Reactivity of diphenylpropynone derivatives toward metal-associated amyloid-β Species

Article abstract of DOI:10.1021/ic302084g

In Alzheimer's disease (AD), metal-associated amyloid-β (metal-Aβ) species have been suggested to be involved in neurotoxicity; however, their role in disease development is still unclear. To elucidate this aspect, chemical reagents have been developed as valuable tools for targeting metal-Aβ species, modulating the interaction between the metal and Aβ, and subsequently altering metal-Aβ reactivity. Herein, we report the design, preparation, characterization, and reactivity of two diphenylpropynone derivatives (DPP1 and DPP2) composed of structural moieties for metal chelation and Aβ interaction (bifunctionality). The interactions of these compounds with metal ions and Aβ species were confirmed by UV-vis, NMR, mass spectrometry, and docking studies. The effects of these bifunctional molecules on the control of in vitro metal-free and metal-induced Aβ aggregation were investigated and monitored by gel electrophoresis and transmission electron microscopy (TEM). Both DPP1 and DPP2 showed reactivity toward metal-Aβ species over metal-free Aβ species to different extents. In particular, DPP2, which contains a dimethylamino group, exhibited greater reactivity with metal-Aβ species than DPP1, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional scaffold that could be utilized to develop chemical reagents for investigating metal-Aβ species in AD.

Full text of DOI:10.1021/ic302084g

Article
pubs.acs.org/IC
Reactivity of Diphenylpropynone Derivatives Toward Metal-
Associated Amyloid‑β Species
Amit S. Pithadia,†,‡ Akiko Kochi,†,‡ Molly T. Soper,† Michael W. Beck,† Yuzhong Liu,† Sanghyun Lee,§
Alaina S. DeToma,† Brandon T. Ruotolo,*,† and Mi Hee Lim*,†,§
§
†Department of Chemistry and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: In Alzheimer’s disease (AD), metal-associated amyloid-β
(metal−Aβ) species have been suggested to be involved in neurotoxicity;
however, their role in disease development is still unclear. To elucidate this
aspect, chemical reagents have been developed as valuable tools for targeting
metal−Aβ species, modulating the interaction between the metal and Aβ, and
subsequently altering metal−Aβ reactivity. Herein, we report the design,
preparation, characterization, and reactivity of two diphenylpropynone
derivatives (DPP1 and DPP2) composed of structural moieties for metal
chelation and Aβ interaction (bifunctionality). The interactions of these
compounds with metal ions and Aβ species were confirmed by UV−vis,
NMR, mass spectrometry, and docking studies. The effects of these
bifunctional molecules on the control of in vitro metal-free and metal-
induced Aβ aggregation were investigated and monitored by gel electro-
phoresis and transmission electron microscopy (TEM). Both DPP1 and DPP2 showed reactivity toward metal−Aβ species over
metal-free Aβ species to different extents. In particular, DPP2, which contains a dimethylamino group, exhibited greater reactivity
with metal−Aβ species than DPP1, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional
scaffold that could be utilized to develop chemical reagents for investigating metal−Aβ species in AD.
molecules with both metal chelation and Aβ interaction
properties (defined as bifunctionality) have been devised.
Some of the compounds have been fashioned based on the
incorporation approach (Figure 1), where a metal chelation site
is directly inserted into a known Aβ imaging agent with
minimal structural modifications, along with consideration of
criteria for possible brain uptake.8,21−25,27−35 These molecules
have been shown to be able to control the interactions and
reactivity of metal−Aβ species in vitro and/or in living cells,
suggesting that the incorporation approach (Figure 1) could be
considered as a promising design strategy to fashion suitable
chemical reagents for uncovering the potential role of metal−
Aβ species in AD development.
Herein, we report the design and preparation of a new class
of bifunctional small molecules (DPP1 and DPP2, Figure 1),
composed of a diphenylpropynone framework, as chemical
reagents for targeting and regulating metal−Aβ species, the
characterization of their interactions with metal ions and Aβ
species, as well as the investigation of their in vitro reactivity
with metal−Aβ species. From our overall results and
observations, the diphenylpropynone scaffold could be a
structural feature for constructing chemical reagents for
investigating metal−Aβ species. Moreover, the dimethylamino
functionality in DPP2 was observed to be an important moiety
INTRODUCTION
■
Alzheimer’s disease (AD) is a fatal neurodegenerative disease
that affects more than 5 million people in the United States.1,2
A pathological hallmark of the diseased brain is an
accumulation of misfolded amyloid-β (Aβ) aggregates.2−8
Monomeric Aβ peptides, generated from the proteolytic
cleavage of the transmembrane amyloid precursor protein,
can further aggregate to produce oligomers, protofibrils, and
eventually fibrils. It is still not completely understood which
conformation of Aβ is associated with AD neuropathogenesis;
however, recent evidence has proposed that soluble oligomers
might be the toxic species due to their ability to interrupt
neurotransmission.4,5,9 In addition to Aβ species, elevated
concentrations of transition metals, such as Fe, Cu and Zn, have
been observed within the deposits of Aβ aggregates.2,7,8,10−15
The possible relationship between metal-associated Aβ species
(metal−Aβ species) and neurotoxicity has been suggested
based on observations that upon binding to Aβ, metal ions
facilitate peptide aggregation as well as enhance oxidative stress
caused by overproduction of reactive oxygen spe-
cies;2,4,5,7,8,12−21 however, this connection has not been clearly
revealed.
