Photodegradation of amyloid β and reduction of its cytotoxicity to PC12 cells using porphyrin derivatives

Article abstract of DOI:10.1039/c4cc03791j

A purpose-designed porphyrin-peptide hybrid effectively degraded amyloid β monomer and oligomers associated with Alzheimer's disease. Degradation was achieved using light irradiation in the absence of any additives and under neutral conditions. Moreover,

Full text of DOI:10.1039/c4cc03791j

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Photodegradation of amyloid b and reduction of
its cytotoxicity to PC12 cells using porphyrin
derivatives†
Cite this: Chem. Commun., 2014,
50, 9543
Received 19th May 2014,
Accepted 4th July 2014
Ayumi Hirabayashi,a Yutaka Shindo,b Kotaro Oka,b Daisuke Takahashia and
Kazunobu Toshima*a
DOI: 10.1039/c4cc03791j
www.rsc.org/chemcomm
A purpose-designed porphyrin–peptide hybrid effectively degraded
amyloid b monomer and oligomers associated with Alzheimer’s
disease. Degradation was achieved using light irradiation in the
absence of any additives and under neutral conditions. Moreover,
the hybrid effectively neutralized the cytotoxicity of amyloid b in
PC12 cells upon photoirradiation.
Alzheimer’s disease (AD)1 is a progressive neurodegenerative
disorder and is the most common form of dementia in
humans. It is characterized by neuronal loss and the presence
of large numbers of senile plaques, consisting of fibrillar
aggregates of 40- and 42-residue amyloid b (Ab) peptides, in
the brain.2 In recent studies, it was suggested that soluble
oligomers of Ab42 are responsible for synaptic dysfunction in
the brains of patients with AD and are the key intermediate
neurotoxic species in the pathology of AD.3 In addition, it was
revealed that the hydrophobic KLVFF region of Ab42 is an
important element responsible for the aggregation of Ab42
(Fig. 1).4 One way of suppressing Ab42 assembly in the brain
is through small molecules with a high affinity for5 or ability to
degrade6 Ab42. Major efforts should be directed toward inhibiting
Fig. 1 Chemical structures of porphyrin derivative 1, porphyrin–peptide
(KLVFF) hybrid 2, and Ab42 peptide.
amyloid formation at very early stages of the disease. In addition, that certain porphyrin derivatives could degrade proteins by
using the degradation approach, even if the inhibitor diffuses emitting reactive oxygen species under photoirradiation condi-
away, the target Ab42 is degraded and remains inactive, resulting tions.9 In addition, certain porphyrins were independently found
in irreversible inhibition of Ab42 function and an apparent to inhibit Ab40 filament formation.10 Based on these findings, we
increase in potency. In this context, we recently disclosed that hypothesized that a porphyrin itself or a porphyrin hybrid
designed and synthesized fullerene derivatives effectively inhib- possessing a KLVFF structure as a key segment for Ab40 aggrega-
ited Ab42 aggregation and degraded Ab42 under photoirradiation tion would effectively degrade Ab42 under photoirradiation
conditions.7 Following our reports, Qu et al. reported the photo- conditions, and could be used to neutralize the cytotoxicity of
degradation of b-sheet amyloid using inorganic molecules, poly- Ab42 in neuron cells. In this communication, we present the
oxometalates, as photocatalysts.8 On the other hand, we found molecular design, chemical synthesis and evaluation of a novel
and artificial light-activatable porphyrin–peptide (KLVFF) hybrid
a Department of Applied Chemistry, Faculty of Science and Technology,
Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
E-mail: toshima@applc.keio.ac.jp; Fax: +81 45-566-1576
b Department of Bioscience and Informatics, Faculty of Science and Technology,
Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc03791j
molecule that can effectively degrade Ab42 and neutralize its
cytotoxicity under photoirradiation.
