) and [Zn(L1-
b)Cl2] (right) showing 50% probability thermal ellipsoids. X-ray crystal-
lographic data are summarized in Supporting Information, Table S1.
in the presence of metal chloride salts, compared to L1-a
(Table 1 and Supporting Information, Figure S1). The optical
features, indicative of the Zn2+-L1-a or Zn2+-L1-b interaction,
were also shown in the solution of isolated metal complexes
[Zn(L1-a)Cl2] or [Zn(L1-b)Cl2] (Figure 2, vide infra) in
CH3CN (Table 1 and Supporting Information, Figure S1). Thus,
UVꢀvis studies described above suggest metal binding of L1-a and
L1-b in solution. Additionally, the investigation for the metal
selectivity of L1-b using UVꢀvis indicated that this ligand was
relatively selective for Cu2+ over other metal ions such as Mg2+,
Ca2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, and Zn2+ in EtOH (Supporting
Information, Figure S2; [L1-b] = 40 μM, [M2+/3+] = 40 μM, at
room temperature). Because of the limited stability of these imine
compounds in aqueous solutions,62 further metal binding proper-
ties such as pH-variable solution speciation36,38 were not studied.
To further understand the metal binding properties, X-ray
quality single crystals of the metal complexes [Zn(L1-a)Cl2] and
[Zn(L1-b)Cl2] (Figure 2) were grown by slow diffusion of Et2O
into a solution of ligand and ZnCl2 (1:1) in CH3CN. Crystal-
lographic data and selected bond lengths and angles are
summarized in Supporting Information, Tables S1 and S2,
respectively. As depicted in Figure 2, the structures present that
two nitrogen atoms of the ligands are responsible for metal
chelation. The overall conformation of the imine ligands L1-a
and L1-b of the Zn complexes exhibited planarity in the solid
state, with distorted tetrahedral geometry around the metal
center. The bond lengths and angles for the Zn complexes did
not vary significantly depending on the presence of the dim-
ethylamino group; however, the slight difference in bond angles
and the ZnꢀN1 bond length between [Zn(L1-a)Cl2] and
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[Zn(L1-b)Cl2] was indicated (Table 2; e.g., ZnꢀN1 (Å) =
2.060(2) versus 2.0446(17), respectively). A Zn2+ complex with
a structure similar to L1-b where the dimethylamino group is
substituted with an iodine atom has been reported.63 The
molecule binds to ZnCl2 in a 1:1 ratio to form a slightly distorted
tetrahedral metal center with bond lengths and angles that are
similar to those of [Zn(L1-a)Cl2] and [Zn(L1-b)Cl2]. This
suggests that neither an electron withdrawing nor electron
donating substituent in the para position of the phenyl ring
significantly alters bond lengths or angles. Overall, X-ray crystal
structure determination confirmed metal chelation by L1-a and
L1-b via two nitrogen donor atoms.
Aβ Interaction with L1-a, L1-b, S0, and S1. To establish the
extent of interaction of L1-a and L1-b with Aβ in the absence of
metal ions, which along with metal chelation affords bifunctionality,
1
high-resolution 2D H-15N TROSY-HSQC NMR experiments
were performed. SDS-d25 was employed in NMR studies of Aβ
to solubilize the peptide and prevent its aggregation.37ꢀ39,47,54,64
When bound to SDS micelles, the Aβ monomer adopts two α-
helical segments involving residues Q15ꢀN27 and G29ꢀM35.47,54
Helicity has also been observed in solution and when in contact with
other biological molecules and has been suggested to precede the
aggregation pathways of Aβ leading to proposed neurotoxic
oligomers.4,6,65ꢀ68 Thus, this system can provide an adequate
model for determining the ability of our compounds to interact
with and target a biologically relevant Aβ conformation. As a
comparison of the interaction of our bifunctional molecules with
Aβ, S0 and S1 that do not contain a metal binding site (Figure 1)
were additionally investigated.
