Photodegradation and inhibition of drug-resistant influenza virus neuraminidase using anthraquinone-sialic acid hybrids

Article abstract of DOI:10.1039/c2cc38742e

The anthraquinone-sialic acid hybrids designed effectively degraded not only non-drug-resistant neuraminidase but also drug-resistant neuraminidase, which is an important target of anti-influenza therapy. Degradation was achieved using long-wavelength UV

Full text of DOI:10.1039/c2cc38742e

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Photodegradation and inhibition of drug-resistant
influenza virus neuraminidase using
anthraquinone–sialic acid hybrids†
Cite this: Chem. Commun., 2013,
49, 1169
Received 12th November 2012,
Accepted 17th December 2012
Yusuke Aoki, Shuho Tanimoto, Daisuke Takahashi and Kazunobu Toshima*
DOI: 10.1039/c2cc38742e
www.rsc.org/chemcomm
The anthraquinone–sialic acid hybrids designed effectively a drug-resistant NA, because even drug-resistant NA must recognize
degraded not only non-drug-resistant neuraminidase but also the sialo-receptor (sialic acid) of host cells for replication. This
drug-resistant neuraminidase, which is an important target of anti- inspired the design of several anthraquinone–sialic acid hybrids,
influenza therapy. Degradation was achieved using long-wavelength 1–3, which consist of an anthraquinone 4 attached to sialic acid
UV radiation in the absence of any additives and under neutral at different positions (Fig. 1).
conditions. Moreover, the hybrids efficiently inhibited neuramini-
dase activities upon photo-irradiation.
After preparation of anthraquinone–sialic acid hybrids 1–3
(see ESI,† Schemes S1–S3), photo-induced degradation assays of
non-drug-resistant NA (influenza A virus (A/California/04/2009)
Influenza, caused by the influenza virus, is a serious disease, and H1N1 substrain)8 were conducted using 1–3 along with 4 under
influenza pandemics continue to be a major medical threat.1 In anti- UV light irradiation (365 nm, 100 W); reaction progress was
influenza therapies, influenza virus neuraminidase (NA) is one of the monitored by SDS-PAGE and immunoblotting. The results are
most important targets. The NA cleaves the linkage between progeny summarized in Fig. 2. Comparisons of lane 1 with lane 2 in
virus from the surface sialo-receptor of host cells, which is essential Fig. 2(a) and lane 1 in Fig. 2(a) with lanes 1 in Fig. 2(b)–(e)
for replication of infective virus.2 Hence, NA has been the subject of showed that neither photo-irradiation of NA in the absence of
attention as a drug target.3 One effective approach for inhibition 1–4 nor treatment of NA with 1–4 in the absence of photo-
of NA activity is to employ small molecules, which have a high irradiation resulted in a change in the SDS-PAGE profile. In
affinity for NA, such as oseltamivir4 and zanamivir.5 However, the contrast, the SDS-PAGE band corresponding to NA in lanes 2 in
emergence of drug-resistant influenza viruses has caused problems Fig. 2(b)–(e) disappeared after exposure to each compound
for effective medical treatment.6 Therefore, new types of drugs for under photo-irradiation, indicating that degradation of NA
treatment of drug-resistant influenza viruses are needed. A recent occurred. Furthermore, for hybrids 2 and 3, the disappearance
study reported that certain anthraquinone derivatives could degrade of the SDS-PAGE band was observed at lower concentrations
proteins upon photo-irradiation under neutral conditions and in the
absence of any additives.7 Here, the fundamental result of this study
was applied to design anthraquinone–sialic acid hybrids that
could effectively degrade NA and inhibit its enzymatic activity
under photo-irradiation. In addition, the hybrids effectively
inhibited drug (oseltamivir)-resistant NA as well as non-drug
(oseltamivir)-resistant NA upon photodegradation.
A
previous study showed that anthraquinone derivatives
degraded proteins upon photo-irradiation in the absence of any
additives and under neutral conditions.7 Sialic acid is a native ligand
for NA, and is essential for NA recognition. Thus, a hybrid molecule
consisting of anthraquinone and sialic acid would be expected
to effectively degrade not only a non-drug-resistant NA but also
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
Fig. 1 Chemical structures of anthraquinone–sialic acid hybrids 1–3, anthraquinone
derivative 4, and model structure of neuraminidase (NA).
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2cc38742e
c
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Fig. 2 Photodegradation of non-drug-resistant NA [influenza A virus (A/Cali-
fornia/04/2009) H1N1 substrain] using 1–4 under long-wavelength UV irradia-
tion. The enzyme (125 mU) was incubated with each compound in Tris buffer
(pH 7.5, 50 mM) at 25 1C for 60 min under irradiation with a UV lamp (365 nm,
100 W) placed 10 cm from the sample. The products were analyzed by SDS-PAGE
and immunoblotting with monoclonal antibody 2F10E12G1. Gels (a–e) represent
(a) enzyme without (lane 1) and with photo-irradiation (lane 2) in the absence of
a compound, (b–e) enzyme with 1, 2, 3, and 4, respectively. In (b–e), lane 1,
enzyme + each compound (100 mM) without photo-irradiation; lanes 2–4,
enzyme + each compound (concentrations 100, 10, and 1 mM, respectively) upon
photo-irradiation.
