The chelation targeting metal-Aβ40 aggregates may lead to formation of Aβ40 oligomers

Article abstract of DOI:10.1039/c1dt00020a

The fluorescent chelator (FC-1) was designed by combining a metal-chelating unit and a ThT-based Aβ aggregate-binding fluorescent unit. FC-1 is a cell membrane-penetrable chelator with a moderate chelation ability to Cu ²⁺ and Zn²⁺ a

Full text of DOI:10.1039/c1dt00020a

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The chelation targeting metal–Ab40 aggregates may lead to formation of
Ab40 oligomers†
Yong Zhang,a,b Li-Yuan Chen,a Wen-Xing Yin,a Jun Yin,a Shi-Bing Zhanga and Chang-Lin Liu*a
Received 6th January 2011, Accepted 8th March 2011
DOI: 10.1039/c1dt00020a
The fluorescent chelator (FC-1) was designed by combining a
metal-chelating unit and a ThT-based Ab aggregate-binding
fluorescent unit. FC-1 is a cell membrane-penetrable chelator
with a moderate chelation ability to Cu2+ and Zn2+ and
can target metal–Ab40 aggregates. Treatment with FC-1
led to enhanced cytotoxicity of the aggregates, because the
aggregates were converted into a pool of oligomers.
chelating ability to Cu2+ and Zn2+ (logKCu–DPA, 9.3; logKZn–DPA, 7.6)
relative to Ab40 (logKCu–Ab40, 5–10; logKZn–Ab40, 3–7).6
FC-1 was synthesized by heterocyclic condensation and halo-
genation reactions, and its Cu2+ or Zn2+ complexes (1 and 2) were
prepared by mixing FC-1 and the corresponding metal chloride
(Scheme S1†). The resulting absorption spectra demonstrate that
the coordination of Cu2+ leads to both a reduction and blue shift
(~40 nm) in the absorption at 350 nm of FC-1 (Fig. S2,3†). The
Job plots showed that FC-1 forms a 1 : 1 complex with Cu2+ or
Zn2+ (Fig. S4†), and this was confirmed by the X-ray crystal
structures of 1 and 2 (Fig. 1).‡ Their maximum excitation (lex)
and emission (lem) wavelengths in the fluorescence spectra all are
360 and 430 nm, respectively (Fig. S5†). FC-1 exhibits a high
fluorescence quantum yield (U ~ 0.86), and its emission intensity
at 430 nm remains nearly constant in the range of pH 5.0–9.0 (Fig.
S6†) and over an incubation time of 0–120 h at 37 ◦C (Fig. S7†).
Moreover, the fluorescent properties of FC-1 are not altered by
addition of the tested metal ions except Cu2+ and Ni2+ at the given
concentrations (Fig. S8†). The fluorescence of FC-1 was quenched
with Cu2+ in the range of 0–200 mM under nearly physiological
conditions (Fig. S9†).
Fluorescence assays showed that FC-1 has a strong affinity
for Ab40 aggregates at any given concentration, like its parent
ThT. After incubation with Ab40 aggregates for 10 min, the
fluorescence of FC-1 at 430 nm was enhanced at least ~4–5
fold, whereas that of ThT at 482 nm was enhanced ~2–3 times
relative to that without Ab40 aggregates under the same conditions
(Fig. S10†). The presence of Cu2+ or Zn2+ resulted in a further
slight enhancement in the fluorescence of FC-1 bound to Ab40
aggregates. The binding of FC-1 to Ab40 aggregates was verified
by fluorescence microscopy (FM). After incubation with FC-1
for 10 min, FM imaging showed that the areas rich in Ab40
aggregates produced a bright blue fluorescence, and the imaging
was not affected by the coordination of Cu2+ or Zn2+ to Ab40
(Fig. S11†). These results, together with the facts delineated above,
indicated that FC-1 is a potential agent that can be used to sense
the formation of Ab aggregates under physiological conditions.