Changes in chemical shifts of Aβ were observed upon the
addition of L1-a and L1-b (Figure 3, ca. 10 equiv, SDS-d25
(200 mM), sodium phosphate buffer (20 mM, pH 7.3), and
D2O (7%, v/v)), suggesting direct Aβ interaction with our small
molecules. This experiment was carried out in the absence of
dimethylsulfoxide (DMSO), which is different from the previous
Table 2. Selected Bond Lengths (Å) and Angles (deg)a
[Zn(L1-a)Cl2]
[Zn(L1-b)Cl2]
Zn1ꢀN1
2.060(2)
2.081(2)
2.2280(6)
2.1998(6)
81.20(8)
107.72(6)
119.14(6)
111.40(6)
117.10(6)
115.48(2)
2.0446(17)
2.0828(18)
2.2162(8)
2.2056(8)
82.24(7)
Zn1ꢀN2
Zn1ꢀCl1
Zn1ꢀCl2
N1ꢀZn1ꢀN2
N1ꢀZn1ꢀCl1
N1ꢀZn1ꢀCl2
N2ꢀZn1ꢀCl1
N2ꢀZn1ꢀCl2
Cl1ꢀZn1ꢀCl2
110.16(6)
117.42(6)
110.06(5)
120.03(6)
113.18(2)
a Numbers in parentheses are estimated standard deviations in the last
significant figures. Atoms are labeled as indicated in Figure 2.
Figure 3. 2D 1H-15N TROSY-HSQC NMR study of the interaction of small molecules with 15N-labeled Aβ1ꢀ40 (900 MHz). (a) NMR spectra of the Aβ
peptide (shown in black; ca. 308 μM in SDS-d25 (200 mM), sodium phosphate buffer (20 mM, pH 7.3, 7% D2O (v/v), 25 °C)) and the peptide with
the addition of 10 equiv of L1-a or L1-b shown in blue (full spectrum of L1-b is shown on the left). Green arrows indicate the direction of resonance shift.
(b) Changes in combined 1H and 15N chemical shifts calculated from eq 1 upon the addition of 10 equiv of compounds to Aβ as a function of peptide
sequence. * Denotes absent or overlapped signals. The amino acid sequence for the Aβ1ꢀ40 peptide is DAEFRHDSGYEVHHQKLVFFAED-
VGSNKGAIIGLMVGGVV.
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Figure 4. Possible conformations of L1-a (light blue), L1-b (yellow), S0 (orange), and S1 (magenta) docked with Aβ1ꢀ40 (PDB 1BA4) by AutoDock4.
The cartoon (left) and surface (right) versions of the peptide indicate interactions with compounds. The N-terminal and inner helix regions of Aβ
(D1ꢀN27) are highlighted in color. Hydrogen bonding is indicated with dashed lines (1.9ꢀ2.2 Å).
report regarding the interaction of L1-b with Aβ monomer.37 Both
compounds exerted aninfluence mainly onthe N-terminal portion
of the peptide (E3ꢀQ15) where shifting occurred the most at
residues E11 and H13. To understand the effect of the dimethy-
lamino functionality on Aβ interaction (L1-a versus L1-b), the
chemical shift patterns were compared. While L1-b interacted
more broadly through the N-terminal region and also with the
inner helix of the peptide (Q15ꢀN27), L1-a had fewer contacts in
these portions of the peptide and did not show as strong of an
interaction (Figure 3). For L1-b, a periodic trend occurred along
the peptide backbone (Figure 3b), similar to our recently reported
heterocyclic bifunctional IMPY derivative.39 The shifting of resi-
dues Q15, V18, and E22 suggest that these residues may lie on a
similar face of the inner helix (Q15ꢀN27 depicted in Figure 4)
and shift because of direct interaction with compound or a change
in conformation upon ligand binding.6,47,54 The absence of the
dimethylamino structural moiety in L1-a, which has been pro-
posed tobe important inAβ interaction,38,59,60 may explain why its
degree of interaction with Aβ is less than that of L1-b. Further-
more, the observation that the residues E11 and H13 were
significantly shifted by our bifunctional molecules is important
because these residues are near the proposed metal binding site of
Aβ (H6, H13, and H14).5,10,11,13ꢀ15,18ꢀ20,22,40 Because of the
close proximity of our compounds to the metal binding residues,
it is possible that L1-a and L1-b may be able to target metal ions
surrounded by Aβ peptides.