To investigate our hypothesis, we utilized porphyrin derivative 1
as a protein photodegrading agent and designed porphyrin–
peptide (KLVFF) hybrid 2 with an appropriate spacer between 1
and the peptide (KLVFF) (Fig. 1). After chemical synthesis of the
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designed hybrid 2 using solid-phase synthesis (see ESI,† Scheme S1),
we first examined the binding abilities of 1 and 2 with Ab42 by
observation of the inhibitory effect using a thioflavin T (Th T)
fluorescence assay.11 The inhibitory concentration (IC50) was
obtained by measuring the Th T fluorescence intensity as a
function of the concentration of 1 or 2. Fig. 2a and b show the
plots obtained, which give IC50 values of 11.0 and 3.56 mM for
1 and 2, respectively. These results indicated that both 1 and 2 had
high affinity for Ab42, and that the affinity of the porphyrin–
peptide hybrid 2 was 3 times stronger than that of 1. Next, the
difference in the Ab42 aggregation-inhibitory abilities of 1 and 2
with and without photoirradiation (365 nm, 100 W) was confirmed
by a Th T fluorescence assay. It was found that the IC50 values of
1 and 2 were 26.9 and 12.9 nM, respectively, with photoirradiation.
These results clearly indicate that 1 and 2 inhibit Ab42 aggregation
much more efficiently under photoirradiation. The inhibitory
ability of 2 was 2 times higher than that of 1 under photoirradia-
tion. Furthermore, it is noteworthy that the inhibitory ability of
2 was 45 times higher than that of the previously reported fullerene
derivative7 under photoirradiation.
Fig. 3 Photodegradation of Ab42 by 1 or 2. A mixture of Ab42 monomer
and oligomers (5.0 mM) was incubated with each compound in 10% DMF/
Tris-HCl buffer (pH 8.0, 20 mM) at 25 1C for 2 h with or without
photoirradiation using a UV lamp (365 nm, 100 W) placed 10 cm from
the sample. Each sample was analyzed using tricine-SDS-PAGE and immuno-
blotting with monoclonal antibody 6E10. Lane 1: Ab42 alone; lane 2: Ab42 with
photoirradiation; lane 3: Ab42 + 1 (50 mM) without photoirradiation; lanes 4–7:
Ab42 + 1 (at a concentration of 50, 15, 5.0 and 1.5 mM, respectively) with
photoirradiation; lane 8: Ab42 + 2 (50 mM) without photoirradiation;
lanes 9–12: Ab42 + 2 (at a concentration of 50, 15, 5.0 and 1.5 mM,
respectively) with photoirradiation.
that porphyrin derivatives 1 and 2 are capable of degrading not
With these favourable results in hand, we next examined the only the Ab42 monomer but also its oligomers upon irradiation
Ab42 photodegradation ability of 1 and 2 against a mixture of with long-wavelength UV light under neutral conditions and in
Ab42 monomer and oligomers12 upon photoirradiation. The the absence of other additives, and that the photodegrading
results of the photodegradation experiment, which was moni- ability of 2 is higher than that of 1. In all cases, it was confirmed
tored by SDS-PAGE and immunoblotting with monoclonal anti- that Ab42 monomer was more effectively degraded than Ab42
body 6E10,12 are shown in Fig. 3. Comparison of lanes 2, 3 and oligomers by the porphyrin derivatives. Because the degradation
8 with lane 1 shows that neither photoirradiation of Ab42 of Ab42 monomer and oligomers by the porphyrin derivatives did
monomer and oligomers in the absence of 1 or 2 (lane 2 in not take place in the absence of light, it was confirmed that UV
Fig. 3), or treatment of Ab42 monomer and oligomers with 1 or 2 light functioned as a trigger to initiate degradation by the
but without photoirradiation (lanes 3 and 8 in Fig. 3), resulted in porphyrin derivatives.
a change in the SDS-PAGE profile. In contrast, lanes 4–6 and
These photodegradation results were also supported by
9–12 show the disappearance of the SDS-PAGE bands corres- analysis using MALDI-TOF MS, as shown in Fig. 4. Thus, after
ponding to both Ab42 monomer and oligomers after exposure to the incubation of Ab42 with 2 under photoirradiation, the MS
1 or 2 and photoirradiation. This indicates that degradation of peak corresponding to Ab42 disappeared. No MS peaks corre-
Ab42 monomer and oligomers took place. These results show sponding to the degradation fragments could be detected due
to the randomness of the degradation,7 leading to many frag-
ments present in very small amounts.
