A prochelator activated by hydrogen peroxide prevents metal-induced amyloid β aggregation

Article abstract of DOI:10.1002/cbic.200900597

(Chemical Equation Presented) The masked marvel: Elevated production of H₂O₂ by deviant species of Cu-Aβ (Cu-amyloid beta) might trigger neurodegeneration in Alzheimer's disease. A new boronic ester-masked prochelator called QBP is activated under conditions that mimic early Alzheimer's pathology in which copper, Aβ, and reductants exacerbate reactive oxygen species formation. Once activated to its unmasked form, the released chelator diminishes copper's pro-oxidant reactivity and inhibits Aβ aggregation.

Full text of DOI:10.1002/cbic.200900597

DOI: 10.1002/cbic.200900597
A Prochelator Activated by Hydrogen Peroxide Prevents Metal-Induced
Amyloid b Aggregation
[
a]
Marina G. Dickens and Katherine J. Franz*
Alzheimer’s is a progressive and fatal brain disease that is the
most common form of dementia. Its characteristic pathology
includes extracellular amyloid plaques that form as a result of
abnormal clearance and/or increased production of amyloid-b
peptides (Ab) that are released from the amyloid precursor
Metal chelating agents have appeared as a compelling strat-
[20]
egy for Alzheimer’s therapies. In particular, 8-hydroxyquino-
line (8HQ) derivatives clioquinol and PBT2 have shown promis-
ing results in mouse models and phase IIa clinical trials of Alz-
[21,22]
heimer’s patients.
These compounds inhibit metal-induced
[
1,2]
+/2+
2+
protein (APP).
Metal ions, particularly Cu
, have been implicated in two processes related to
Ab pathology: peptide aggregation and formation of reactive
and Zn , but
Ab aggregation and ROS generation. While these reports en-
courage further development of metal-targeted compounds
for neurodegenerative disease, there remain significant con-
2
+/3+
also Fe
[
3]
[23,24]
oxygen species (ROS).
It is speculated that both APP and Ab might have normal
cerns about manipulating metal distribution in the brain.
Given the complexity of the metallobiology in Alzheimer’s, it is
particularly challenging to design metal-binding agents that
can mitigate the damaging effects of metals while preserving
their beneficial properties. In our laboratory, we are developing
[
4,5]
roles in copper homeostasis.
It has also been shown in vitro
that Ab can act as an antioxidant by quenching free radicals
[
6,7]
and/or by chelating copper.
Other evidence, however, sug-
gests that Ab–Cu complexes are pro-oxidant and directly cul-
prochelators that are designed to bind metals only under con-
[
8]
[25–27]
pable of neurotoxicity. In vitro, Ab in the presence of copper
ditions of oxidative stress.
The indications that elevated
[9–11]
or iron and reducing agents like ascorbate produces H O ,
production of H O by deviant Cu–Ab interactions might trig-
2 2
2
2
which can subsequently react with the reduced metal ions to