To further compare the effect of both the addition of two
nitrogen atoms and the presence of the dimethylamino group in
our molecules, the compounds without a metal binding site, S0
and S1, were also studied by NMR. Because of the low solubility
of S0 and S1 in the buffered conditions above, DMSO was
employed to deliver the compounds to the Aβ solution.46 Both
compounds and DMSO alone (ca. 2 equiv of compound)
appeared to have an influence on the shifting of residues but
shifting trends were different from L1-a and L1-b (Supporting
Information, Figure S3). Upon addition of both S0 and S1 to the
solution of Aβ, residues E11 and H13 did not shift as much as
DMSO alone suggesting these compounds may compete for
contacts with the peptide although direct comparison is difficult
to discern because of the possibility of compounds interacting
both with the peptide and/or counteracting the DMSO effect
(Supporting Information, Figure S3). With the consideration of
the DMSO effect on the Aβ monomer, S0 and S1 showed a
different ability to recognize Aβ under these conditions com-
pared to the bifunctional compounds L1-a and L1-b. Contrasting
L1-a and L1-b from S0 and S1, the dimethylamino functionality
may not be solely responsible for interaction within this molec-
ular scaffold. The combination of placing two nitrogen atoms
into the stilbene framework with the dimethylamino group
revealed that minor changes in chemical structure might better
control Aβ interaction via hydrogen bonding and hydrophobic
contacts. From the compounds investigated, the choice of
molecular scaffold and functionality are important variables in
selecting effective Aβ interacting molecules to be used as a basis
for rational structure-based design. Overall, along with metal
binding studies, our NMR investigations above suggest that the
bifunctional stilbene derivatives can interact with both metal ions
and Aβ demonstrating their bifunctionality.
Docking Studies of L1-a, L1-b, S0, and S1 with Aβ. While
the residue-specific influence of our compounds (L1-a and L1-b)
on the Aβ monomer was determined by 2D NMR spectroscopy,
visualizationofthe structural details of their contact modes with the
peptide backbone or residue side chains would provide more
insights into the structure-interaction relationship between small
molecules and the peptide. To consider possible conformations of
our compounds with Aβ, docking studies were conducted using
AutoDock4.38,51ꢀ53 A previously determined solution NMR struc-
ture of Aβ1ꢀ40 in SDS micelles (PDB 1BA4) was chosen, and the
molecules were evaluated with 10 conformations of Aβ.54 Among
them, five conformations of Aβ docked with L1-a and L1-b were
consistent with our NMR results. Based on the lowest energy
clusters with highest occurrence, the conformations of compounds
with Aβ were selected (Supporting Information, Table S2).
In agreement with NMR observations discussed above, the
overall docking results suggested that our bifunctional small
molecules may contact the N-terminal region and inner helix of
the peptide near the proposed metal binding site (Figure 4 and
Supporting Information, Figure S4).47,54 Most of the conforma-
tions presented that the compounds were oriented closer to the
hydrophilic metal binding region while one conformation
showed ligands docked near the hydrophobic inner helix
(conformation C, Supporting Information, Figure S4). The
docking results for L1-a and L1-b illustrated similar binding
in three of the conformations analyzed, suggesting that these
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Figure 5. Control of Zn2+-induced Aβ aggregation by small molecules. Top: Scheme of the inhibition and disaggregation experiments. Bottom: TEM
images of samples (a) from the inhibition experiment [fresh Aβ treated with ZnCl2 followed by incubation with the compounds (L1-a and L1-b)]; (b)
from the disaggregation experiment [Zn2+-induced Aβ aggregates by 24 h incubation and subsequent 24 h treatment with L1-a or L1-b]. (c) Results of
(a) visualized by native gel electrophoresis followed by Western blot (anti-Aβ antibody 6E10): (1) [Aβ + ZnCl2]; (2) [1 + L1-a]; (3) [1 + L1-b].
Conditions: [Aβ] = 25 μM, [ZnCl2] = 25 μM, [compound] = 50 μM, pH 7.4, 24 h, 37 °C, constant agitation.
molecules may behave similarly with common hydrogen bonding
contacts with the peptide backbone and side chain residues.
Along with hydrogen bonding interaction, positioning of the
dimethylamino group of L1-b either toward the hydrophilic
N-terminal region or the hydrophobic helical region revealed
various amphiphilic contacts with the peptide potentially ac-
counting for the greater chemical shift changes exhibited in the
NMR experiment. For S0 and S1, interactions were indicated in
the hydrophilic turn and along the inner helix of the peptide as
well. More variation between these two molecules (S0/S1) was
observed in the different conformations tested although some
conformations showed similar binding modes of these com-
pounds as were seen for L1-a and L1-b (Figure 4 and Supporting
Information, Figure S4).