Based on these results, we then examined the inhibition
effect of porphyrin derivatives 1 and 2 on Ab-mediated cyto-
toxicity in neuron-like cells with and without photoirradiation
Fig. 2 Variation in Th T fluorescence intensity as a function of the
concentration of 1 or 2 with and without photoirradiation (365 nm,
100 W, 10 cm, 2 h). Ab42 (40 mM) was incubated with each compound
(300–0.01 mM) in 10% DMF/Tris-HCl buffer (pH 8.0, 20 mM) at 37 1C for
24 h, then Th T (6.67 mM) was added to each sample. Fluorescence was
measured using a Safire (TECAN) microplate reader using excitation and
emission wavelengths of 430 nm and 491 nm, respectively. Data were
analyzed using GraphPad Prism to obtain IC50 values using log (com-
pound) vs. normalized response-variable slope. (a) Dose–response curves
showing fractional binding of Th T to Ab42 fibrils in the presence of 1 with
(red line) or without (blue line) photoirradiation. (b) Dose–response curves
showing fractional binding of Th T to Ab42 fibrils in the presence of 2 with
(red line) or without (blue line) photoirradiation.
Fig. 4 MALDI-TOF MS profile of Ab42 peptide obtained by photo-
degradation using the peptide–porphyrin hybrid 2. Ab42 peptide (5.0 mM)
was incubated with 2 (50 mM) in 10% DMF/Tris-HCl buffer (pH 8.0, 20 mM)
at 25 1C for 2 h with (b) or without (a) photoirradiation using a UV lamp
(365 nm, 100 W) placed 10 cm from the mixture. The resulting products were
analyzed by MALDI-TOF MS (matrix: 3,5-dimethoxy-4-hydroxycinnamic acid).
A peak was observed at m/z 4516.2, indicating Ab42 peptide in (a); this peak
was not observed in (b).
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manner (lanes 8, 10, 12 and 16, 18, 20, respectively, in Fig. 5), and
that the inhibition ability of 1 and 2 with photoirradiation was
higher than without photoirradiation. In addition, the inhibition
ability of 2 was higher than that of 1 and much higher
(B30 times) than that of the previously reported fullerene deri-
vative.7 Importantly, these results strongly suggest that the photo-
degradation of Ab42 by 1 and 2 took place in PC12 cells in a highly
target-selective manner, allowing PC12 cells to survive even in the
presence of toxic Ab42.
In conclusion, we have developed a novel method for the
inhibition of the cytotoxic effects of Ab42 in neuron-like PC12 cells
using porphyrin derivatives in conjunction with photo-irradiation
to degrade Ab42 monomer and oligomers. Pin-point photoirradia-
tion permits high-resolution control over a defined area and
low interference with normal biological functions. The results
Fig. 5 Cell culture assay. PC12 cells were seeded into 96-well plates presented here will aid in the molecular design of artificial protein
(4 Â 103 per well). Cells were pre-incubated with Ab42 oligomer (0.5 mM)
photodegradation agents which will, in turn, help provide a means
of controlling the specific functions of certain proteins.
This research was supported in part by Scientific Research
(B) (No. 23310153 and 26282212) and Scientific Research on
and 1 or 2 for 15 minutes at 25 1C with or without photoirradiation using a
UV lamp (365 nm, 30 W) placed 15 cm from the sample; the samples were
then further incubated at 37 1C for 24 h. The cell viability was determined
using the MTT viability assay. Lanes 1 and 2: PC12 alone without and with
photoirradiation, respectively; lanes 3 and 4: PC12 + Ab42 oligomer (0.5 mM) Innovative Areas ‘‘Chemical Biology of Natural Products’’
without and with photoirradiation, respectively; lanes 5 and 6: PC12 + 1
(1.0 mM) without and with photoirradiation, respectively; lanes 7 and 8:
PC12 + 1 (0.3 mM) + Ab42 oligomer (0.5 mM) without and with photoirradia-
tion, respectively; lanes 9 and 10: PC12 + 1 (0.03 mM) + Ab42 oligomer
(No. 24102528 and 26102738) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (MEXT).
(0.5 mM) without and with photoirradiation, respectively; lanes 11 and 12:
PC12 + 1 (0.003 mM) + Ab42 oligomer (0.5 mM) without and with photo-
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