To gain a better understanding of the involvement of metal−
Aβ species in AD pathogenesis, recent advancements in the
development of chemical reagents to specifically target metal−
Aβ species and modulate their interaction and reactivity have
been made.2,7,8,21−33 Among them, rationally designed small
Received: September 25, 2012
Published: November 15, 2012
© 2012 American Chemical Society
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Figure 1. Incorporation approach (top) and structures of small molecules (bottom). Left to right: 3-(4-(dimethylamino)phenyl)-1-(4-
iodophenyl)prop-2-yn-1-one; DPP1 = 3-phenyl-1-(pyridin-2-yl)prop-2-yn-1-one; DPP2 = 3-(4-(dimethylamino)phenyl)-1-(pyridin- 2-yl)prop-2-
yn-1-one.
Scheme 1. Synthesis of DPP2
NaHCO3 (2 mL), diluted with EtOAc (10 mL), and washed with
brine (2 × 25 mL). The aqueous layer was extracted with EtOAc (2 ×
15 mL), and the combined organic solutions were dried over MgSO4
and filtered, followed by removal of the solvent in vacuo. The crude
product was purified by silica gel chromatography (CH2Cl2:EtOAc =
9:1) to yield an orange product (202 mg, 0.81 mmol, 28%). 1H NMR
(400 MHz, CDCl3)/δ (ppm): 8.77 (d, J = 4.8 Hz, 1 H), 8.14 (d, J =
7.6 Hz, 1 H), 7.82 (td, J = 7.6, 1.6 Hz, 1 H), 7.57 (d, J = 8.4 Hz, 2 H),
7.44 (m, 1 H), 6.59 (d, J = 8.4 Hz, 2 H), 2.98 (s, 6 H). 13C NMR (100
MHz, CDCl3)/δ (ppm): 177.6, 153.7, 151.8, 149.7, 136.8, 135.6,
126.9, 123.3, 111.4, 105.5, 100.2, 89.3, 39.9. HRMS: Calcd for [M
+H]+, 251.1179; found, 251.1176.
Parallel Artificial Membrane Permeability Assay Adapted for
Blood-Brain Barrier (PAMPA-BBB). PAMPA-BBB experiments were
carried out using the PAMPA Explorer kit (pION Inc., Billerica, MA)
with modification to previously reported protocols.29,40−42 Each stock
solution was diluted with pH 7.4 Prisma HT buffer (pION) to a final
concentration of 25 μM (1% v/v final DMSO concentration) and 200
μL were added to the wells of the donor plate (number of replicates =
12). BBB-1 lipid formulation (5 μL, pION) was used to coat the
polyvinylidene fluoride (PVDF, 0.45 μM) filter membrane on the
acceptor plate. The acceptor plate was placed on top of the donor
plate forming a sandwich and the brain sink buffer (BSB, 200 μL,
pION) was added to each well of the acceptor plate. The sandwich was
incubated for 4 h at ambient temperature without stirring. UV−vis
spectra of the solutions in the reference, acceptor, and donor plates
were measured using a microplate reader. The PAMPA Explorer
software v. 3.5 (pION) was used to calculate the −logPe values for the
compounds. CNS designations were assigned by comparison to
compounds that were identified in previous reports.29,40−42
for potentially enhancing Aβ interaction of the compound, as
reported previously,29,31,36 offering noticeable reactivity with
metal−Aβ species.
EXPERIMENTAL SECTION
■
Materials and Procedures. All reagents were purchased from
commercial suppliers and used as received unless otherwise stated.
The compound, 3-phenyl-1-(pyridin-2-yl)prop-2-yn-1-one (DPP1),
was prepared following previously reported methods.37−39 Aβ1−40
(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV)
was purchased from AnaSpec (Fremont, CA). An Agilent 8453 UV−
visible (UV−vis) spectrophotometer was used to measure the optical
spectra. Transmission electron microscopy (TEM) images were
recorded with a Philips CM-100 transmission electron microscope.
A SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale,
CA) was employed for the measurement of absorbance for 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
parallel artificial membrane permeability (PAMPA) assays. Nuclear
magnetic resonance (NMR) spectra of small molecules and for Zn2+
binding studies were obtained on a Varian 400 MHz NMR
spectrometer. Mass spectrometric measurements for compounds
were conducted by a Micromass LCT electrospray time-of-flight
mass spectrometer. Mass spectrometric studies for investigating the
interaction of DPP1 and DPP2 with the peptide were carried out on a
Waters Synapt G2 ion-mobility mass spectrometer (Milford, MA).
