Platinum-based inhibitors of amyloid-? as therapeutic
agents for Alzheimer’s disease
Kevin J. Barnham*†‡§, Vijaya B. Kenche*†‡, Giuseppe D. Ciccotosto*†‡, David P. Smith*¶, Deborah J. Tew*†‡, Xiang Liu*†,
Keyla Perez*†‡, Greg A. Cranston*†‡, Timothy J. Johanssen*†‡, Irene Volitakis*†, Ashley I. Bush*†, Colin L. Masters*†?,
Anthony R. White*†?, Jeffrey P. Smith*†, Robert A. Cherny*†, and Roberto Cappai*†‡
*Department of Pathology,†The Mental Health Research Institute,‡Bio21 Institute of Molecular Science and Biotechnology, and?Centre for Neuroscience,
University of Melbourne, Parkville, Victoria, 3010, Australia
Edited by Peter J. Sadler, University of Warwick, Coventry, United Kingdom, and accepted by the Editorial Board March 2, 2008 (received for review
January 23, 2008)
Amelyoid-? peptide (A?) is a major causative agent responsible for
Alzheimer’s disease (AD). A? contains a high affinity metal binding
site that modulates peptide aggregation and toxicity. Therefore,
identifying molecules targeting this site represents a valid thera-
peutic strategy. To test this hypothesis, a range of L-PtCl2 (L ?
1,10-phenanthroline derivatives) complexes were examined and
shown to bind to A?, inhibit neurotoxicity and rescue A?-induced
synaptotoxicity in mouse hippocampal slices. Coordination of the
complexes to A? altered the chemical properties of the peptide
inhibiting amyloid formation and the generation of reactive oxy-
gen species. In comparison, the classic anticancer drug cisplatin did
not affect any of the biochemical and cellular effects of A?. This
implies that the planar aromatic 1,10-phenanthroline ligands L
confer some specificity for A? onto the platinum complexes. The
potent effect of the L-PtCl2 complexes identifies this class of
compounds as therapeutic agents for AD.
in Alzheimer’s disease (AD) (1), although the neurotoxic mech-
anism(s) and pathway(s) involved remain unresolved (2). Given
its central role in AD, diverse therapeutic strategies that target
the generation, disaggregation, and clearance of A? are being
pursued. Our sequence activity studies have shown that altering
the metal binding activity of A? inhibits its neurotoxic activity
(3, 4). Methylation of the imidazole side chains of His-6, -13, and
-14, which constitute the high affinity metal binding site (5),
changed A?–metal interactions and A?:cell binding and ren-
dered the peptide nontoxic (3, 4). An important conclusion from
these studies is that agents, which can target the metal binding
thirty years, and annual sales exceed one billion U.S. dollars. The
structure of the prototypic anti-cancer drug, cisplatin [cis-
Pt(NH3)2Cl2] (1) is shown in Fig. 1a. Despite this success, there
is scant evidence that Pt-based drugs are viable therapeutics for
other diseases. The anticancer Pt compounds have been termed
‘‘DNA alkylators,’’ because they bind to the nucleobases of DNA
(guanine in particular). These interactions apparently depend on
the specific formation of hydrogen bonds between the am(m)ine
ligands bound to the Pt and the DNA (6).
Given the importance of the histidine residues in the A? metal
binding site (7), we targeted the imidazole side chains as a
strategy to inhibit A?’s neurotoxic activity. Imidazole side chains
are excellent ligands for a variety of metal ions, including Pt(II).
Our strategy was to design a specific class of ligand that would
explicitly target Pt(II) to the histidine residues of A?. Pt(II)
complexes with 1,10-phenanthroline ligands (Fig. 1a) have been
well characterized. The ligands themselves have an intrinsic,
albeit very weak (approximately millimolar) affinity for A? (8).
