Target-selective photo-degradation of verotoxin-1 and reduction of its cytotoxicity to Vero cells using porphyrin-globotriose hybrids

Article abstract of DOI:10.1039/c3cc42957a

Designed and synthesized porphyrin-globotriose hybrids effectively degraded verotoxin-1, which causes severe bloody diarrhoea and fetal hemolytic uremic syndrome (HUS). Degradation was achieved using long-wavelength UV or visible light irradiation in the

Full text of DOI:10.1039/c3cc42957a

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Target-selective photo-degradation of verotoxin-1 and
reduction of its cytotoxicity to Vero cells using
porphyrin–globotriose hybrids†
Cite this: Chem. Commun., 2013,
49, 6027
Received 22nd April 2013,
Accepted 15th May 2013
Atsushi Okochi, Shuho Tanimoto, Daisuke Takahashi and Kazunobu Toshima*
DOI: 10.1039/c3cc42957a
www.rsc.org/chemcomm
Designed and synthesized porphyrin–globotriose hybrids effectively photo-irradiation conditions. Using this approach, even if the
degraded verotoxin-1, which causes severe bloody diarrhoea and fetal inhibitor diffuses away, the target protein is degraded and
hemolytic uremic syndrome (HUS). Degradation was achieved using remains inactive, resulting in irreversible inhibition of protein
long-wavelength UV or visible light irradiation in the absence of any function and an apparent increase in potency. In our previous study,
additives and under neutral conditions. Moreover, the hybrids neutra- porphyrin derivative 3 was found to degrade proteins by emitting
lized the cytotoxicity of verotoxin upon photo-irradiation.
reactive oxygen species under photo-irradiation conditions.8 Based
on these findings, we hypothesized that a protein photo-degrading
Verotoxin is an exotoxin produced by entero-hemorrhagic agent possessing a Gb3 structure as a recognition moiety for the B
Escherichia coli (EHEC). There have been many outbreaks of subunit of verotoxin would selectively degrade verotoxin under
EHEC over the past several decades.1 Verotoxin causes severe bloody photo-irradiation conditions and could be used to neutralize the
diarrhea and fetal hemolytic uremic syndrome (HUS).2 In this cytotoxicity of verotoxin. In this communication, we present the
context, the development of specific inhibitors for verotoxin would molecular design, chemical synthesis and biological evaluation
be useful for clinical use and would provide an efficient counter- of novel and artificial light activatable porphyrin–Gb3 hybrid
measure against outbreaks of EHEC. Verotoxin is classified into two molecules that can effectively degrade verotoxin and neutralize
subgroups, verotoxin-1 (VT-1) and verotoxin-2 (VT-2).3 The toxin its cytotoxicity under photo-irradiation.
consists of a pentameric B subunit, which is responsible for the
To investigate our hypothesis, we utilized porphyrin derivative 3
recognition of cell surface oligosaccharide units, and an A subunit, as a protein photo-degrading agent and designed porphyrin–Gb3
which damages ribosomal RNA once internalized by the cell.4 Cell hybrids 4–8 (Fig. 1). After chemical synthesis of the designed
surface recognition is a necessary step in allowing the toxin to be hybrids 4–8 with an appropriate spacer between 1 and 3 (see ESI,†
internalized and fulfill its enzymatic function. In mammalian cells, Scheme S1 and Fig. S1), we first examined the photo-induced
globotriaosyl ceramide, a glycolipid which contains globotriose protein degradation activities of porphyrin derivative 3 and
(Gb3; Gala(1–4)-Galb1–4)-Glc, Fig. 1), is the recognition element porphyrin–Gb3 hybrid 4 as representative samples using VT-1 under
responsible for the binding of the B subunit.5 The conventional aerobic conditions. The progress of the reaction was monitored by
approach for neutralizing the cytotoxicity of verotoxin is to SDS-PAGE.9 The results are shown in Fig. 2. It is clear (Fig. 2(a)) that
synthesize analogues which possess several or a large number porphyrin derivative 3 had no degradation activity towards
