DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication
by Preventing a Cyclophilin A Induced Cis-Trans
Isomerisation in Domain II of NS5A
Lotte Coelmont1, Xavier Hanoulle2, Udayan Chatterji3, Carola Berger4, Joke Snoeck1, Michael Bobardt3,
Precious Lim3, Inge Vliegen1, Jan Paeshuyse1, Gre ´goire Vuagniaux5, Anne-Mieke Vandamme1, Ralf
Bartenschlager4, Philippe Gallay3, Guy Lippens2, Johan Neyts1*
1Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium, 2Centre National de Recherche Scientifique
– Unite ´ Mixte de Recherche 8576, Universite ´ de Lille 1, Villeneuve d’Ascq, France, 3Department of Immunology and Microbial Science, The Scripps Research Institute, La
Jolla, California, United States of America, 4Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany, 5Pharmacology and Screening Unit,
Debiopharm, Lausanne, Switzerland
DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both
in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans
isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons
(genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically ,2
weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E
in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-
fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding
sequence from the DEB025resreplicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was
observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025resreplicons. Unlike
WT, DEB025resreplicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between
CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025resreplicon. NMR titration
experiments with peptides derived from the WT or the DEB025resdomain II of NS5A corroborated this observation in a
quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320
revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level
resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025
treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the
high barrier to resistance.
Citation: Coelmont L, Hanoulle X, Chatterji U, Berger C, Snoeck J, et al. (2010) DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a
Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A. PLoS ONE 5(10): e13687. doi:10.1371/journal.pone.0013687
Editor: Wang-Shick Ryu, Yonsei University, Republic of Korea
Received August 9, 2010; Accepted October 5, 2010; Published October 27, 2010
Copyright: ? 2010 Coelmont et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported in part by grant G.0728.09N from the "Fonds voor Wetenschappelijk Onderzoek, Vlaanderen (FWO)". J. Paeshuyse and J. Snoeck
are supported by a post-doctoral position of the FWO. Part of this study was sponsored by Debiopharm (Lausanne, Switzerland). G. Vuagniaux is an employee of
Debiopharm. R. Bartenschlager is supported by a grant from the "Deutsche Forschungsgemeinschaft" (BA 1505/2). P. Gallay is supported by U.S. Public Health
Service grants no. AI087746 (P.A.G.). The funder Debiopharm had a role in study design, decision to publish and preparation of the manuscript, but not in data
collection and analysis.
Competing Interests: Part of this study was sponsored by Debiopharm (Lausanne, Switzerland). G. Vuagniaux is an employee of Debiopharm. The above does
not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
Worldwide more than 170 million people are chronically
infected with HCV and at increased risk to develop liver cirrhosis
and/or hepatocellular carcinoma . The current standard of
care consists of a combination of pegylated interferon alpha
(pegIFN-a) and ribavirin (RBV), administered for a period of 24 to
48 weeks depending on HCV genotype. This therapy is however
associated with serious side effects and sustained virological
response rates are unsatisfactory, particularly for HCV genotype
1 infection . Direct acting antivirals (DAA), i.e. molecules that
target for example the HCV NS3 protease and NS5B polymerase
have been discovered and several are in clinical development .
Alternatively, host cell factors that are essential for efficient HCV
replication can be targeted. The immunosuppressive drug CsA has
been reported to exert anti-HCV activity in vitro and in vivo [4–6].
The anti-HCV activity of CsA has been shown to be linked to its
ability to interact with cyclophilins (Cyps), which were identified as
crucial cellular cofactors for HCV replication. Cyps display a
peptidyl-prolyl cis-trans isomerase activity (PPIase) that catalyzes
the cis-trans isomerisation of the prolyl peptide bond preceding
proline residues. Data concerning the Cyp subtype essential for
HCV replication are controversial. CypB has been suggested to
act as a functional regulator of the HCV RNA polymerase by
enhancing its RNA-binding affinity and subsequently increasing
the RNA polymerase activity . Others reported that CypA, B
PLoS ONE | www.plosone.org1October 2010 | Volume 5 | Issue 10 | e13687
and C are indispensible for HCV replication . More recently
there is a growing consensus that in particular CypA is a crucial
factor during HCV replication [9–11]. A number of point
mutations in NS5B and NS5A have been reported to be associated
with in vitro resistance to CsA [12–15]. Direct interactions between
CypA and NS5B or NS5A have been observed [11,16–18].