Because the ligands 2,9-dimethyl-4,7-diphenyl-1,10-phenanthro-
line and 4,7-diphenyl-1,10-phenanthroline interact predomi-
significant body of data indicates the amyloid-? peptide
nantly with the aromatic residues Phe-4, Tyr-10, and Phe-19 (8),
it was proposed that the ligand-A? contacts were mediated
through ?–? stacking interactions (8). The A? amino acid
sequence (Fig. 1b) shows that the histidine residues (6, 13, and
14) and the aromatic residues are located in the hydrophilic
N-terminal domain. The residue most perturbed by the ligand
binding, Tyr-10, is located in the middle of the sequence that
spans the metal binding site of A?, whereas Phe-4 and Phe-19
flank the metal binding site. Therefore, we have postulated that
Pt(II)-1,10-phenanthroline complexes will also target the N-
region and so modify the behavior of the A? peptide.
We have evaluated three platinum phenanthroline derivatives,
G.A.C., T.J.J., I.V., A.R.W., and J.P.S. performed research; A.I.B., C.L.M., R.A.C., and R.C.
contributed new reagents/analytic tools; K.J.B., R.A.C., and R.C. analyzed data; and K.J.B.
wrote the paper.
§To whom correspondence should be addressed. E-mail: email@example.com.
9JT, United Kingdom.
© 2008 by The National Academy of Sciences of the USA
proposed to be targeted by L-PtCl2, are highlighted, as are the aromatic
residues Phe-4, -19, and -20 and Tyr-10, which are critical to the interactions
with the polyaromatic ligands L.
Pt complexes and A? sequences. (a) The structures of L-PtCl2com-
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roline)Cl2 (3), and Pt(4,7-diphenyl-1,10-phenanthroline disul-
fonate)Cl2(4) (Fig. 1a) ability to inhibit the metal-dependent
biochemical and cellular actions of A?, while using the proto-
typic anticancer drug cisplatin (1) (Fig. 1a) as a negative control.
The work demonstrates that Pt(II)-1,10-phenanthroline com-
plexes coordinate to the histidine residues of A? and act as
potent inhibitors of A?–metal chemistry and synaptotoxic
activity. These complexes have potential as therapeutic agents
Selection of Platinum Complexes to Target A?. Pt compounds
(L-PtCl2; L ? 1,10-phenanthroline ligand) 2–4 (Fig. 1a) were
selected for testing, because, after loss of chloro ligands, they are
predicted to coordinate to the histidine residues of the A?
peptide and alter its physiochemical and biological properties.
Cisplatin (1) lacks the organic scaffold predicted to be necessary
to target the Pt(II) metal center to the histidine residues of A?
and should have little effect on A? properties.
Platinum Complexes Bind to A?. Mass spectrometry. To determine
whether the platinum complexes bind to A?, the L-PtCl22–4 and
cisplatin compounds were incubated with A?1–42 and the
product solutions analyzed by surface-enhanced laser desorp-
tion/ionization time-of-flight (SELDI-TOF) mass spectrometry,
using 4G8 as the capture antibody. The A? peptide alone gave
a single peak at 4,515 ? 1 Da, corresponding to its expected mass
(4,515 Da) (Fig. 2a). Incubation of A?42 overnight with 2
produced a second peak at 4,890 ? 1 Da (Fig. 2a). The increase
in mass of 375-Da units corresponds to the formation of a
L-Pt-A? adduct in which the two chloro ligands of 2 have been
displaced as the compound coordinated to A?. The mass spectra
from the incubation of A? with cisplatin, 3 and 4 showed that
similar adducts are formed by these compounds (data not
NMR spectroscopy. As a ‘‘soft’’ metal, Pt has a preference for
ligands with ‘‘soft’’ donor atoms. Within the A? sequence,
potential Pt binding sites include the ‘‘soft’’ sulfur atom of
Met-35 and the imidazole nitrogen atoms of the three histidine
side chains that are considered an intermediate between ‘‘hard’’
and ‘‘soft’’ ligand. In keeping with these preferences, cisplatin
has a high affinity for sulfur containing ligands, such as gluta-
thione, but, because of its relatively slow kinetics, it is still able
to form complexes with nitrogen-based ligands, such as with the
nucleobases of DNA (9). To confirm that the complexes L-PtCl2
coordinated the histidine residues of A?, their interaction with
A?40 was monitored by1H NMR. A?40 was used in these
experiments, because its greater solubility and slower aggrega-
tion rates over A?42 allowed NMR experiments to be per-
formed. Spectra recorded before and after the addition of 2 to
solutions of A?40 revealed a strong perturbation of the peaks
corresponding to the C4H and C2H of the imidazole side chains
of His-6, -13, and -14, consistent with the complex coordinating
to these residues (Fig. 2b). The assignment of these peaks as
being due to the imidazole side chains was confirmed by 2D1H
TOCSY NMR spectra and are consistent with data published in
ref. 10. There was little perturbation of the peak due to C?H3of
Met-35, indicating no significant interaction with this residue.