of Gb3 moieties to permit multivalent interaction with verotoxin. VT-1 whether the mixture of VT-1 and 3 was irradiated with a
Recently, Gb3-conjugated molecules6 and Gb3-grafted polymers7 long-wavelength UV lamp (365 nm, 100 W) or not. In Fig. 2(b) using
were reported as inhibitors of the B subunit of verotoxin. 4, comparison of lanes 3 and 4 with lane 2 shows that neither photo-
Unfortunately, no effective clinical agents which can selectively bind irradiation of VT-1 in the absence of 4 or treatment of VT-1 with 4 in
to verotoxin and neutralize its cytotoxicity have been developed to the absence of photo-irradiation resulted in a change in the SDS-
date, in part because conventional inhibitors generally bind to PAGE profile. In contrast, lane 5 shows the disappearance of the
verotoxin reversibly, so the protein regains its activity when the bands corresponding to both the A and B subunits of VT-1,
inhibitors diffuse away. An alternative strategy is to utilize a indicating that porphyrin–Gb3 hybrid 4 caused degradation of
photosensitizer to degrade proteins when triggered by specific VT-1 upon photo-irradiation. These results clearly demonstrated
that interaction between 4, which possesses a Gb3 moiety, and
VT-1 at the B subunit is important to effectively degrade VT-1. In
addition, to confirm the protein selectivity of 4 in photo-degradation,
Department of Applied Chemistry, Faculty of Science and Technology,
Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
a mixture of VT-1, bovine serum albumin (BSA) and hen egg
E-mail: toshima@applc.keio.ac.jp; Fax: +81 45-566-1576
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc42957a lysozyme (Lyso) was subjected to the photo-degradation conditions.
c
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Fig. 2 Photo-degradation of VT-1 using 3 and 4 under long-wavelength UV or
visible light irradiation. Each protein (0.5 mM) was incubated with 3 or 4 in 0.1 M
Tris–HCl buffer (pH 7.9, 0.1 M NaCl) containing 3% DMF at 25 1C for 2 h under
irradiation with a UV lamp (365 nm, 100 W) or a visible light lamp (diffuse
sunlight, 100 W) placed 10 cm from the sample. The products were analysed by
tricine–SDS-PAGE. The gels were stained with Sypro Ruby protein gel stain. Gels
(a–d) represent (a) 3 + VT-1 with UV, (b) 4 + VT-1 with UV, (c) 4 + VT-1 + BSA + Lyso
with UV, (d) 4 + VT-1 with visible light. Lanes 1, size marker; lanes 2, proteins
alone; lanes 3, proteins upon photo-irradiation; lanes 4, proteins + compound
(15 mM) without photo-irradiation; lanes 5–8, proteins + compound (at concen-
trations of 15, 5, 1.5 and 0.5 mM, respectively) upon photo-irradiation.
with lanes 1 in Fig. 3(a)–(e) showed that neither photo-irradiation of
VT-1 in the absence of each compound or treatment of VT-1 with
each compound in the absence of photo-irradiation affected VT-1.
Furthermore, no significant degradation of VT-1 B subunits was
observed when VT-1 was treated with 6 or 7 under photo-irradiation
conditions (Fig. 3(c) and (d)). In contrast, lanes 4 and 5 in Fig. 3(a),
(b) and (e) show the disappearance of or reduction in the SDS-PAGE
band corresponding to the VT-1 B subunit after exposure to 4, 5 or 8
under photo-irradiation, which indicated that degradation of
the VT-1 B subunit had occurred. In addition, for hybrid 5, the
disappearance of the SDS-PAGE band was observed at lower
Fig. 1 Model structure of verotoxin (PDB ID: 1DM0) and chemical structures of
globotriose (1), methyl globotrioside 2, porphyrin derivative 3 and designed
porphyrin–globotriose hybrids 4–8.
Neither BSA nor Lyso were degraded even in the presence of 4
upon photo-irradiation at the concentration range examined
(Fig. 2(c)). In addition, whether visible light (diffuse sunlight,
100 W) was used or UV, the photo-degrading ability of 4 was
quite similar (Fig. 2(d)). This outcome indicated that designed
hybrid 4 could be activated even using visible light, which is
more harmless and permeable than UV.