Several CsA-analogues, i.e. NIM811 , DEB025 and SCY-
635 , are currently in preclinical and clinical development.
These molecules retain the binding affinity for Cyp, but do not
inhibit calcineurin, which is the molecular target underlying the
immunosuppressive activity of CsA . We reported earlier on
the potent in vitro anti-HCV activity of DEB025  and on the
particular characteristics of its anti-HCV activity . During
phase I clinical studies in HCV/HIV-coinfected patients, DEB025
monotherapy (1200 mg BID) resulted in a mean maximal decrease
in HCV viral load of 23.6 log10IU/ml after 15 days of therapy
. The efficacy and safety of DEB025 was further evaluated in a
phase II study in which HCV genotype 1, 2, 3 and 4 treatment–
naı ¨ve patients were randomized to receive escalating doses of
DEB025 (200, 600, 1000 mg/day) combined with pegIFN-a-2a or
monotherapy of either drugs. In patients with genotype 1 and 4,
therapy based on the 1000 mg/day dose of DEB025 resulted in a
decrease in viral load of 24.75 log10IU/ml. Viral load reduction
in genotype 2 and 3 patients was even more pronounced with a
decrease up to 25.89 log10IU/ml . Whereas lower doses
regimens are very well tolerated, the 1000 mg dose was associated
with isolated and transient hyperbilirubinemia which returned to
baseline level after treatment cessation. Subsequently, DEB025
was investigated in combination with pegIFN-a-2a and RBV in
HCV genotype 1 null/partial responders to pegIFN/RBV for 29
days. DEB025, at doses of 400 mg (with initial loading dose) and
800 mg daily, resulted, when combined with pegIFN/RBV, in a
viral load reduction of 21.96 to 22.38 log10IU/ml, respectively
We here report on the in vitro selection and characterization of
HCV subgenomic replicons (GT1b) resistant to DEB025. We
propose a mechanism by which the cyclophilin inhibitors prevent
HCV replication and how resistant replicons may interfere with
In vitro resistance selection and cross-resistance profiles
of DEB025 and CsA
Huh 9–13 cells (Con1-GT1b) were cultured (in G418-
containing medium) in the presence of an initial concentration
of 0.21 mM of DEB025 or 0.42 mM of CsA. Higher concentrations
of DEB025 resulted in rapid elimination of the HCV replicon
from the cells. Replicon-containing cells were continuously
passaged in these double-selection media. When cultures had
adapted to replication in the presence of a particular concentration
of DEB025 (i.e. no more massive cell death) the concentration was
increased in steps of 0.21 mM (typically every 3 passages). Finally,
replicon-containing cells were obtained that replicated in the
presence of 2.05 mM DEB025 (approximately 65-fold the EC50-
value of DEB025), a concentration that completely inhibits WT
replicon replication, or 2.5 mM CsA (approximately 10-fold the
EC50-value of CsA). As on average 35 passages (,20 weeks) and
40 passages (,23 weeks) of selective pressure were needed, for
DEB025 and CsA respectively, the barrier to resistance appears to
be high for cyclophilin inhibitors. DEB025 and CsA proved cross-
resistant, as the DEB025resculture was markedly less sensitive
to both DEB025 (EC50: 2.0560.56 mM) and CsA (EC50:
3.6760.78 mM) as compared to WT (EC50: 0.0360.01 mM and
0.2360.03 mM, respectively) (Figure 1). Likewise the CsAres
culture proved markedly less sensitive to both DEB025 and CsA
(EC50: 0.4660.26 mM for DEB025 and 3.7760.63 mM for CsA).