Although the SELDI-TOF MS data indicated that cisplatin was
also able to form adducts with A?; the1H NMR spectrum of
solutions of 1 with A? showed that the nature of these adducts
is different from those formed with 2. A reduction in intensity
of the peak due C?H3of Met-35 was observed together with a
broadening of the spectrum (Fig. 2b). These observations are
consistent with the formation of A? oligomers and multiple
L-PtCl2Complexes Alter A? Secondary Structure: Circular Dichroism
Spectroscopy. Aging aqueous solutions of A?42 in the presence
of equimolar Cu2?results in a conformational change from a
predominately random coil conformation to a ?-sheet confor-
mation (Fig. 3) (3, 11). The presence of compounds 2–4 in aging
A?42/Cu2?solutions induced a change in peptide conformation
(Fig. 3) with a blue shift away from the 215-nm minimum
normally associated with classic ?-sheet structures. Although the
spectra of the A?42/Cu2?/L-PtCl2solutions could not be fitted
to any of the classic secondary structure peptide conformations,
the observed differences are consistent with different confor-
mations. An alternative explanation for the changes observed is
that compounds 2–4 have aromatic ligands and, as such, will
absorb in the UV region and when coordinated to the peptide
will be in a chiral environment and, therefore, being optically
1H NMR Spectra of A?40 before and after the addition of 1 and 2. The spectra show that 2 is able to perturb the resonances because of the C4H protons of the
imidazole side chains of the three histidine residues, whereas cisplatin does not (see box centered at 7.15 ppm). Conversely, the peak due to the S-CH3protons
of Met 25 at 2.12 ppm was significantly reduced in intensity after incubation with cisplatin, whereas incubation with 2 does not significantly affect this peak.
The peak marked with asterisk was due to acetate impurity present in the peptide samples.
Pt complexes coordinate A?. (a) SELDI-TOF Mass spectrum of A?42 incubated (2). The mass spectrum shows the formation of an A? drug adduct. (b)
www.pnas.org?cgi?doi?10.1073?pnas.0800712105 Barnham et al.
region. Either explanation is consistent with the compounds
coordinating to A?.
L-PtCl2 Complexes Inhibit A? Aggregation. A? toxicity is strongly
correlated with peptide aggregation. The time-dependent ag-
gregation of A? into amyloid fibrillar structures can be followed
by using ThT, which gives a characteristic fluorescence signal
when bound to amyloid (12). Compounds 2–4 all inhibited ThT
fluorescence in a dose-dependent manner (Fig. 4), indicating
that the formation of amyloidogenic structures was inhibited.
This was substantiated by negative staining EM, which showed
the generation of amphorous aggregates rather than amyloid
fibrils (data not shown). Both ThT and EM showed that cisplatin
(1) did not inhibit amyloid formation (data not shown).