Preparation of 3-(4-(Dimethylamino)phenyl)-1-(pyridin-2-yl)-
prop-2-yn-1-one (DPP2). DPP2 was synthesized by slight modifica-
tions to a previously reported procedure (Scheme 1).39 To a solution
of 4′-dimethylaminophenyl acetylene (0.42 g, 2.9 mmol) in dry
tetrahydrofuran (THF, 5 mL) was added n-butyllithium (0.72 mL, 2.9
mmol, 2.5 M solution in hexanes) dropwise by a syringe over 5 min at
−40 °C. The reaction mixture was allowed to stir at −40 °C for 10
min and then warmed to −15 °C. After 30 min, the mixture was
cooled to −78 °C (dry ice/acetone) and a solution of Weinreb’s amide
(0.40 g, 2.4 mmol in 9 mL of dry THF) was introduced through a
syringe. The reaction mixture was allowed to react at −78 °C for 10
min and then warmed to room temperature and followed by 1 h
stirring. The reaction was quenched by adding saturated aqueous
Determination of Solution Speciation for DPP1, DPP2, and the
Cu2+−DPP2 Complex. The pKa values for DPP1 and DPP2 were
determined by UV−vis variable-pH titrations as previously
reported.27,29−33,43,46 To establish the pKa values, a solution (100
mM NaCl, 10 mM NaOH, pH 12) of DPP1 (40 μM) or DPP2 (20
μM) was titrated with small aliquots of HCl. At least 30 spectra were
recorded in the range of pH 2−10. Similarly, a solution containing
DPP2 (20 μM) and CuCl2 in a ratio of 2:1 was titrated with small
additions of HCl and at least 30 spectra were recorded over the range
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of pH 2−7. The acidity and stability constants were calculated by using
the HypSpec program (Protonic Software, UK).44 Speciation diagrams
for DPP1, DPP2, and Cu2+−DPP2 complexes were modeled by the
HySS2009 program (Protonic Software).45
Amyloid-β (Aβ) Peptide Experiments. Aβ1−40 was used in all Aβ
experiments. Aβ1−40 peptide (1 mg) was dissolved with ammonium
hydroxide (NH4OH, 1% v/v, aq), aliquoted, lyophilized, and stored at
−80 °C. A stock solution (ca. 200 μM) was prepared by redissolving
Aβ with NH4OH (1% v/v, aq, 10 μL) followed by dilution with
ddH2O. All Aβ solutions were prepared following previously reported
procedures.28−32 The buffered solutions (20 μM HEPES, pH 6.6 (for
Cu2+ samples) or pH 7.4 (for metal-free and Zn2+ samples), 150 μM
NaCl) were used for both inhibition and disaggregation studies. For
the inhibition experiment, Aβ (25 μM) was first treated with a metal
chloride salt (CuCl2 or ZnCl2, 25 μM) for 2 min followed by addition
of a compound (DPP1 or DPP2, 50 μM in DMSO, 1% v/v final
DMSO concentration). The resulting samples were incubated at 37 °C
for 4, 8, or 24 h with constant agitation. For the disaggregation
experiment, Aβ and a metal chloride salt (CuCl2 or ZnCl2) were
initially incubated at 37 °C for 24 h with steady agitation. The
compound was added afterward followed by additional 4, 8, or 24 h
incubation at 37 °C with constant agitation.
Native Gel Electrophoresis/Sodium Dodecyl Sulfate-Polyacryla-
mide Gel Electrophoresis (SDS-PAGE) with Western Blotting. The
Aβ peptide experiments (as described above) were analyzed by both
native gel electrophoresis and SDS-PAGE with Western blotting using
an anti-Aβ antibody (6E10).28−32,53 Each sample containing 25 μM Aβ
(10 μL) was separated using either a 10−20% gradient Tris-tricine gel
(Invitrogen, Grand Island, NY) or SDS gel (4% stacking gel; 10%
resolving gel; nonreducing conditions). The gel was transferred to a
nitrocellulose membrane and blocked overnight with bovine serum
albumin (BSA, 3% w/v, Sigma, St. Louis, MO) dissolved in Tris-
buffered saline (TBS, Fisher, Pittsburgh, PA) containing 0.1% Tween-
20 (TBS-T, Sigma). The membrane was treated with 6E10 (1:2,000;
2% BSA in TBS-T, Covance, Princeton, NJ) for 4 h at room
temperature. The membrane was probed with a horseradish
peroxidase-conjugated goat anti-mouse secondary antibody (1:5000;
Cayman Chemical, Ann Arbor, MI) in 2% BSA in TBS-T solution for
1 h at room temperature. The protein bands were visualized using the
Thermo Scientific Supersignal West Pico Chemiluminescent Subtrate
(Rockford, IL).
Transmission Electron Microscopy (TEM). Samples for TEM were
prepared following a previously reported method.28−31,33,53 Glow-
discharged grids (Formar/Carbon 300-mesh, Electron Microscopy
Sciences, Hatfield, PA) were treated with samples from either
inhibition or disaggregation experiments (5 μL) for 2 min at room
temperature. Excess sample was removed with filter paper and washed
with ddH2O five times. Each grid was stained with uranyl acetate (1%
w/v, ddH2O, 5 μL) for 1 min. Uranyl acetate was blotted off and grids
were dried for 15 min at room temperature. Images of samples were
taken by a Philips CM-100 transmission electron microscope (80 kV,
25,000x magnification).