The fluorescence of FC-1 bound to metal–Ab40 aggregates
was observed to decrease over an incubation time of 10 min–
2 days compared to the controls of metal-free Ab40 aggregates
(Fig. 2), showing that FC-1 may chelate the metal ions in the
aggregates with incubation, and the chelation of FC-1 may
promote dissociation of the aggregates. To obtain evidence in
support of chelation of the copper, the time course of interactions
Increasing evidence suggests that the aggregation of amyloid b-
peptides (Ab) and the production of reactive oxygen species (ROS)
by copper–Ab species are two important neurotoxic events in
Alzheimer’s disease (AD).1 Because the Ab aggregation mediated
by the metal ions including Cu2+ and Zn2+ is reversible, the
dissociation of metal–Ab aggregates could be controlled by metal
chelators.2 Thus, chelation therapy using metal-specific chelating
ligands, which reduce the damaging effect of some metal ions
while not disrupting the beneficial properties of others, seems to
be a reasonable choice for AD treatment.2,3 A variety of chelators
including carbohydrate conjugates, clioquinol (CQ), thioflavin-T
(ThT)-based derivatives, cyclen, and prochelators activated by Cu–
Ab species have been designed and tested in vitro.4 The evidence
showed that the chelators dissociate the metal–Ab aggregates and
inhibit ROSgeneration to a certain extent by targeting the metal
ions in Ab species. Based on this consideration, we designed a flu-
orescent chelator (FC-1) by combining a metal-chelating unit and
a ThT-based Ab aggregate-binding fluorescent unit to promote
the dissociation of metal–Ab aggregates (Scheme 1 and Fig. S1†).
The fluorescent moiety of FC-1 was produced by removing four
methyl groups from lipophilic ThT,5 a fluorescent dye with a high
affinity for protein amyloid fibrils. The appreciable selectivity and
reasonable affinity for the metal ions in Ab species are requisite for
the ligands in chelation therapy to maintain the metal homeostasis
in the brain of AD patients. Thus, the ligand di-(2-picolyl)amine
(DPA) was selected as the chelating moiety.2a DPA has a moderate
aKey Laboratory of Pesticide & Chemical Biology, Ministry of Education,
School of Chemistry, Central China Normal University, Wuhan, 430079,
P. R. China. E-mail: liuchl@mail.ccnu.edu.cn
bSchool of Chemical and Materials Engineering, Huangshi Institute of
Technology, Huangshi, 435003, P. R. China
† Electronic supplementary information (ESI) available: Experimental
details. CCDC reference numbers 759320–759321 for FC-1, 1 and 2,
respectively. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1dt00020a
4830 | Dalton Trans., 2011, 40, 4830–4833
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Scheme 1 The design of multifunctional fluorescent chelator FC-1.
Fig. 1 An ORTEP view of the structures of complexes 1 and 2. The solvent molecules and hydrogen atoms have been omitted for clarity.
To confirm the incomplete dissociation of metal–Ab40 aggre-
gates, turbidity experiments and bicinchoninic acid (BCA) protein
tests were performed. The former showed that upon incubation
of Ab40 aggregates with FC-1 a reduction in the absorbance at
405 nm compared to Ab40 without FC-1 resulted (Fig. S14†),
while the latter showed that the content of soluble Ab40 is
increased upon incubation with FC-1 (Fig. S15†), but significant
resolubilization of metal–Ab40 aggregates was not observed in
either case.Moreover, CD spectra showed that the conformation
of the metal–Ab40 aggregates is altered by incubation with FC-1
(Fig. S16†). These results indicated that the FC-1 binding leads
to incomplete dissociation and to conformational change of the
metal–Ab40 aggregates.