Different orientations of L1-a, L1-b, S0, and S1 were visible in
the dockinginvestigations, withgeneral interactionnearthe turnof
the peptide (near the metal binding site and inner helix) indicating
the utility ofthe stilbeneframework for recognizingthe Aβ peptide
(Figure 4 and Supporting Information, Figure S4). The individual
differences between the ligands docked to the Aβ conformations
displayed several possible contacts that may exist in solution, such
as hydrogen bonding and hydrophobic contacts that along with
NMR imply more favorable interaction of L1-a and L1-b over S0
and S1 with the Aβ peptide. Therefore, these docking results give
further support for direct Aβ interaction of our bifunctional small
molecules in addition to the NMR investigations.
electrophoresis followed by Western blotting using anti-Aβ
antibody 6E10.37ꢀ39,55ꢀ58 Both inhibition and disaggregation
studies were carried out (forward and backward reactions as
shown in Figure 5 and Supporting Information, Figure S5). For
inhibition experiments, fresh Aβ (25 μM) and CuCl2 or ZnCl2
(25 μM) were treated with the compounds (50 μM) for 24 h
(Figure 5 and Supporting Information, Figure S5a). In the
disaggregation experiments, the samples containing Aβ and
metal chloride salts were incubated for 24 h to allow Aβ
aggregates to form, and then agitated with each compound for
24 h (Figure 5 and Supporting Information, Figure S5a).
The presence of metal ions (Cu2+ or Zn2+) triggered the
formation of large, aggregated Aβ species (Figure 5 and Support-
ing Information, Figure S5a). On the other hand, upon treatment
with our compounds, metal-induced Aβ aggregation was notice-
ably inhibited according to TEM and native gel analysis.37ꢀ39,58
As shown in Figure 5, Aβ species that had low MW (e32 kDa)
and could enter the gel matrix were visible when our bifunctional
compounds were introduced into metal-treated Aβ samples. In
particular, much less metal-mediated Aβ aggregation was seen
with L1-b over L1-a. Based on our previous report, at the same
conditions, the traditional metal chelators CQ, phen (Figure 1),
and EDTA resulted in the generation of high MW Aβ aggregates,
which was observed by the same methods.37,38 The control
molecule (S1) that does not contain a metal binding site did not
block metal-induced Aβ aggregation,37,38 suggesting that disrup-
tion of metal-Aβ interaction was one of the important parameters
to block metal-mediated Aβ aggregation with our bifunctional
compounds. In addition, the effect of L1-b on the metal-free Aβ
Effect of L1-a and L1-b on Metal-Induced Aβ Aggregation.
The regulation of L1-a and L1-b on metal-associated Aβ
aggregation processes was investigated by TEM and native gel
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Figure 6. An NMR investigation of L1-a and L1-b interacting with Zn2+-bound Aβ1ꢀ40. 2D 1H-15N TROSY-HSQC NMR spectra (900 MHz, 200 mM
SDS-d25, 20 mM sodium phosphate, pH 7.3, 7% D2O (v/v), 25 °C) of 15N-labeled Aβ peptide (ca. 308 μM, black) and upon the addition of ZnCl2 (ca. 1
equiv, red) followed by the addition of 10 equiv of either L1-a (purple) or L1-b (blue) after 1 h incubation.
aggregation was studied (Supporting Information, Figure S5b).
No apparent control of Aβ aggregation by L1-b was observed
under the metal-free condition, implying that the reactivity of
L1-b is specific for metal-Aβ species over metal-free Aβ species.
Taken together, TEM and Western blot results suggest that the
bifunctional molecules could prevent metal-induced Aβ fibrillo-
genesis generating smaller sized Aβ species more effectively than
with traditional metal chelating agents. Therefore, inhibition
studies demonstrate that our bifunctional small molecules could
function as modulators of metal-induced Aβ aggregate forma-
tion, which supports the hypothesis that metal ions could play
important roles in Aβ aggregation in AD.
ability of L1-b to compete for Zn2+ binding from Aβ, the
compound (40 μM) was added to a solution containing equal
concentrations of ZnCl2 and Aβ (20 μM, 200 mM SDS, 20 mM
sodium phosphate buffer, pH 7.3). The resulting UVꢀvis
spectrum showed the generation of Zn2+-L1-b species with
optical bands near 450 nm, which is comparable to those from
only the metalꢀligand complex (Supporting Information, Figure
S6a). Because of the small optical change upon addition of Zn2+
into the solution of L1-a (Supporting Information, Figure S1),
the UVꢀvis study of Aβ, Zn2+, and L1-a was not performed.