L-PtCl2Complexes Inhibit Copper Redox Chemistry. We showed that
A? in the presence of Cu2?is able to generate H2O2catalytically
(13, 14). To ascertain whether the Pt complexes could inhibit
A?:Cu2?-mediated redox chemistry, the complexes were titrated
into an A?:Cu preparation, and H2O2generation was measured
with a fluorimetric assay (13). The IC50values for compounds
1–4 are reported in Table 1. Although cisplatin had no observ-
able effect, compounds 2–4 inhibited Cu2?-mediated H2O2
production with an IC50in the nanomolar range. This level of
8-hydroxyquinoline), which inhibits A?:Cu redox chemistry by
directly targeting the Cu rather than the peptide component of
the A?–metal complex (14).
L-PtCl2Complexes Inhibit A? Neurotoxicity. Having demonstrated
that the L-PtCl2complexes could bind to the A? peptide and
change its chemical and structural properties, we assessed their
ability to inhibit A? toxicity in primary mouse cortical neuronal
cell cultures. Treatment of the neurons with 10 ?M A?42 for 4
days reduced cell viability to 65% as measured by the MTS assay
(Fig. 5). Coincubation of 10 ?M A?42 with 2–4 at either 10 ?M
or 5 ?M significantly increased cell viability (Fig. 5). Compound
3 completely restored neuronal viability, whereas cisplatin 1 was
inactive at either 5 or 10 ?M. Compounds 2–4 were not toxic at
the concentrations tested (data not shown).
L-PtCl2Complexes Rescue A?: Inhibition of Long-Term Potentiation.
Long-term potentiation (LTP) in the rodent hippocampal slice
is a measure of synaptic plasticity that focuses on activity-
dependent persistent increases in synaptic strength and is con-
sidered to be the biochemical basis of learning and memory (15,
16). Synthetic and cell derived A? can inhibit LTP in vitro and
in vivo and supports the role of A? in promoting the learning and
memory loss that occurs in AD (17, 18). The high-frequency
stimulation of a mouse hippocampal slice gives an LTP ranging
from 148% (Fig. 6b) to 135% (Fig. 6c). Incubating the hip-
pocampal slice with 2 ?M A?42 for 30 min before the stimulus
for LTP significantly reduced LTP from 148% to 124% (Fig. 6b)
and from 135% to 105% (Fig. 6c). Compound 3 was chosen as
the L-PtCl2complex to be tested in the LTP assay as it had the
best handling properties with respect to high solubility and low
heterogeneity. Conversely, 2 has low solubility, and 4 is heter-
ogeneous because of the multiple positions occupied by the
sulfonate groups on the bathophenanthroline ligand. The addi-
tion of 4 ?M compound 3 to the A?42 solution completely
reversed the A?42-inhibition of LTP (Fig. 6 a and b). In
comparison, 4 ?M cisplatin did not affect A?42-dependent
inhibition of LTP (Fig. 6c), supporting the specificity of the
at 37°C for 17 h with equimolar Cu alone (solid line) or in the presence of 2
(dotted line), 3 (dashed line), or 4 (dot-dash line).
Pt 1,10-phenanthroline complexes inhibit amyloid formation in a dose-
subtraction of the blank and by taking A?42 in the absence of Pt complexes as
unity. Data are displayed in arbitrary units. Open squares, 2, ?1 arbitrary unit;
open circles, 3, ?0 arbitrary units; open triangles, 4, ?1 arbitrary unit.
Inhibition of amyloid formation. The ability of the Pt complexes to
Table 1. IC50values for the inhibition of copper-mediated
hydrogen peroxide generation by A?
Compound IC50, ?M
10 ?M A?42 for 4 more days. The platinum compounds 2, 3, and 4 but not 1
#, P ? 0.001 versus A?42. n ? 3–6 samples per group. Each sample group was
done in triplicate. Results are shown as mean ? SE.
Inhibition of A?42 induced neurotoxicity. Primary cortical neurons
Barnham et al.
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effect of 3 being mediated through its organic scaffold. Com-
pound 3 alone did not affect LTP, consistent with specific
targeting of the A?42-dependent inhibition of LTP.