Fig. 3 Relation between concentration of 2 or 3 and non-drug-resistant NA
[influenza A virus (A/California/04/2009) H1N1 substrain] activity (a) without
photo-irradiation and (b) with photo-irradiation (UV lamp, 365 nm, 100 W,
10 cm, 15 or 60 min). The curve represents the concentration–inhibition curve
for the estimated IC50 value. Assays were performed in Tris buffer (pH 7.5, 50 mM) at
25 1C. Nonlinear regression analysis with Prisms version 5 (Graphpad Software, Inc.)
was used for curve fitting of the substrate cleavage reaction.
(Fig. 2(c) and (d)). These results demonstrated that anthraquinone
derivatives are capable of degrading the target enzyme NA upon
irradiation with long-wavelength UV light. In addition, certain
anthraquinone–sialic acid hybrids effectively photodegraded
NA upon photo-irradiation.
Reaction progress was monitored by SDS-PAGE and results are
summarized in Fig. 4. Comparisons of lane 1 with lane 2 in
Fig. 4(a) and lane 1 in Fig. 4(a) with lanes 1 in Fig. 5(b) and (c)
showed that neither photo-irradiation of drug (oseltamivir)-
resistant NA in the absence of the hybrid nor treatment of drug
(oseltamivir)-resistant NA with 2 or 3 in the absence of photo-
irradiation resulted in a change in the SDS-PAGE profile. In
contrast, lanes 2–4 in Fig. 4(b) and (c) show the disappearance
of or reduction in the SDS-PAGE band corresponding to drug
(oseltamivir)-resistant NA after exposure to each compound
under photo-irradiation, which indicates that degradation of
drug (oseltamivir)-resistant NA occurred. These results showed
that anthraquinone–sialic acid hybrids 2 and 3 were capable of
degrading not only a non-drug-resistant NA but also a drug-
resistant NA upon irradiation with long-wavelength UV light.
To confirm that 2 and 3 inhibited drug (oseltamivir)-resistant
NA upon photo-irradiation, an enzyme inhibition assay was
performed (Fig. 5). Results showed that 2 and 3 possessed
moderate inhibitory activity against the drug (oseltamivir)-resistant
NA without photo-irradiation (IC50: 394 and 148 mM, respectively).
In contrast, IC50 values were 82.3 and 8.77 mM for 2 and 3,
respectively, with 15 min photo-irradiation. Furthermore, IC50
values were 6.58 and 2.09 mM for 2 and 3, respectively, upon
60 min photo-irradiation. The enzyme inhibitory activities of 2 and
3 increased 60- and 71-fold, respectively, upon photo-irradiation. In
contrast, for oseltamivir (carboxylic acid form), IC50 values were
0.003 and 0.454 mM against non-drug (oseltamivir)-resistant NA
To investigate the mechanism behind the photodegradation
of NA, electron paramagnetic resonance (EPR) studies were
conducted.9 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used
as aꢁspin-trapping agent for the detection of superoxide anions
ꢀ
(O2 ) or hydroxyl radicals (ꢀOH) (see ESI,† Fig. S1). Results
showed that photo-irradiation of 1–4 in the presence of DMPO
gave products with EPR spectra characteristic of the DMPO–
hydroxy radical spin adduct DMPO–ꢀOH. The results also confirmed
that no peaks corresponding to DMPO–ꢀOH were detected, either
upon treatment of DMPO with each compound without photo-
irradiation or upon photo-irradiation of DMPO in the absence
of each compound. Thus, the ꢀOH, produced by reaction of the
photo-excited anthraquinone moieties and O2, was the major
contributor to photodegradation of NA by these anthraquinone
derivatives.10
To confirm that 1–4 bind to non-drug-resistant NA and inhibit its
activity, an enzyme inhibition assay using 20-(4-methylumbelliferyl)-
a-D-N-acetylneuraminic acid as a substrate11 hydrolyzed by NA was
conducted (Fig. 3).12 As a result, although 1 and 4 did not inhibit
enzymatic activity of NA (IC50: >500 mM), 2 and 3 showed moderate
inhibitory activity against NA (IC50: 364 and 240 mM, respectively),
indicating that 2 and 3 bind to the active site of NA. The difference
in the inhibitory effects of 2 and 3 on NA in the presence or
absence of photo-irradiation was also examined. The IC50 values
were 91.8 and 18.1 mM for 2 and 3, respectively, upon 15 min
photo-irradiation. The enzyme inhibitory activities of 2 and 3
increased 4.0- and 13-fold, respectively, with photo-irradiation.