ger neurodegeneration suggested to us that prochelators acti-
[
12,13]
produce OHC via the Fenton reaction [Eq. (1)].
ated H O generation appears at an early stage during in vitro
Metal-medi-
vatable by H O2 could be beneficial for managing a metal
2
burden at locations of disease progression without stimulating
widespread metal redistribution. Here, we present a boronic
ester-masked 8-hydroxyquinoline derivative called QBP that
converts to 8HQ in the presence of H O . Once converted to
2
2
[
10,11]
Ab aggregation,
which supports the notion that soluble
Ab–Cu species are responsible for the oxidative damage that is
one of the earliest pathological events in Alzheimer’s dis-
2
2
[
14]
ease. Furthermore, copper has been shown to intensify Ab
8HQ, it is available for coordinating metal ions, as shown in
[
9,10,15]
2+
2+
2+
toxicity in primary cortical neurons.
Like Cu , Zn also
Scheme 1 for Cu . The protecting group is ultimately released
[28,29]
promotes Ab aggregation in vitro, but the Zn-induced aggre-
as pinanediol and nontoxic boric acid.
[
16–18]
gates could be neuroprotective.
þ
2þ
ꢀ
Cu þ H O ! Cu þ OH þ OHC
ð1Þ
2
2
One hypothesis to reconcile the seemingly contra-
dictory evidence related to metals, Ab, and oxidative
stress is that metal binding and Ab aggregation
might represent an initial, protective response to
dampen production of ROS. Excessive H O and an
2
2
overburden of copper could eventually push the
system into a vicious cycle that switches Ab–Cu ac-
[
19]
tivity from antioxidant to pro-oxidant. During this
2
+
stage, metal exchange with Zn could promote fur-
ther Ab aggregation as a defense against copper-
induced damage. While chelating agents are known
2 2
Scheme 1. Oxidation of QBP by H O and subsequent binding of copper by 8-hydroxy-
quinoline (8HQ).
to reverse metal-induced aggregates, this model suggests that
disaggregating plaques alone could have the unintended con-
QBP was synthesized by reacting commercially available
quinoline boronic acid (QBA) with pinanediol in a Dean Stark
apparatus. The X-ray crystal structure is shown in Figure 1. QBP
is stable in aqueous solution between pH 5–8 over the course
of 10 h, although some hydrolysis to QBA occurs at lower and
higher pH values, as monitored by UV/Vis and mass spectrom-
etry (data not shown).
[
19]
sequence of exacerbating oxidative damage.
[
a] M. G. Dickens, Dr. K. J. Franz
Department of Chemistry, Duke University
P.O. Box, 90346 Durham, NC 27708 (USA)
Fax: (+1)919-660-1605
With the phenol of 8HQ masked by the pinanediol boronic
ester, the QBP prochelator should have little to no affinity for
metal ions. Comparison of the UV/Vis spectra in Figure 2 of
E-mail: katherine.franz@duke.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900597.
ChemBioChem 2010, 11, 59 – 62
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
59