Cytotoxicity (MTT Assay). The murine neuro-2a (N2a) neuro-
blastoma cell line was purchased from the American Type Culture
Collection (ATCC, Manassas, VA). The cell line was maintained in
media containing 45% Dulbecco’s modified Eagle’s medium (DMEM)
and 50% OPTI-MEM (GIBCO, Grand Island, NY), supplemented
with 5% fetal bovine serum (FBS, Sigma), 2 mM glutamine, 100 U/
mL penicillin, and 100 mg/mL streptomycin (GIBCO). The cells were
grown and maintained at 37 °C in a humidified atmosphere with 5%
CO2. Cell viability upon treatment of compounds was determined
using a MTT assay (Sigma). N2a cells were seeded in a 96 well plate
(15 000 cells in 100 μL per well) and treated with various
concentrations of DPP1 and DPP2 (2.5−50 μM, final 1% v/v
DMSO). After 24 h incubation at 37 °C, 25 μL MTT (5 mg/mL in
phosphate buffered saline, PBS, pH 7.4, GIBCO) was added to each
well and the plates were incubated for 4 h at 37 °C. Formazan
produced by the cells was dissolved overnight at room temperature by
addition of a solution (100 μL) containing N,N-dimethylformamide
(DMF, 50% v/v, aq, pH 4.5) and sodium dodecyl sulfate (SDS, 20%
w/v). A microplate reader was used to measure the absorbance (A600).
Metal Binding Studies. The interaction of DPP1 and DPP2 with
Cu2+ and Zn2+ was determined by UV−vis and 1H NMR spectroscopy,
respectively, based on previously reported procedures.28−33,46
A
solution of ligand (20 μM in EtOH) was prepared, treated with 1 to
20 equiv of CuCl2, and incubated at room temperature for 2.5 h (for
DPP1) or 5 min (for DPP2). The optical spectra of the resulting
solutions were measured by UV−vis. The interaction of DPP1 or
1
DPP2 with ZnCl2 was observed by H NMR. ZnCl2 (1 equiv) was
added to a solution of DPP1 or DPP2 (4 mM) in CD3CN. The metal
selectivity of both compounds was investigated by measuring the
optical changes upon addition of 1 equiv of CuCl2 to a solution of
ligand (DPP1 = 40 μM; DPP2 = 20 μM in EtOH) pretreated with 1
or 25 equiv of a divalent metal chloride salt (MgCl2, CaCl2, MnCl2,
FeCl2, CoCl2, NiCl2, or ZnCl2). The Fe2+ samples were prepared
anaerobically (all solutions were purged with N2). Quantification of
the metal selectivity was calculated by comparing and normalizing the
absorption values of metal−ligand complexes at 360 nm (for DPP1)
or 580 nm (for DPP2) to the absorption at these wavelengths before
and after the addition of CuCl2 (AM/ACu). Cu2+ binding of DPP2 in
the presence of Aβ was examined by UV−vis. Aβ (25 μM) was treated
with CuCl2 (25 μM) in 20 mM HEPES (2-[4-(2-hydroxyethyl)-
piperazin-1-yl]ethanesulfonic acid), pH 6.6, 150 mM NaCl for 2 min
at room temperature. DPP2 (50 μM) was added to the resulting
solution, followed by 0.5−4 h incubation. For comparison, the optical
spectra of DPP2 (50 μM) were measured in the absence and presence
of CuCl2 (25 μM; 0.5−4 h incubation) without Aβ at pH 6.6.
Aβ Interaction of DPP1 and DPP2 by Mass Spectrometry. The
interaction of DPP1 or DPP2 with Aβ1−40 was investigated by
nanoelectrospray ionization-mass spectrometry (nESI-MS) that was
carried out on a Synapt G2 quadrupole-ion mobility-mass spectrom-
etry system. Samples were prepared by mixing stock solutions of
DPP1 or DPP2 (prepared in DMSO) and Aβ1−40 (dissolved in 100
mM ammonium acetate, pH 6.8) to generate desired final
concentrations of the peptide and the compound. Mixtures were
incubated on ice or at room temperature for 2 or 4 h, respectively, and
then analyzed. To produce protein complex ions, an aliquot of the
sample (ca. 5 μL) was sprayed from the nESI emitter using a capillary
voltage of 1.4 kV, with the source operating in positive ion mode and
the sample cone operated at 50 V. To normalize nESI-MS data for
nonspecific and electrospray artifact interactions that could occur at
high concentrations, data were acquired for Aβ samples containing
thioflavin-T (ThT), a compound known to have no affinity for soluble
forms of the Aβ peptide,47 under identical concentration conditions as
our DPP1 and DPP2 experiments. Any ThT binding observed was
assumed to be due to either nonspecific binding or the electrospray
process, and subtracted from the intensities of the DPP1 and DPP2
interactions observed.48 This procedure was performed over a broad
range of concentrations. The mass spectra were acquired with the
following settings and tuned to avoid ion activation and to preserve
noncovalent protein−ligand complexes:49 backing pressure, 7.3 mbar;
IMS pressure reading, 3.09 mbar; ToF analyzer pressure, 1.14 × 10−6
mbar.