In addition, when the photo-irradiation time was increased to
60 min, the IC50 values were 5.18 and 1.87 mM for 2 and 3,
respectively. The enzyme inhibitory activities of 2 and 3 increased
70- and 128-fold, respectively, upon photo-irradiation. These
results demonstrate that 2 and 3 inhibit the enzymatic activity
of non-drug-resistant NA much more efficiently under photo-
irradiation. In addition, the inhibitory activity can be controlled
by adjusting both the compound’s concentration and the photo-
irradiation time. Next, the photodegradation activity of 2 and 3
against drug (oseltamivir)-resistant NA (influenza A virus
(A/California/04/2009) H1N1 (H274Y) substrain) was investigated.8
Fig. 4 Photodegradation of drug-resistant NA [influenza A virus (A/California/
04/2009) H1N1 (H274Y) substrain] using 2 and 3 under long-wavelength UV
irradiation. The enzyme (125 mU) was incubated with each compound in Tris
buffer (pH 7.5, 50 mM) at 25 1C for 60 min under irradiation with a UV lamp
(365 nm, 100 W) placed 10 cm from the sample. Products were analyzed by SDS-
PAGE and immunoblotting with monoclonal antibody 2F10E12G1. Gels (a–c)
represent (a) enzyme without photo-irradiation (lane 1) and with photo-irradia-
tion (lane 2) in the absence of a compound; (b) and (c) enzyme with 2 and 3,
respectively. In (b) and (c), lane 1, enzyme + each compound (100 mM) without
photo-irradiation; lanes 2–4, enzyme + each compound (concentrations 100, 10,
and 1 mM, respectively) upon photo-irradiation.
c
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not only non-drug-resistant NA but also a drug-resistant NA was
developed using photo-irradiation and the hybrids. The present
photodegradation method using a hybrid molecule consisting
of a protein photodegradation agent and a native ligand for
the specific enzyme allows development of new strategies for
inhibiting drug-resistant enzymes with a wide range of applica-
tions. In addition, the results presented here contribute to the
molecular design of novel artificial protein photodegrading
agents and agents for controlling the functions of proteins
involved in many diseases or infections. Additional studies with
respect to the target-selectivity are currently underway in our
laboratory.
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).
Fig. 5 Relation between concentration of 2 or 3 and drug-resistant NA [influ-
enza A virus (A/California/04/2009) H1N1 (H274Y) substrain] activity (a) without
photo-irradiation and (b) with photo-irradiation (UV lamp, 365 nm, 100 W,
10 cm, 15 or 60 min). The concentration–inhibition curve represents the esti-
mated IC50 value. Assays were performed in Tris buffer (pH 7.5, 50 mM) at 25 1C.
Nonlinear regression analysis with Prisms version 5 (Graphpad Software, Inc.)
was used for curve fitting of the substrate cleavage reaction.
and drug (oseltamivir)-resistant NA, respectively (Fig. 6a). The
inhibitory activity for drug (oseltamivir)-resistant NA decreased
151-fold compared to that for non-drug (oseltamivir)-resistant
NA. Furthermore, the IC50 values for 2,3-didehydro-2-deoxy-N-
acetylneuraminic acid (DANA),2a,13 which is the lead drug of
oseltamivir and zanamivir, were 6.45 and 7.69 mM against non-
drug (oseltamivir)-resistant NA and drug (oseltamivir)-resistant NA,
respectively (Fig. 6b). The inhibitory activity for drug (oseltamivir)-
resistant NA was similar to that for non-drug (oseltamivir)-resistant
NA. The results revealed that, although the inhibition activities of 2
and 3 against drug (oseltamivir)-resistant NA were slightly
weaker than that of oseltamivir, 2 and 3 inhibited the activity
of drug (oseltamivir)-resistant NA much more efficiently under
photo-irradiation. In addition, 2 and 3 inhibited not only non-
drug-resistant NA but also a drug-resistant NA at the same level.
Furthermore, the inhibitory activities of 3 against non-drug
(oseltamivir)-resistant NA and drug (oseltamivir)-resistant NA
upon photo-irradiation were greater than those of the lead drug
of oseltamivir, DANA.
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degraded not only non-drug-resistant NA but also a drug-
resistant NA upon irradiation with long-wavelength UV light
in the absence of any additives and under neutral conditions.
Thus, a new and innovative method for effective inhibition of
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Fig. 6 Relation between concentration of oseltamivir or DANA and non-drug-
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resistant NA [influenza A virus (A/California/04/2009) H1N1 (H274Y) substrain]
activity. (a) Oseltamivir and (b) DANA. The concentration–inhibition curve repre-
sents the estimated IC50 value. Assays were performed in Tris buffer (pH 7.5,
50 mM) at 25 1C. Nonlinear regression analysis with Prisms version 5 (Graphpad
Software, Inc.) was used for curve fitting of the substrate cleavage reaction.
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1169--1171 1171