The pathological accumulation of A? in the brain is a major
hallmark of AD. Genetic studies from early onset cases of AD
identify alterations in A? metabolism as being directly linked to
the disease (19). Although the mechanism of A? neurotoxicity
is still unknown, it is generally accepted that the A? peptide is
a valid target for therapeutic development. Because it is un-
structured in the native state, the de novo design of effective
inhibitors of A? is problematic. We have adopted a strategy of
using metal compounds that target A? specifically by taking
advantage of its intrinsic affinity for metal ions. The three
histidine residues His-6, -13, and -14 are the A? metal binding
ligands (5). Methylation of the imidazole side chains altered Cu
binding and inhibited A? toxicity (3, 4).
We used Pt(II) complexes to target the A?–metal binding site.
Pt(II) compounds are stable and essentially redox inert when
present in biological systems. The slow kinetics associated with
substitution reactions at the Pt(II) center means that, once
bound to a target, the Pt(II) metal is difficult to displace. The
specificity of the interaction between Pt anticancer drugs and
DNA has been attributed largely to the ability of the am(m)ine
ligands to form hydrogen-bonds to guanine nucleotides of DNA.
To promote specific binding to A? by L-PtCl2 2–4 complexes,
the 1,10-phenanthroline ligand L was designed to target the
N-terminal domain of A?. This was based on the observation
that polyaromatic compounds bind to A? and inhibit its aggre-
gation (20). Moreover, the classic amyloid-binding fluorescent
dyes Congo red and thioflavin T are also polyaromatic com-
pounds. L-PtCl2complexes are highly stable and the likelihood
of the chelating ligand, L, dissociating from the metal is remote.
We demonstrated that the metal free ligands, L, bind weakly to
A? via interactions with the aromatic residues Phe-4, Tyr-10 and
Phe-19 on A? (8). Importantly for our strategy, these residues
span the metal binding residues His-6, -13, and -14.
To establish that the 1,10-phenanthroline ligands were con-
ferring the necessary specificity of action on the Pt complexes,
we tested the compound’s ability to inhibit key activities of A?
and compared them with cisplatin, which lacks the polyaromatic
ligand (Fig. 1a). The SELDI-TOF mass spectra and NMR
spectra (Fig. 2) indicated that the L-PtCl2complexes bound to
A? as a stable adduct primarily at the histidine residues. In
contrast, although cisplatin (1) did form adducts with A?, it
coordinated predominantly at the sulfur atom of Met-35. The
ability of the L-PtCl2complexes to promote coordination to A?
via the histidine residues demonstrates that the 1,10-
phenanthroline ligands target these residues as predicted.
The coordination of the L-PtCl2complexes to A? significantly
altered the chemical and biophysical properties of the peptide.
This is reflected in the altered secondary structure indicated by
CD spectra (Fig. 3), inhibition of amyloid formation (Fig. 4), and
A?:Cu2?-mediated redox chemistry (Table 1). An interesting
observation from these biophysical/chemical studies is that,
although the Pt-free 1,10-phenanthroline ligands have a very low
millimolar affinity for A? (8), the corresponding Pt complexes
circles), LTP was significantly inhibited by treating the slices for 30 min with 2 ?M A? (open circles). Time-matched treatment of control slices in 4 ?M 3 alone
did not affect LTP (closed triangles), but inhibition of LTP by A? was completely reversed by cotreatment with 4 ?M 3 (open triangles). (b) Bar graphs showing
LTP levels quantified as the baseline-percentage of the fEPSP averaged from 55 and 60 min after tetanus ? SE. Treatment in ACSF alone produced LTP levels of
148 ? 10%, whereas treatment with A? significantly reduced these levels to 122 ? 7%. Treatment with 3 gave LTP of 155 ? 10% that was not significantly
different from control but was significantly greater than slices treated with A? alone. Most importantly, treatment with A? and 3 together restored the levels
of LTP to 145 ? 9% and was not significantly different from controls. (c) Bar graphs from a similar set of experiments showing that cisplatin does not affect LTP
or its inhibition by A?. ACSF alone produced LTP levels of 135 ? 10%. Treatment with 2 ?M A? significantly reduced these levels to 104 ? 10% of baseline.