Docking Studies. Flexible ligand docking studies using AutoDock
Vina50 for DPP1 and DPP2 were conducted against the Aβ1−40
monomer from the previously determined aqueous solution NMR
structure (PDB 2LFM).51 Ten conformations were selected from 20
conformations within the PDB file (1, 3, 5, 8, 10, 12, 13, 16, 17, and
20). The MMFF94 energy minimization in ChemBio3D Ultra 11.0
was used to optimize the structures of the ligands for the docking
studies. The structural files of DPP1, DPP2, and the peptide,
generated by AutoDock Tools and imported into PyRx,52 were used to
run AutoDock Vina. The search space dimensions were set to contain
the entire peptide. The exhaustiveness for the docking runs was set at
1024. Docked poses of the ligands were visualized with Aβ using
Pymol.
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RESULTS AND DISCUSSION
■
Design Consideration, Preparation, and Character-
ization of Diphenylpropynone Derivatives for Targeting
and Modulating Metal−Aβ Species. The diphenylpropy-
none scaffold was shown to have high binding affinity (ca. 6
nM) for Aβ aggregates; thus, it has been utilized for the design
of an Aβ plaque imaging probe (Figure 1).54 Based on the
incorporation approach, a nitrogen donor atom was installed
into this framework to generate a metal chelation site with an
oxygen donor atom from the carbonyl group, which afforded
two bifunctional molecules (DPP1 and DPP2, Figure 1).
Furthermore, a minor structural difference between DPP1 and
DPP2 (i.e., a dimethylamino functionality, suggested to be
critical for Aβ interaction)29,31,36 was included to understand a
structure-eactivity relationship. DPP1 was synthesized as
previously established.37−39 The new compound, DPP2, was
prepared by slight modifications to a previously reported
method (Scheme 1).39
To predict potential druglike and BBB penetration properties
of the structural scaffolds of DPP1 and DPP2, we calculated
values of Lipinski’s rules and logBB.7,34,35,40,41 As shown in
Table 1, the theoretical values indicate that both compounds
Table 1. Values (MW, clogP, HBA, HBD, PSA, logBB, and
−logPe) of DPP1 and DPP2
Figure 2. Solution speciation studies of DPP1 and DPP2. Top: UV−
vis spectra of DPP1 (40 μM, left) and DPP2 (20 μM, right) in the
range of pH 2−10. Middle: Solution speciation diagrams for DPP1
(left) and DPP2 (right) (FL = fraction of compound with given
protonation). Bottom: Acidity constants (pKa) of L (L = DPP1 or
DPP2). Charges are omitted for clarity. Error in the parentheses is
shown in the last digit. Conditions: I = 0.10 M NaCl; room
temperature.
Lipinski’s rules and
a
calculation
DPP1
DPP2
others
MW
207
2.47
2
250
2.63
3
≤450
≤5.0
≤10
a
clogP
HBA
HBD
PSA
0
0
≤5
30.0
0.0618
33.2
0.0390
≤90 Å2
logBB
>0.3 (readily cross the
BBB)
indicated a single acidity constant (pKa) for DPP1 (pKa =
2.035(5)) and two pKa values for DPP2 (pKa1 = 7.106(1) and
pKa2 = 2.959(4)). These pKa values suggest that monoproto-
nated, diprotonated, and neutral species exist in solution of
DPP2 depending on pH (from 2 to 10), while the
monoprotonated and neutral forms of DPP1 are present in
this pH range (Figure 2). In addition, the generated solution
speciation diagrams of DPP1 and DPP2 exhibit that their
neutral forms are relatively predominant at physiologically
relevant pH (i.e., 7.4) (DPP1, 100%; DPP2, ca. 65%).
Metal Binding Properties of DPP1 and DPP2. Metal
binding properties of DPP1 and DPP2 (specifically, Cu2+ and
Zn2+) were studied by UV−vis and NMR spectroscopy. Upon
the addition of CuCl2 (1−20 equiv) to a solution of DPP1 and
DPP2 in EtOH, new optical bands were observed, indicative of
Cu2+ binding to the ligand (Figure 3a). In particular, in the
presence of Cu2+, DPP2, which has a dimethylamino group,
showed a distinguishable optical shift from 407 to 525 nm.
NMR was employed to investigate the interaction of DPP1 or
DPP2 with Zn2+. When 1 equiv of ZnCl2 was introduced in a
solution of DPP1 or DPP2 (in CD3CN), noticeable downfield
chemical shifts of the pyridyl protons were recorded (Figure
3b), demonstrating that Zn2+ binding to the ligand occurred
through the pyridyl N-donor atom.46 Overall, both UV−vis and
NMR studies confirmed Cu2+ and Zn2+ binding to DPP1 and
DPP2.
<−1.0 (poorly
distributed in the
brain)
b
−logPe
CNS
4.2 0.1
4.2 0.1
CNS+
CNS+
−logPe < 5.4 (CNS+)
−logPe > 5.7 (CNS−)
c
prediction
a
MW, molecular weight; clogP, calculated logarithm of the octanol−
water partition coefficient; HBA, hydrogen-bond acceptor atoms;
HBD, hydrogen-bond donor atoms; PSA, polar surface area; logBB =
−0.0148 × PSA + 0.152 × clogP + 0.130. The values of −logPe were
measured by the parallel artificial membrane permeability assay
b
c
(PAMPA). Compounds categorized as CNS+ have the ability to
permeate through the BBB and target the CNS. In the case of
compounds assigned as CNS− they have poor permeability through
the BBB and therefore, their bioavailability into the CNS is considered
to be minimal.
are druglike and possibly BBB permeable. To verify this
prediction for BBB penetration of compounds, we performed in
vitro PAMPA-BBB following a previously reported proce-
dure.29,40−42 Permeability values (−logPe) were measured to be
4.2 ( 0.1) for both DPP1 and DPP2 (Table 1). On the basis
of empirical classification of BBB-permeable molecules (i.e.,
verapamil),29,40−42 DPP1 and DPP2 would also be likely to
cross the BBB.