Carrying out 2D TROSY-HSQC experiments under similar
conditions suggested that more than simple metal chelation by
our small molecules from the peptide may occur. As expected
from previous reports, addition of 1 equiv of ZnCl2 to an NMR
solution of Aβ (308 μM, 200 mM SDS-d25, 20 mM sodium
phosphate buffer, pH 7.3, 7% D2O (v/v), 25 °C) caused a large
portion of the 2D NMR spectrum to disappear because of line
broadening as a result of Zn2+ binding (Figure 6).20,69,70 With the
treatment of 10 equiv of compounds, the 1H and 15N cross peaks
absent upon introduction of Zn2+ were recovered with 30 min
incubation of L1-a and L1-b (Figure 6). Additionally, cross peak
shifts were observed for both L1-a and L1-b that indicated small
variations from the metal free samples in the N-terminal and
inner helix portions of the peptide (Figure 6 and Supporting
Information, Figure S6b). These observations suggest that the
interaction of these bifunctional molecules with Zn2+-Aβ occurs
via a combination of metal chelation by the compound from Aβ
and interaction of free compound and/or the metal complex with
Aβ.38 Metal chelation was also supported by optical spectra
indicating Zn2+ binding to the compound (Supporting Informa-
tion, Figure S6a) as discussed above.
The ability of L1-a and L1-b to disassemble preformed metal-
mediated Aβ aggregates was also explored. Both compounds L1-a
and L1-b were able to alter the structure of metal-triggered Aβ
aggregates affording their disaggregation (Figure 5 and Supporting
Information, Figure S5).37 Similar to the inhibition study, L1-b
provided greater effect toward disaggregation of preformed metal-
Aβ aggregates than L1-a. Different from the bifunctional mol-
ecules, our previous studies showed that the traditional metal
chelators phen and EDTA did not disaggregate preformed metal-
associated Aβ aggregates, though changes in morphology were
observed.37,38,55 In the case of CQ, partial disaggregation of Zn2+-
treated Aβ aggregates was recorded.55 Therefore, on the basis of
the results from both inhibition and disaggregation experiments,
our bifunctional molecules L1-a and L1-b were better able to
control metal-mediated Aβ aggregation processes (formation of
metal-induced Aβ aggregates as well as their disaggregation) than
the traditional metal chelators. The compound L1-b that indicated
greater bifunctionality (vide supra) exhibited more effective con-
trol of metal-induced Aβ aggregation, compared to L1-a. These
observations imply that the bifunctionality of our compounds
leads to enhanced reactivity with metal-Aβ species (structure-
interaction-reactivity relationship).
Based on reactivity studies of compounds with metal-Aβ
species by TEM (vide supra), disruption of both metal-peptide
and peptideꢀpeptide interactions could be a driving force for
preventing metal-induced Aβ aggregation. From previously
reported NMR observations, the traditional metal chelator
EDTA was shown to remove Cu2+ or Zn2+ from Aβ;71 but from
our previously reported TEM results discussed above, EDTA and
other orthodox metal chelators did not deter metal-induced Aβ
aggregation.37,38 Our NMR and optical results involving
Investigation of the Reaction of Zn2+-Treated Aβ with L1-
a and L1-b. To understand possible mechanisms underlying the
modulation of metal-triggered Aβ events by our small molecules,
spectroscopic investigations were conducted using high-resolu-
tion 2D NMR and UVꢀvis on Zn2+-treated Aβ solutions in
the absence and presence of compounds. To first determine the
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Zn2+-Aβ species and compounds indicated that the observed
interference on metal-Aβ reactivity could be due to the removal
of the metal ion from metal-Aβ species (through metal chelation
by the ligand or ligand-induced metal-Aβ conformational
changes) and/or the potential formation of ternary type com-
plexes involving Aβ, Zn2+, and compound (through the interac-
tion of ligand with metal-Aβ species or ligandꢀmetal complexes
with the peptide).38 Thus, small variations in the stilbene scaffold
could not only affect ligand binding to Aβ but also indicate the
ability to tune interactions with metal-Aβ species.