Figure 1. X-ray structure of QBP. Thermal ellipsoids are shown at 50% proba-
bility. Hydrogen atoms have been omitted for clarity.
Figure 3. Turbidity assay, as monitored by the difference in absorbance at
05 nm between the sample and its matched control that does not contain
Ab. Samples containing 10 mm Ab were incubated with 10 mm Cu(Gly) or
ZnCl in the presence or absence of 20 mm 8HQ for 1 h at 378C. The A405
4
2
2
readings were taken 1 h after mixing (black, left-hand bars). The %soluble
peptide remaining after 1 h was determined by a BCA protein concentration
assay (gray, right-hand bars) on centrifuged samples.
2
+
samples, with Zn causing a more profound effect. The pres-
2
+
ence of 2 equiv of 8HQ inhibits the Cu -induced aggregation
2
+
and significantly reduces Zn -induced aggregation. After an
hour of incubation, the aggregated peptide was removed by
centrifugation and the amount of soluble peptide remaining
was determined by a BCA assay. The gray bars in Figure 3
Figure 2. UV/Vis spectra of 100 mm QBP in PBS pH 7.4 in the absence (b)
and presence (b) of 50 mm Cu(Gly)
QBP/Cu sample with 4 mm H (c). The resulting spectrum is nearly
identical to that of independently prepared [Cu(8HQ) ] (c).
2
, and 60 min after treatment of the
2+
show that samples treated with Cu alone have only 60%
2
O
2
soluble peptide, while those containing 8HQ have greater than
2
2
+
9
0% soluble Ab. Similar results are seen for Zn , with greater
than 80% of the peptide remaining soluble when 8HQ is pres-
ent. These data corroborate the turbidity assay and show that
8HQ present at the outset of metal–Ab incubations prevents
aggregation. If 8HQ is added to preformed metal–Ab aggre-
gates, the turbidity of the solutions decreases (see the Sup-
porting Information); this demonstrates that 8HQ can both
prevent and reverse Ab aggregation under these conditions.
QBP, on the other hand, does not interfere with metal-in-
duced Ab aggregation. The black bars in Figure 4 show that
the presence of QBP does not change the turbidity of solutions
2
+
QBP alone or in the presence of Cu for an hour reveals no
change in spectral features and validates the assumption that
2
+
QBP does not interact with Cu in its prochelator form. Addi-
tion of H O , however, causes a new spectrum to appear that
2
2
matches that of [Cu(8HQ) ], consistent with the reaction in
2
Scheme 1.
In order to determine the rate of conversion of QBP to 8HQ
by H O , reactions were monitored spectrophotometrically
2
2
under pseudo-first-order conditions of excess H O . The ob-
2
2
2
+
served rate constants (kobs) were plotted against peroxide con-
containing Ab alone or Ab plus Cu . These results show that
QBP itself neither induces nor prevents metal-induced Ab
aggregation, as predicted based on its lack of metal-binding
ability.
ꢀ
1
ꢀ1
centration to give a rate constant k of 0.25m
s
(see the
Supporting Information).
Chelators, including those based on the 8HQ motif, have
been shown to have a dramatic effect on metal-induced Ab
In order to show that the 8HQ that is generated in situ from
the reaction of QBP and H O is capable of reversing Ab aggre-
[
30]
aggregation. Here, we monitored the metal-induced aggre-
gation of Ab by two complementary methods: light scattering,
which reports the change in solution turbidity as a result of
precipitate formation, and soluble protein concentration,
which ascertains the fraction of protein that did not precipitate
from solution. These methods provide a preliminary assess-
ment of aggregation propensity, but do not report on detailed
2
2
gation, H O2 (1 mm) was added to each of the samples in
2
2
+
Figure 4 that already contained Cu -aggregated Ab. The gray
bars in Figure 4 show that H O alone neither reduces the tur-
2
2
2
+
bidity of samples containing Ab and Cu , nor does it increase
2
+
the turbidity of samples containing Ab alone or Ab/Cu /8HQ.
These results show that H O itself does not influence the ag-
2
2
[
31]
morphological changes of aggregates or fibrils. In the light
scattering assay, turbidity is assessed as the difference in ab-
sorbance at 405 nm between the sample and its matched con-
trol that contains the same components but without Ab. The
black bars in Figure 3 show that, as previously observed by
gregation state of Ab. In contrast, samples that contain Ab/
2
+
Cu /QBP show a significant decrease in turbidity 30 min fol-
lowing H O addition. This result is consistent with conversion
2
2
2
+
of QBP to 8HQ, which can subsequently bind Cu and reverse
Ab aggregation. Given the concentrations of QBP and H O
2
2
[
32,33]
2+
2+
others,
both Cu and Zn increase the turbidity of Ab
present in the samples and the rate constant for prochelator-
6
0
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ChemBioChem 2010, 11, 59 – 62