Along with BBB permeability, the solution speciation of
DPP1 and DPP2 was determined through UV−vis variable-pH
titration experiments (I = 0.10 M NaCl; room temperature; pH
2−10).27,29−33,43,46 As summarized in Figure 2, titration results
To further identify binding stoichiometry and affinity, the
solution speciation investigation of the Cu2+−DPP2 complexes
was carried out through UV−vis variable-pH titration experi-
ments (1:2 [Cu2+]/[DPP2]; I = 0.10 M NaCl, room
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pH 7 are a mixture of Cu(LH) and CuL complexes in a ratio of
3:2. Free Cu2+ was shown up to pH 7, indicating pCu = 6.6 at
pH 6.6 (pCu = −log[Cuunchelated]) (Figure 4).27,29,31−33,43,46
The pCu value suggests the approximate dissociation constant
(Kd) of Cu2+−DPP2 to be ca. high nanomolar. When
compared to the reported Kd values of Cu2+−Aβ (picomolar
to nanomolar),2,5,7,8,12,14,21 DPP2 may interact with Cu2+ from
soluble Cu2+−Aβ species. In order to test if Cu2+ binding of
ligand occurs in the presence of Aβ, a solution containing
DPP2 with Cu2+-treated Aβ was monitored by UV−vis. The
new spectral features that coincided with those of the Cu2+−
DPP2 complex without Aβ were observed, suggesting an
interaction of DPP2 with Cu2+ in the presence of Aβ (see
Figure S1 in the Supporting Information). Cu2+ binding of
DPP2 with Aβ occurred more slowly than that without Aβ,
proposing that Aβ might interfere with metal binding to the
ligand. Taken together, our spectroscopic studies present the
capability of DPP1 and DPP2 to chelate Cu2+ and Zn2+, as well
as the potential interaction of DPP2 with Cu2+ in the presence
of Aβ species, which may be associated with its noticeable
reactivity toward metal−Aβ species (vide infra).
The metal selectivity of DPP1 and DPP2 was also
determined by competitive reactions with Cu2+ over bio-
logically relevant divalent metal ions (Mg2+, Ca2+, Mn2+, Fe2+,
Co2+, Ni2+, and Zn2+), which was monitored by UV−vis. As
depicted in Figure S2 in the Supporting Information, DPP1
and DPP2 displayed selectivity for Cu2+ over Mg2+, Ca2+, Mn2+,
and Zn2+. Binding of both ligands to Co2+ and Ni2+ was also
observed. Considering the lower abundance of Co2+ and Ni2+
than Cu2+ in biological systems,55,56 the overall metal selectivity
of DPP1 and DPP2 may be sufficient to be used for targeting
and interacting with Cu2+−Aβ species in heterogeneous
biological environments like the brain.
Aβ Interaction with DPP1 and DPP2 Studied by MS
and Docking Studies. The interaction of DPP1 and DPP2
with Aβ1−40 in the absence of metal ions was probed by ESI-
MS, tuned to preserve noncovalent protein−ligand interac-
tions.49 At a low Aβ concentration (10 μM), a small signal
corresponding to the interaction between DPP2 (30 μM) and
the Aβ monomer in the 3+ charge state could be detected,
whereas no interaction between DPP1 (60 μM) and the
peptide was observed under these conditions (see Figure S3 in
the Supporting Information). At high concentrations of the
peptide (100 μM) and compounds (600 μM), both DPP1 and
DPP2 interacted with Aβ species to different extents (Figure
5a). Data for DPP1 indicated that the molecule interacted
broadly with Aβ monomers and oligomers in 1:1, 2:1, and 3:1
Aβ to ligand ratios. In the case of DPP2, a stronger preference
toward larger Aβ oligomers was shown, but with similar
stoichiometries as DPP1. The total bound intensities recorded
from MS data, and those from individual oligomeric species
were shown in Figure 5b and Table S1 in the Supporting
Information. The intensities shown were normalized for both
nonspecific interactions and artifactual complexes formed
during the electrospray process using Aβ:ThT binding data as
a control, and ion mobility separation was used to separate
oligomers that overlapped in m/z.6,49,57 From these data, it was
clear that, at high concentrations, a higher proportion of DPP1
was bound to Aβ species than DPP2, but that both could be
classified as having weak Aβ affinity in solution (low mM Kd).