Regulation of Cu-Aβ-Induced ROS Production by L1-a and
L1-b. The production of ROS by redox active metal-Aβ species
and subsequent oxidative stress may lead to neurotoxicity in
AD.2,5,10ꢀ17,21ꢀ25 In addition to targeting metal-Aβ species for
modulating their aggregation properties, control of ROS forma-
tion by our bifunctional small molecules must be considered.
Employing the HRP/Amplex Red assay,23,32,37,38,40 amounts of
H2O2 produced by Cu-Aβ species in the absence and presence of
our bifunctional molecules (L1-a and L1-b) was determined.
Inhibition of Cu-Aβ-triggered H2O2 formation by L1-a and L1-
b was observed (reduction of H2O2 formation by 64((1.1)% (for
L1-a) and 70((0.75)% (for L1-b)37). Other metal chelating
agents CQ and EDTA also exhibited decreased H2O2 production
under the same conditions.37,38 In the absence of Aβ, free Cu ions
generated about 94% of the amount of H2O2 as was produced in
the presence of the peptide. The ligands werealso able to lowerthe
level of H2O2 generated by peptide-free Cu by about 52% for L1-a
and 56% for L1-b ([CuCl2]/[ligand] = 1:2), relative to Cu-Aβ.
Taken together, the prevention of H2O2 formation by peptide-free
or -bound Cu in vitro by our stilbene derivatives was observed,
which may suggest their use as ROS regulators.
Cytotoxicity of L1-a and L1-b with and without Metal
Chloride Salts As Well As Their Ability to Modulate Metal-Aβ
Neurotoxicity in Human Neuroblastoma Cells. First, cell
viability in the presence of our compounds at various concentra-
tions was evaluated in human neuroblastoma M17 and AS cells
after 24 or 72 h incubation. The cell survival rates were monitored
by the MTT assay.37,38 After 24 h of incubation with our bifunc-
tional small molecules, no toxic effects were observed in either M17
or AS cells. After 72 h, cytotoxicity of L1-a was not visible up to 100
μM for either cell line. For both cell lines (M17/AS), L1-b (up to
100 μM) showed 66((3.2)/84((4.2)% survival, respectively
(Supporting Information, Figure S7a). Considering the previously
studied cell viability of CQ and phen (30((0.3)/34((0.8)% and
37((0.5)/44((0.2)% in M17 and AS cell lines, respectively),38
toxicity of our compounds may be minimal up to 100 μM.
Furthermore, the cytotoxicity of L1-a and L1-b with CuCl2 or
ZnCl2 (5ꢀ120 μM) at 1:1 and 1:2 metal/compound ratios for 24 h
was examined in the M17 cell line. Cell survival (ca. 96ꢀ100%) was
observed from cells treated with up to 20 μM CuCl2 or ZnCl2 and
20/40 μM compounds (Supporting Information, Figures S7b and
S7c). Above 40 μM metal chloride salt and 40/80 μM compounds,
the cell viability decreased. When CuCl2 or ZnCl2, (120 μM) and
compounds (L1-a or L1-b; 120 or 240 μM) were incubated with
cells, about 40ꢀ50% cell survival was indicated. From these
cytotoxicity studies, our bifunctional small molecules were mini-
mally toxic at concentrations of 40 μM or lower in the presence of
up to 20 μM metal ions. Thus, based on these cytotoxicity studies,
testing our compounds’ effect on metal-Aβ species in a cellular
environment could be explored.
Figure 7. Regulation of metal-associated Aβ neurotoxicity in M17 cells
for 24 h using the MTT assay. Cell viability (%) upon incubation of (1)
Aβ; (2) [Aβ + CuCl2]; (3) [Aβ + CuCl2 + L1-a]; (4) [Aβ + CuCl2 +
L1-b]; (5) [Aβ + ZnCl2]; (6) [Aβ + ZnCl2 + L1-a]; (7) [Aβ + ZnCl2 +
L1-b]. Condition: [Aβ] = 20 μM, [CuCl2 or ZnCl2] = 20 μM,
[compound] = 40 μM. Cell viability (%) shown in the figure is relative
to that of cells containing 1% DMSO (v/v). Cytotoxicity from cells
treated with metal ions and compounds were not indicated at this
condition (Supporting Information, Figures S7b and S7c).