Treatment with 4 ?M cisplatin did not affect LTP levels compared with control and were measured at 134 ? 7%. Cotreatment of slices with A?and cisplatin
together did not affect the inhibition of LTP by A?, producing LTP of 96 ? 6%.
www.pnas.org?cgi?doi?10.1073?pnas.0800712105Barnham et al.
are potent inhibitors of A?. This reflects that Pt chemistry is
kinetically, rather than thermodynamically, controlled. There-
fore, even though the low affinity of L for A? should result in
short residency times with the intended target, the kinetic
inertness of Pt complexes means that, once bound to the target,
the L-Pt:A? adducts are very stable and will not dissociate. The
synergistic effect of coupling the low affinity ligand and kinet-
ically stable Pt is highlighted by the lack of activity by the
cisplatin in inhibiting either amyloid formation or A?:Cu2?
redox chemistry. The inability of cisplatin to inhibit A?:Cu2?-
mediated redox chemistry is interesting, because cisplatin will
coordinate to Met-35, and this residue has been implicated in the
redox chemistry of A? (21, 22).
A key test of the L-PtCl2complexes potential efficacy is their
We examined the compounds ability to rescue A? induced
toxicity in primary neuronal cell cultures and A? induced
inhibition of LTP in hippocampal neuronal slices. All three
L-PtCl2complexes rescued A? induced toxicity in the primary
cortical neurons, whereas cisplatin was inactive. The mechanism
of A? toxicity is still unclear, with proposed mechanisms de-
pendent on a variety of A? biophysical and chemical properties,
such as peptide aggregation, and the ability of A? to coordinate
metal ions, such as copper and zinc (2). Changes in the CD
L-PtCl2 complexes altered the structural properties of A?.
Because the L-PtCl2 complexes coordinate to the histidine
residues of A?, they occupy the zinc and copper binding site on
A? and so inhibit metal-mediated phenomena, such as ROS
Having established that the L-PtCl2 complexes inhibit A?
neurotoxicity, we investigated whether an example of this class
of compound could rescue A?-induced inhibition of LTP.
Compound 3 was chosen as the L-PtCl2complex to be tested in
this assay, because it had the best handling properties with
respect to high solubility and low heterogeneity. Conversely, 2
has low solubility, and 4 is heterogeneous because of the multiple
positions occupied by the sulfonate groups on the bathophenan-
throline ligand. Electrophysiological recordings of LTP in the
hippocampal slice measure the level of synaptic strengthening
after high-frequency stimulation of a population of synaptically
connected neurons. Because changes in synaptic strength are
thought to underlie the learning and memory processes, the LTP
assay and A? inhibition of LTP is an established method for
investigating A? synaptotoxicity (17, 18) and assessing putative
AD-therapeutic compounds. Compound 3 completely reversed
it did not significantly alter the levels of LTP (Fig. 6). Impor-
tantly, cisplatin did not affect either LTP or rescue A?-inhibition
of LTP, thus supporting the specificity of 3. These cellular results
demonstrate the potency, specificity, and efficacy of this com-
pound in reducing the neurotoxic and synaptotoxic activities
The data presented here indicate that the 1,10-phenanthroline
complexes of Pt(II) coordinate to the histidine imidazole side
chains of A? and alter the biochemical and biophysical proper-
ties of the peptide. Importantly, these alterations to the physical
properties of A? potently inhibit the peptides neurotoxic and
synaptotoxic actions. The inactivity of cisplatin indicates that the
aromatic 1,10-phenanthroline scaffold coordinated to Pt(II)
conferred the necessary specific targeting of the Pt to the
histidine residues of A?. The results achieved with the L-PtCl2
complexes support the future development of this class of
compound as therapeutic agents for AD to ensure they effi-
ciently cross the blood–brain barrier. The potent effects of these
Pt complexes define the histidine residues of A? as a viable
therapeutic target to inhibit the neurotoxic and synaptotoxic
actions of A?.