The previous experiment contained a relatively high concen-
tration (1 mm) of H O that was added in a single bolus. Sever-
2
2
2
+
al groups have shown that combinations of Ab, Cu and re-
[10,34]
ductants produce significant amounts of H O2 from O2.
2
Therefore, to investigate whether these conditions of more
biologically relevant H O production are sufficient for activat-
2
2
2
+
ing QBP, samples of Ab, Cu , ascorbic acid, and either 8HQ or
QBP were monitored for H O2 production with the Amplex
2
Red/horseradish peroxidase (HRP) assay. Figure 6 shows the
Figure 4. Turbidity assay, as monitored by the difference in absorbance at
05 nm between the sample and its matched control that does not contain
4
2
+
Ab. Samples contain combinations of 10 mm Ab, 10 mm Cu (provided as
Cu(Gly) ), 20–100 mm QBP, or 10–20 mm 8HQ, as indicated. The A405 readings
were taken 1 h after mixing (black bars), and then again 30 min after addi-
tion of 1 mm H (gray bars).
2
2
O
2
to-chelator conversion, this reaction is predicted to generate
–45 mm 8HQ, depending on the initial QBP concentration. The
highest concentration is certainly sufficient for complete bind-
Figure 6. Hydrogen peroxide detection by the Amplex Red/HRP assay for
9
samples containing various combinations of 200 nm Ab, 200 nm Cu(Gly)
0 mm ascorbic acid (AA), 2–200 mm QBP, 200–400 nm 8HQ, in a total volume
of 50 mL buffer (50 mm HEPES, 150 mm NaCl). Samples were incubated at
78C for 1 h before 50 mL of Amplex Red/HRP reagent was added. Fluores-
2
,
1
2
+
ing of Cu in a 1:2 complex, although as shown in the figure,
even the lower concentration is effective.
3
cence readings were taken with lex =485 and lem =590 nm.
Confirmation that [Cu(8HQ) ] is generated in the reaction of
2
Ab/Cu/QBP/H O described in Figure 4 comes from mass spec-
2
2
tral detection of m/z 352, which is consistent with [Cu(8HQ)2].
concentration of H O detected by Amplex Red after 1 h incu-
2
2
Further evidence comes from the UV/Vis spectrum of the reac-
bation of 200 nm Cu(Gly) , 200 nm Ab, 10 mm ascorbic acid and
2
tion mixture (Figure 5). The complex [Cu(8HQ) ] has a charac-
either 8HQ or QBP over a range of concentrations in sodium
2
2
+
teristic absorbance band at 375 nm, which is clearly visible in
phosphate buffer at pH 7.4. Samples that contain Cu and as-
corbate (with or without Ab) provide just over 300 nm detecta-
ble H O when the Amplex Red reagents are added at the end
2
+
samples that contain Ab, Cu and 8HQ. Samples that contain
Ab/Cu/QBP/H O also show this characteristic peak, verifying
2
2
2
2
that [Cu(8HQ) ] has indeed been generated. This absorbance
of the 1 h incubation period. This result shows that Ab neither
2
band tails into the 405 nm region used for monitoring turbidi-
ty, but its contribution was appropriately accounted for by
subtracting the matched control from the sample value, ensur-
ing that residual A405 reported in Figures 3 and 4 can be attrib-
uted to turbidity.
prevents nor promotes H O2 production by copper under
2
these conditions when compared to copper in the presence of
[12]
glycine as a carrier ligand, a result also seen by others. In
contrast, 8HQ inhibits H O production, even when present at
2
2
2
+
only a 0.25 equiv of Cu . When 8HQ is present at a 2:1 ratio
2
+
to Cu , the amount of detectable H O2 diminishes to one
2
third of that detected in the absence of 8HQ. This result con-
2
+
firms that 8HQ coordinates Cu in a manner that diminishes
its ability to catalyze the formation of H O2 from O in the
2
2
presence of reductant.
When QBP is added to the reaction mixture in place of 8HQ,
similar results are obtained. As shown in Figure 6, samples that
2
+
contained 200 mm QBP along with Cu , ascorbate, and Ab
result in detection of ~100 nm H O , which is a third of the
2
2
concentration obtained in the absence of chelator.
If the Amplex Red/HRP reagents are included at the begin-
ning of the incubations, then up to 500 nm H O is detected
2
2
(
data not shown). These data suggest that H O2 degrades
2
during the incubation period, possibly via Fenton reaction or
hydrolysis. This experiment also provides the maximum
amount of H O produced by the Cu/Ab/ascorbic acid system
Figure 5. UV/Vis analysis of H
2
O
2
-treated Ab samples from Figure 4. b:
; c: Ab+Cu+QBP+H
Ab+H ; a: Ab+Cu+8HQ+H
2
O
2
2
O
2
2 2
O .
2
2
ChemBioChem 2010, 11, 59 – 62
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
61

under these conditions. Given this number and the rate con-
stant for the QBP-to-8HQ conversion, the 200 mm QBP sample
in Figure 6 gives a calculated yield of 90 nm 8HQ. The results
in Figure 6 are consistent with this prediction, as they indeed
show that 200 mm QBP provides protection against H O for-
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bioinorganic chemistry · copper · hydrogen peroxide
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