out the reactions of metal ions, Aβ, and compounds in the
human neuroblastoma M17 cell line.37,38 Our cell studies pre-
sented 70((2.4) or 80((4.3)% of cells survived upon 24 h
incubation with Cu2+- or Zn2+-treated Aβ (Figure 7). Interest-
ingly, as described in Figure 7, when the compound with the
dimethylamino functionality (L1-b) was added to Cu2+- and
Zn2+-Aβ-treated cells, cell survival rates increased to about 9037
and 100%, respectively. Compared to L1-b, L1-a induced a slight
increase in the survival of cells treated with Cu2+- or Zn2+-treated
Aβ (77% and 86% cell viability for the samples of Cu2+ and Zn2+,
respectively). Under the same conditions traditional metal
chelators such as CQ, EDTA, and phen were not able to recover
the cell survival from metal-Aβ neurotoxicity.37,38 Overall, these
cell-based studies employing our bifunctional compounds de-
monstrate their capability to regulate metal-Aβ neurotoxicity in
living cells, which is expected based on their reactivity in vitro
(anti-Aβ aggregation as well as ROS regulation). These cell
studies also agree with the overall observation that assessing the
ability of our bifunctional compounds to interact with metal-
Aβ species is a key factor to comprehend greater reactivity
toward metal-induced Aβ events (structure-interaction-reactivity
relationship).
’ SUMMARY AND PERSPECTIVE
Small molecules that have the ability to target and modulate
metal-Aβ species are greatly desired and could be instrumental in
determining how metal-associated pathways may be involved
with AD neuropathogenesis. To achieve this, we have taken the
rational structure-based design approach to develop bifunctional
compounds that are capable of chelating metal ions as well as
interacting with Aβ species. Our group has previously applied
this strategy to the chemical structures of two known Aβ
aggregate indicators, p-I-stilbene and IMPY, that were altered
through installation of heteroatoms for metal coordination.
These small, neutral, lipophilic imaging agents have demon-
strated attributes that render them beneficial for this application.
To investigate how our bifunctional molecules influence the
neurotoxicity induced by metal-associated Aβ species, we carried
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According to the promising behavior of our first series of
compounds, a further generation based on the same frameworks
was devised. Importantly, the entire structure was likely a
valuable contribution to targeting Aβ while simple structural
modifications could improve their interaction and reactivity with
Aβ and metal-Aβ species. Here, we focus on two compounds that
serve as an extension to our previous studies of stilbene-like
molecules; these studies supplement our understanding of how
simple modifications such as the inclusion of two nitrogen atoms
for metal chelation and the presence or absence of the dimethy-
lamino functionality can influence interaction/reactivity. The
bifunctionality (metal chelation and Aβ interaction) of the
compounds (L1-a and L1-b) was verified by spectroscopic and
other methods including UVꢀvis, high-resolution 2D NMR, and
X-ray crystallography. Further biochemical studies displayed the
potential direct interaction of our small molecules with metal-Aβ
species through their ability to regulate reactivity (metal-induced
Aβ aggregation and neurotoxicity) in vitro and in living cells.
Together with our previous work, the studies presented here
validate the selection of the stilbene compound as a model
interaction framework as it importantly interacts with Aβ pep-
tides in various aggregated forms and displays minimal toxicity in
human neuroblastoma cells. In our studies, the structural moi-
eties, such as dimethylamino functionality, are crucial parameters
for a structure-interaction-reactivity relationship that can be
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: mhlim@umich.edu.
’ ACKNOWLEDGMENT
This work was supported by start-up funding from the Uni-
versity of Michigan, the Alzheimer’s Art Quilt Initiative (AAQI), as
well as the Alzheimer’s Association (NIRG-10-172326) (to M.H.L.)
and NIH (DK078885 and RR023597 to A.R.). We acknowledge
funding from NSF Grant CHE-0840456 for X-ray instrumenta-
tion. J.J.B. is grateful for the Murrill Memorial Scholarship from
the Department of Chemistry at the University of Michigan. K.N.
conducted this work as an intern from the Western Reserve
Academy, OH, U.S.A. and is currently at Hankuk Academy Of
Foreign Studies, Korea. We thank Kermit Johnson, Dr. Sub-
ramanian Vivekanandan, and Dr. Ravi P. R. Nanga for help with
900 MHz NMR experiments and data analysis, as well as Allana
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