Materials and Methods
Pt complexes 1 and 2 were purchased from Aldrich; 3 and 4 were prepared as
described in ref. 23. Peptides were obtained from AusPep and from the W. M.
Keck Laboratory (Yale University, New Haven, CT).2H2O was obtained from
Cambridge Isotope Laboratories. Stock solutions of 2, 3, and 4 were prepared
by dissolving known amounts of the compounds in DMSO to give a final
concentration of 4 mM.
A? Peptide Preparation. Dry A?1-42 or A?1-40 peptide was weighed and
dissolved in hexafluro-2-isopropanol (HFIP) and incubated at 25°C for 1 h to
remove any preformed aggregates. It was then aliquotted into known
sonicated in a water bath containing ice for 15 min. The solution was then
centrifuged in a bench-top centrifuge at 16,000 ? g for 20 min, and the
supernatant was stored on ice until used. Initial peptide concentrations were
determined by spectrophotometry at 214 nm, using an extinction coefficient
of 75,887 liters?mol?1?cm?1.
NMR Spectroscopy. Samples for NMR were run in aqueous PBS with 10%2H2O
added. Samples containing A?40 were run at 0.3 mM. The compounds were
incubated with A? at 30°C for 2 h. NMR spectra were recorded on Bruker
DRX-600 and AMX-500 spectrometers as described in ref. 5.
ProteinChip arrays (Ciphergen Biosystems). Two microliters of antibody (4G8)
in PBS (0.25 mg/ml) was added to the spots of the PS10 chip, which was
incubated in the humidified chamber at 4°C overnight. The antibody was
removed, and blocking buffer (0.5 M ethanolamine in PBS) was added (5 ?l).
The array was incubated for 30 min. The blocking buffer was removed, and
each spot was washed with 5 ?l of 0.5% Triton X-100/PBS (wash buffer) for 5
5 min. A 60-?l sample was added to each spot, and the array was incubated at
room temperature for 3 h. The samples were removed, and each spot was
washed twice with 60 ?l of wash-buffer for 5 min. Each spot was washed with
60 ?l of PBS twice for 5 min and then washed with 60 ?l of 1 mM Hepes twice
for 1 min. The array was air-dried. One microliter of sinapinic acid (SPA) [50%
saturated in 50% (vol/vol) acetonitrile and 0.5% in TFA] was applied to each
spot twice. The array was air-dried between each application. All incubations
and washes were performed on a shaking table. Chips were analyzed in a
PBSIIC protein chip reader; SELDI-TOF MS and peaks were analyzed by using
Ciphergen ProteinChip software, Version 3.1.
CD Spectroscopy. A? peptide was prepared as described above and incubated
at 37°C. The final peptide concentration was determined to be 26 ?M, and it
26 ?M 2, 3, or 4.
Synchrotron radiation circular dichroism spectra were collected on station
U.K.). Data were collected at 37°C in a 0.02-cm fused silica cell with a peptide
concentration of 26 ?M. Data were collected between 195 and 260 nm in
0.5-nm increments with 1-s accumulation at each wavelength. Background
readings of buffer in the absence of peptide were subtracted. Spectra were
smoothed by using a Fourier transform.
at 50, 10, 5, 1, 0.5, and 0.1 ?M in 100% DMSO to create a 100? stock solution.
The final DMSO concentration was 1%. Compound was added to 200 ?l of
A?1-42 at 10 ?M in PBS (prepared as described above) and incubated at 37°C
with agitation at 200 oscillations per minute for 24 h. Samples where run in
triplicate. For determination of fibril growth end points, peptide samples (20
fluorescence emission at 500 nm over 10 sec when excited at 442 nm, using a
Hidex Oy Plate CHAMELEMON II plate reader in 96-well black plates.