To further verify the ability of FC-1 to dissociate the metal–
Ab40 aggregates, the morphology of metal–Ab40 aggregates were
examined by TEM (transmission electron microscopy).7,8 The
Ab40 aggregates freshly prepared in the absence and presence of
Fig. 2 The influence of metal ions, Ab40 aggregates or metal–Ab40
Cu2+ or Zn2+ exhibit well-organized fibrils under TEM (Fig. 3a–
aggregates on the emission (arbitrary units) of FC-1 at 430 nm (excitation
d). After treatment with FC-1 for 24 h, the resulting metal–
Ab40 aggregates become small and amorphous under TEM
(Fig. 3e,f), showing that FC-1 converts the aggregates into a pool
of oligomers. The result was consistent with the above-observed
facts that FC-1 can lead to the incomplete dissociation of metal–
Ab40 aggregates.
at 360 nm). The Ab40 and metal–Ab40 aggregates were prepared by
incubating 10 mM Ab40 for 2 days at 37 ◦C in the absence and presence of 10
mM Zn2+ or Cu2+. FC-1 (10 mM) was added into samples containing 10 mM
Zn2+ or Cu2+, Ab40 aggregates and metal–Ab40 aggregates and incubated
for 10 min, 1 h, 1 and 2 days at 37 ◦C with agitation, respectively.
The inhibition of metal-mediated Ab40 aggregation by FC-1
was also explored under the same conditions. After the addition
of FC-1 into samples containing Ab40 and Cu2+ or Zn2+ followed
by agitation for 5 min, the resulting mixtures were incubated for
48 h. The resulting Ab40 species were observed to be similar in
morphology to those formed via the FC-1-mediated dissociation
of metal–Ab40 aggregates (Fig. S17†), showing that FC-1 inhibits
the formation of the Ab40 fibrils by chelating Cu2+ or Zn2+.
The cell membrane penetration of FC-1 was detected by adding
FC-1 into HeLa cell cultures. When excited by UV light (360–
370 nm), the collected cells emit blue fluorescence under FM after
incubation with FC-1 (Fig. S18b†). Moreover, FC-1 bound to the
between FC-1 and Cu2+-Ab40 aggregates was monitored by UV-
vis absorption spectroscopy (Fig. S12†). The data showed that
the absorption of FC-1 at 350 nm was reduced over a period
of incubation of 2–48 h, as indicated in Fig. S3.† However, the
fluorescence of FC-1 was not quenched to that of its complexes
over these incubation periods (Fig. S13†), indicating that the
dissociation of metal–Ab40 aggregates may be incomplete, likely
because the moderate chelation ability of FC-1 limits the complete
removal of the metal ions from the aggregates, and the competition
between FC-1 and Ab40 for Cu2+ or Zn2+ can reach an equilibrium
after a given incubation period.
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Fig. 3 TEM images of Ab40 samples. a and b, Ab40 aggregates produced
by incubating 50 mM Ab40 at pH 6.6 and pH 7.4, respectively; c and d,
Cu2+– and Zn2+–Ab40 aggregates prepared by incubating 50 mM Ab40
plu◦s 50 mM Zn2+ or Cu2+. These incubations were carried out for 2 days at
37 C. e and f, Cu2+– and Zn2+–Ab40 aggregates after treatment with 50
mM FC-1 for 1 day at 37 ◦C with agitation.
Fig. 4 Cytotoxicity of Ab40 and Cu2+–Ab40 aggregates with and without
FC-1 toward HeLa cells after incubation for 24 h ([Ab40] = 5 mM;
[Ab40]/[Cu2+]/[FC-1] = 1 : 1 : 1). Results represent means s.d. (n = 3).
*, P < 0.05; **, P < 0.01.
reduce the cytotoxicity of the aggregates or not is worthy of further
investigation.
metal–Ab40 aggregates was also observed inside the cells (Fig.