Therefore, a weak Aβ/compound interaction was captured by
MS. Normalized intensity MS data suggest that DPP2 binding
was almost exclusively driven through Aβ multimer interactions
Figure 3. Cu2+ or Zn2+ binding of DPP1 and DPP2. (a) UV−vis
spectra of DPP1 (left) and DPP2 (right) with CuCl2 (1−20 equiv) in
EtOH at room temperature (incubation for 2.5 h (for DPP1) and 5
min (for DPP2)). (b) H NMR spectra of DPP1 (left, black) or
DPP2 (right, black) with ZnCl2 (red) in CD3CN at room temperature
1
([compound] = 4 mM; [ZnCl2] = 4 mM).
temperature). Based on the pKa values of DPP2 and these
titration results, stability constants for the Cu2+−DPP2
complexes were obtained (Figure 4, M + LH ⇌ M(LH) (log
β1 = 12.99(9)); M + L ⇌ ML (log β2 = 5.85(3)); M = Cu2+, L
= DPP2). A solution speciation diagram was generated from
these stability constants, suggesting that the major species at
Figure 4. Solution speciation investigation of the Cu2+−DPP2
complexes. Top left: UV−vis spectra (pH 2−7) for the Cu2+−DPP2
complexes ([Cu2+]/[L] = 1:2; [Cu2+]total = 10 μM; 7 h incubation with
ligand (L) prior to pH titration, L = DPP2; room temperature). Top
right: Solution speciation diagram of the Cu2+−DPP2 complexes (FCu
= fraction of free Cu and Cu complexes). Bottom: Stability constants
(log β) of the Cu2+−DPP2 complexes. Charges are omitted for clarity.
aError in the parentheses is shown in the last digit. bThe species
containing CuL2 was introduced into the calculation model yielding a
good fit to the data.
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Figure 5. Interactions of DPP1 and DPP2 with Aβ. (a) MS data for the complexes of Aβ1−40 and DPP1 or DPP2 ([Aβ] = 100 μM; [compound] =
600 μM; M = monomer, D = dimer, and T = trimer). Many binding stoichiometries were detected, including 1:1 (star), 2:1 (square), and 3:1
(triangle). (b) A histogram showing the total bound MS signal intensity, normalized for nonspecific interactions and ESI-MS artifacts, for each
binding stoichiometry observed in (a). (c) Docking studies of DPP1 (orange) and DPP2 (green) with Aβ1−40 (PDB 2LFM) by AutoDock Vina.
Poses for both compounds were overlapped in this conformation (other conformations, see Figure S4 in the Supporting Information). The helical
region of Aβ (H13-D23) is highlighted in color (tan) in both the cartoon (left) and surface (right) representations.
(Figure 5b). Overall, our MS results suggest that although both
compounds could interact with Aβ species at high concen-
trations, DPP2 was able to bind Aβ species at both low and
high concentrations.
(6E10), whereas morphological changes were identified by
transmission electron microscopy (TEM).26,28−33,53
The inhibition studies, as shown in Figure 6, demonstrated a
different reactivity of DPP1 or DPP2 toward metal-induced Aβ
species over metal-free Aβ species. In the case of metal-involved
Aβ aggregation, Aβ species with a wide range of MW were
indicated with DPP2 for both Cu2+- and Zn2+-treated samples
upon longer incubation (Figure 6a, lanes 6 and 9). In the
samples containing DPP1 (lanes 5 and 8), less intense gel
bands were detected across the longer incubation time,
suggesting that further Aβ aggregation may have occurred.
The reaction of DPP1 or DPP2 with metal-free Aβ also
exhibited a different distribution of various-sized Aβ species
(Figure 6a, lanes 2 and 3). Aβ species formed with compounds
in both metal-mediated and metal-free conditions were not
completely denatured by SDS implying that these molecules
may generate different Aβ assemblies (Figure 6b). TEM images
of metal-induced Aβ species incubated with DPP2 for 24 h
revealed smaller amorphous aggregates compared to DPP1;
some of the metal-free Aβ species treated with DPP1 and
DPP2 presented similar morphology to those untreated with
compounds (Figure 6c). Overall, DPP1 and DPP2 displayed
their ability to recognizably modulate metal-induced Aβ
aggregation to different extents.
To visualize the potential interaction between Aβ and
DPP1/DPP2, docking studies by AutoDock Vina50 were
performed using the previously determined NMR structure
(PDB 2LFM)51 of Aβ1−40 monomer. Typically, both
compounds were positioned between the α-helix and the
unstructured N-terminal side of Aβ (Figures 5c and Figure S4
in the Supporting Information). Most docked structures
showed a nonspecific orientation of the ligand with respect to
the surface features of Aβ. Our preliminary docking studies
support the potential interaction of the compounds with Aβ
monomer.
Effects of DPP1 and DPP2 on Metal-Free and Metal-
Induced Aβ Aggregation in Vitro. Confirming metal
binding and Aβ interaction properties (bifunctionality) of
DPP1 and DPP2, their influence on in vitro metal-free and
metal-induced Aβ aggregation was studied.26−33 Two different
experiments (inhibition and disaggregation) were performed to
investigate whether DPP1/DPP2 can control the formation of
metal-free and metal-induced Aβ aggregates (inhibition, Figure
6) or transform preformed metal-free and metal-induced Aβ
aggregates (disaggregation, see Figure S5 in the Supporting
Information). Various-sized Aβ species from both studies were
monitored by native gel electrophoresis and SDS-PAGE
followed by Western blotting with an anti-Aβ antibody
Furthermore, for the disaggregation experiment (see Figure
S5 in the Supporting Information), DPP2-treated metal-
triggered Aβ aggregates presented different-sized Aβ species
than DPP1-treated samples, indicating that DPP2 could alter
the properties of preformed Aβ aggregates to a greater extent
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Figure 6. Inhibition experiment (scheme, top). Analysis of various-sized Aβ species by (a) native gel electrophoresis and (b) SDS-PAGE
(nonreducing conditions) with Western blot using an anti-Aβ antibody (6E10). (c) TEM images of the 24 h incubated samples. Conditions: [Aβ] =
25 μM; [CuCl2 or ZnCl2] = 25 μM; [compound] = 50 μM; pH 6.6 (for Cu2+ samples) or 7.4 (for metal-free and Zn2+ samples); 4, 8, or 24 h
incubation; 37 °C; constant agitation.