Inhibition of H2O2Generated by A?:Cu. H2O2production by A? peptides was
measured by using a fluorimetric assay described in ref. 13. Dichlorofluores-
cein diacetate (DCF) (Molecular Probes) was dissolved (5 mM) in 100% di-
methyl sulfoxide (argon purged for 2 h at 20°C), deacetylated with 0.25 M
NaOH for 30 min, and neutralized (pH 7.4) to a final concentration of 1 mM.
Horseradish peroxidase (HRP) stock solution was prepared to 1 ?M in PBS (pH
7.4). The reactions were carried out in PBS (pH 7.4) under ambient conditions
in a 96-well plate (250 ?l per well) containing freshly prepared synthetic
Barnham et al.
May 13, 2008 ?
vol. 105 ?
no. 19 ?
peptide (up to 1 ?M), Cu-Gly (up to 2 ?M), reducing agents (up to 10 ?M), Download full-text
was included to prevent reactions with contaminating concentrations (?0.2
?M) of free Cu2?. The concentrations of A? used varied to bring readout
values into a convenient target range. A? was used at 200 nM. Studies were
completed on the day of reagent preparation. Reactions were conducted in
the dark to avoid photodynamic effects. The signal specific for H2O2was the
decrease in fluorescence of parallel samples coincubated with catalase (4,000
units/ml; 10 ?M). Fluorescent readings were recorded by a Packard Fluoro-
count plate reader (485-nm excitation, 530-nm emission) against a standard
curve of reagent-grade H2O2in PBS (pH 7.4).
A? Neurotoxicity. Primary neuronal cultures and Cell viability assay were
Statistical comparisons between groups were done with Student’s t test.
anesthesia by halothane inhalation in accordance with University of Mel-
bourne animal ethics guidelines. Brains were rapidly removed and chilled in
mM KCl, 2 mM MgSO4, 2 mM CaCl2, 10 mM D-glucose, 1.25 mM NaH2PO4, and
26 mM NaHCO3 and was gassed with 95% O2 and 5% CO2 (pH 7.35)
(HCl?NaHCO3). A?1–42 (3) and cisplatin were dissolved in DMSO and prein-
cubated on the slices for 30 min in ACSF vehicle with DMSO levels controlled
at 0.3%. All experiments were interleaved and conducted at room tempera-
ture. Field potential recordings were made in 350-?m transverse sections of
CA1 region. Recordings were made with an NPI microelectrode amplifier in
bridge-mode, connected to a 20? preamplifier with a 10-kHz low-pass eight-
pole Bessel filtration (Krohn–Hite; model 3381 filter/amplifier). Signals were
digitized with a Digidata 1322A A/D converter (Axon Instruments) and stored
in pClamp software, Versions 8.2 or 9.0 (Axon Instruments). Baseline stimula-
tion intensity was calibrated at the beginning of each experiment to produce
responses of 20–30% of the maximum slope of the field excitatory post
at the test intensity in substitution for the test stimulus. Data were analyzed
between the peak of the presynaptic fiber volley and the peak of the fEPSP.
Normalized fEPSP slopes were expressed as the percentage of the average
slope of the baseline between 30 to 20 min before tetanus. Long-term
potentiation was quantified by averaging the normalized data 55–60 min
after tetanus for each slice. Results were presented as the means ? SE, and
statistical significance was determined by using an unpaired t test at the 95%
Experimental Treatment Protocol for Brain Slices. Slices were transferred to a
24-well plate and preincubated in gassed ACSF at room temperature with or
iological recording. A?42 was dissolved in HFIP, dried, resuspended in DMSO,
and stored in small aliquots at ?20°C. A?42 aliquots were rapidly thawed and
used immediately. Compound 3 and cisplatin were dissolved in DMSO imme-
diately before use. Final DMSO concentrations were controlled at 0.3% in all
ACKNOWLEDGMENTS. We thank Tony Wedd and Paul Donnelly for helpful
carried out with the support of the Daresbury Synchrotron Radiation Source
and the assistance of David Clarke. This work was funded by the National
Health and Medical Research Council, Wellcome Trust Grant WT069851MA,
and Technology Organisation through the Access to Major Resource Facilities
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