S18c†). These results indicate that FC-1 is cell plasma membrane
penetrable. In theory, like CQ which has been tested in phase II
clinical trials,4b FC-1 fulfils common drug-like criteria and crosses
the blood-brain barrier (BBB)according to Lipinski’s rules (MW
£ 450, HBD £ 5, HBA £ 10, TPSA £ 90, clogP £ 5) and calculated
logBB for BBB penetration (logBB = -0.0148 TPSA + 0.152
clogP + 0.130) (Table S1†).9
Finally, the cytotoxicity of the metal–Ab40 aggregates treated
with FC-1 was assessed in HeLa cells using MTT assays (Fig. 4).
The data shows that FC-1, its Cu2+ complex and the adduct
of FC-1 with Cu2+-free Ab40 aggregates all exhibit less toxicity
than the Cu2+–Ab40 aggregates treated with FC-1 under the same
conditions. Moreover, the toxicity of the Cu2+–Ab40 aggregates
was increased upon incubation with FC-1, affording <60% cell
survival after an incubation time of 24 h. This can be attributed
to the observed fact that the chelation of FC-1 targeting the
Cu2+–Ab40 aggregates led to formation of oligomers, because Ab
oligomers, including the currently reported Ab oligomers which
exhibit a morphology similar to those observed above by TEM
(Fig. 3), have been found to be the neurotoxic form of Ab.10
Thus, this result is not unexpected, although some chelators have
been reported to be capable of reducing cytotoxicity of metal–Ab
aggregates.4e,7
Acknowledgements
This work was supported by NSFC (No. 20971049), and in part
by the PCSIRT (No. IRT0953).
Notes and references
‡ Crystal data for FC-1: C25H19N4S, M = 407.51, monoclinic, a = 10.0651(6)
◦
◦
˚
˚
˚
A, b = 6.0997(4) A, c = 16.8565(10) A, a = 90.00 , b = 91.991(12) , g =
◦
3
˚
90.00 , V = 1034.26(11) A , T = 298(2) K, space group P2(1), Z = 2, 6275
reflections measured, 4010 independent reflections (Rint = 0.0318). R1
=
0.0657 (I > 2s(I)), the final wR(F2) was 0.1336 (all data). Crystal data for
1: C25H20Cl2CuN4S·1/6C2H3N·4/3H2O, M = 574, triclinic, a = 8.5380(9)
◦
◦
˚
˚
˚
A, b = 19.9727(19) A, c = 22.537(2) A, a = 92.188(2) , b = 91.350(2) , g =
◦
3
¯
˚
97.128(2) , V = 3809.1(7) A , T = 294(2) K, space group P1, Z = 1, 23 106
reflections measured, 15 447 independent reflections (Rint = 0.0305). R1 =
0.0540 (I > 2s(I)), the final wR(F2) was 0.1612 (all data). Crystal data
for 2: C25H20N4SCl2Zn·1/2CH3OH, M = 560, monoclinic, a = 33.495(3)
◦
◦
˚
˚
˚
A, b = 8.9680(7) A, c = 17.7961(13) A, a = 90.00 , b = 94.3510(10) , g =
◦
3
˚
90.00 , V = 5330.3(7) A , T = 297(2) K, space group C2/c, Z = 4, 14 346
reflections measured, 4936 independent reflections (Rint = 0.0195). R1
0.0493 (I > 2s(I)), the final wR(F2) was 0.1750 (all data).
=
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In conclusion, the ThT-based fluorescent chelator FC-1 exhibits
stable fluorescence under conditions similar to physiological
environments. The cell plasma membrane penetrable agent fulfils
common drug-like criteria, and binds to metal–Ab40 aggregates
with a high affinity. Through chelating the metal ions, FC-1
leads to not only the dissociation of metal–Ab40 aggregates into
oligomers, but also inhibits the metal-mediated Ab40 aggregation.
The incomplete dissociation leads to enhanced cytotoxicity of the
metal–Ab40 aggregates treated with FC-1. This result is quite
unexpected. Therefore, although some chelators were found to be
capable of reducing the cytotoxicity of metal–Ab aggregates,4e,7
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©