than DPP1 (see Figure S5a in the Supporting Information). In
the metal-free conditions, more various-sized Aβ species were
indicated in the presence of DPP2 than DPP1 (in particular, at
4 h, Figure S5a, lanes 2 and 3). The Aβ species generated with
compounds in both metal-triggered and metal-free conditions
were relatively stable in the presence of SDS (see Figure S5b in
the Supporting Information). As shown in Figure S5c in the
Supporting Information, DPP2 was able to reorganize
preformed structured metal−Aβ aggregates to amorphous
species more noticeably than DPP1. Taken together, the
results from both the inhibition and disaggregation experiments
presented that DPP1 and DPP2 could regulate metal-involved
Aβ aggregation over metal-free aggregation in vitro differently.
Moreover, the structural variation (i.e., dimethylamino
functionality) may enhance the contact with Aβ species via
hydrogen bonding and/or hydrophobic interactions,29,31,36
which may afford greater reactivity toward metal-Aβ species.
metal chelation site and a diphenylpropynone framework (for
Aβ interaction), which could possibly serve as chemical
reagents to target and modulate metal−Aβ species in vitro.
Their bifunctionality (metal chelation and Aβ interaction) was
confirmed by physical methods and preliminary docking
studies. Biochemical and TEM studies revealed that DPP1
and DPP2 could modulate metal-induced Aβ aggregation in
vitro. Notably, DPP2, which has a dimethylamino group,
exhibited more apparent reactivity toward metal−Aβ species,
compared to DPP1. This suggests that the interaction and
reactivity of molecules with metal−Aβ species can be tuned by
such structural variations, proposing a structure-interaction-
reactivity relationship. DPP1 and DPP2, however, would be
limited in their biological applications since they displayed
cytotoxicity in living cells at low micromolar concentrations
(see Figure S6 in the Supporting Information). Overall, the
promising in vitro reactivity of these potentially BBB-permeable
molecules toward metal−Aβ species warrants pursuit of
structural modifications that would improve the viability of
diphenylpropynone derivatives in biological settings, followed
by more detailed characterization by MS and molecular
SUMMARY AND PERSPECTIVE
■
Following the incorporation approach, we have developed two
bifunctional small molecules (DPP1 and DPP2) composed of a
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modeling. Our studies have demonstrated the capability of two
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ASSOCIATED CONTENT
■
S
* Supporting Information
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J.; Rath, N. P.; Kim, J.; Mirica, L. M. J. Am. Chem. Soc. 2012, 134,
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Table S1 and Figures S1−S6 (PDF). This material is available
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(27) Rodríguez-Rodríguez, C.; San
́
chez de Groot, N.; Rimola, A.;
Alvarez-Larena, A.; Lloveras, V.; Vidal-Gancedo, J.; Ventura, S.;
Vendrell, J.; Sodupe, M.; Gonzalez-Duarte, P. J. Am. Chem. Soc. 2009,
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AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: bruotolo@umich.edu (B.T.R.), mhlim@
umich.edu (M.H.L.).
■
́
́
́
(28) Hindo, S. S.; Mancino, A. M.; Braymer, J. J.; Liu, Y.;
Vivekanandan, S.; Ramamoorthy, A.; Lim, M. H. J. Am. Chem. Soc.
2009, 131, 16663−16665.
Author Contributions
‡These authors contributed equally.
(29) Choi, J.-S.; Braymer, J. J.; Nanga, R. P. R.; Ramamoorthy, A.;
Lim, M. H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 21990−21995.
(30) Choi, J.-S.; Braymer, J. J.; Park, S. K.; Mustafa, S.; Chae, J.; Lim,
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(31) Braymer, J. J.; Choi, J.-S.; DeToma, A. S.; Wang, C.; Nam, K.;
Kampf, J. W.; Ramamoorthy, A.; Lim, M. H. Inorg. Chem. 2011, 50,
10724−10734.
This study was supported by the Alzheimer’s Art Quilt
Initiative, the Alzheimer’s Association (NIRG-10-172326),
and the Ruth K. Broad Biomedical Foundation (to M.H.L.),
as well as start-up funding from the University of Michigan (to
B.T.R.). A.K., M.T.S., and A.S.D. are grateful for the Research
Excellence Fellowship from the Department of Chemistry at
the University of Michigan, the NIH Training Grant (T32
CA140044), and the NSF Graduate Research fellowship,
respectively. We thank Dr. Xiaoming He for synthetic
assistance and Nathan Merrill for the NMR measurements
for Zn2+ binding studies.
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