In addition, the photo-degradation efficiency was found to be
dependent on photo-irradiation time. Importantly, lanes 5 and 6 in
Fig. 2(b)–(d) indicate that the VT-1 B subunit was degraded prior to
the VT-1A subunit. These results suggested that reactive oxygen
species, which have very short lifetimes, caused severe degradation
of the VT-1 B subunit due to the high affinity between 4 and the
VT-1 B subunit. Our previous study revealed that hydroxyl radicals
(ꢀOH) and singlet oxygen (1O2) are key players in the photo-
degradation of proteins using porphyrins.8 No other SDS-PAGE
bands corresponding to degraded peptide fragments or the
aggregated product were observed after photo-irradiation with 4.
This suggests that the degradation reaction occurred non-site-
Fig. 3 Photo-degradation of VT-1 B subunits using 4–8 under visible light
irradiation. VT-1 (70 nM) was incubated with 4–8 in PBS (pH 7.4) containing
specifically and that VT-1 was degraded into small peptide fragments 10% DMF at 25 1C for 2 h under irradiation with a visible light lamp (diffuse
that were too small to observe by SDS-PAGE analysis.10,11
Next, to compare the efficacy of photo-degradation of designed
hybrids 4–8, highly sensitive immunoblot analysis using the anti-
sunlight, 100 W) placed 45 cm from the sample. The products were analysed by
tricine–SDS-PAGE and immunoblotting with monoclonal antibody. Gels (a–e)
represent the results using 4, 5, 6, 7, and 8, respectively. Lanes 1, protein alone;
lanes 2, protein upon photo-irradiation; lanes 3, protein + compound (70 nM)
VT-1 B subunit antibody was performed using a visible light lamp.
without photo-irradiation; lanes 4–7, protein + compound (at concentrations of
The results are summarized in Fig. 3. Comparisons of lanes 2 and 3 70, 21, 7, and 2.1 nM, respectively) upon photo-irradiation.
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Table 1 EC100 of 2–8 to VT-1 with or without 30 min of photo-irradiation using
chemical synthesis and biological evaluation provide signifi-
cant information about the molecular design of novel and
artificial protein photo-degrading agents for controlling the
functions of proteins involved in diseases.
This research was supported in part by the High-Tech
Research Center Project for Private Universities: Matching Fund
Subsidy, 2006–2010, Scientific Research (B) (No. 20310140 and
23310153) and Scientific Research on Innovative Areas
‘‘Chemical Biology of Natural Products’’ from the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(MEXT) and Grant-in-Aid for JSPS fellows (22-5423).
a visible light lamp (diffuse sunlight, 100 W)
Compound
2
3
4
5
6
7
8
VIS(À) (mM)
VIS(+) (mM)
>100
>100
>20
>20
1.9
0.32
1.6
0.19
3.4
0.93
22
1.1
19
0.62
concentrations (Fig. 3(b)). These results indicated that hybrid 5
exhibited the most potent photo-degrading activity towards VT-1
among the tested hybrids 4–8. The photo-degrading activities of 4
and 8 were similar, whereas hybrids 6 and 7 showed the weakest
activities among the hybrids 4–8. These results indicated that the
photo-degrading activity of the hybrids did not simply increase as
the number of Gb3 units increased; rather, two GB3 units were
most effective, and their optimal position was when positioned cis
to each other.
Notes and references
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To confirm that hybrids 4–8 reduce the cytotoxicity of VT-1 to
Vero cells, cell proliferation after treatment of VT-1 with hybrids
4–8, and with 2 and 3, was measured by staining with crystal
violet. The effective concentrations (EC100: a minimum concen-
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In conclusion, the present work demonstrates not only the
molecular design and chemical synthesis of novel porphyrin–
Gb3 hybrids, but also their target-selective protein-degradation
profiles and potency for reducing the cytotoxicity of VT-1 to
Vero cells under photo-irradiation conditions. The described
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c
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Chem. Commun., 2013, 49, 6027--6029 6029