However, both of them retained WT-activity against replicons
resistant to DAA inhibitors of different classes . Conversely,
HCV polymerase inhibitors (29-C-methylcytidine and 49-azidocy-
tidine) and protease inhibitors (BILN2061 and VX-950) retained
WT-antiviral activity against DEB025resand CsAresreplicons ,
indicating a lack of cross-resistance between DEB025 and DAAs of
In vitro resistance to DEB025 is mediated via the viral
To study whether the factor that determines drug resistance was
associated with the viral genome or with the host cell, RNA
isolated from DEB025res, CsAresand WT cultures (the latter
cultured in parallel with the resistance selection) was transfected
into naı ¨ve Huh7-Lunet cells by means of lipofection. Following
three weeks of G418 selective pressure stable cell lines were
obtained and subsequently phenotyped. DEB025 proved on
average 10-fold less active in cells stably transfected with RNA
isolated from the DEB025resculture (EC50: 0.4460.07 mM), as
compared to the cell line transfected with WT replicon (EC50:
0.0460.01 mM) (Figure 2A). CsA proved on average 3.5-fold less
active in the cell line obtained following transfection with RNA
isolated from the CsAresculture, as compared to WT (EC50:
1.0860.12 mM and 0.3160.05 mM, respectively) (Figure 2B).
Furthermore, cross-resistance between DEB025 and CsA was
confirmed. Of note, when stable cell lines were generated in the
absence of DEB025 or CsA, the mutant replicons retained their
original mutations and did not revert back to the WT sequence
(data not shown). Since the cell lines transfected with RNA from
DEB025resand CsArescultures proved also resistant to the drugs,
although not to the full extent as the originally selected resistant
cultures; it is concluded that resistance is at least in part associated
with the viral genome, although cellular changes may also play a
role as discussed below.
The P-glycoprotein [P-gp, the product of the multidrug
resistance (MDR) gene, is an ATP-dependent pump that extrudes
certain drugs from the cell] content of these cell lines was
determined by means of FACS analysis. P-gp was on average 9-
fold more abundant on the DEB025rescells and 7.5-fold more on
the CsArescells, as compared to WT cells (data not shown).
Whether these cellular changes play any role in resistance to
DEB025 and CsA remains to be determined. We will focus our
attention in this manuscript on the resistance associated with the
NS5A D320E confers low-level resistance to DEB025
To identify the variations in the viral genome associated with
the observed resistant phenotype, we sequenced the entire
subgenomic region encoding the non-structural proteins of the
replicon RNA from DEB025resand CsArescultures and compared
them to the WT sequence of non-resistant control cells. Four
mutations were identified (both by population as well as clonal
sequencing) with one residing in the NS3 serine protease gene
(A241P) and three in domain II of the NS5A gene (R262Q,
R318W and D320E) (Figure 3A).
We determined the prevalence of these mutations in HCV
genotypes (GT) 1a, 1b, 2a, 2b, 3 and 4 in the European HCV
database (euHCVdb)  (Figure 3B) and Los Alamos HCV
database (Table S2). The A241P variant in NS3 is observed in
none of the sequences in the database. All genotypes have an A at
this position, except for genotype 3, which has an S as wild type
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amino acid. In the NS5A gene, Q262 and W318 are equally
absent in all sequences. All genotypes have the R at position 318.
For position 262 genotype 1 and 3 have the R (as the replicon),
while most of the genotype 2 and 4 sequences lack this position
(based on an alignment with all genotypes). The D320E variant is
present in a small number of sequences of genotype 1b, 2a, 2b and
4; all genotypes bear the D as wild type amino acid.
To study the contribution of the identified mutations to the
resistant phenotype, single mutations and combinations thereof
were reintroduced into a WT GT1b subgenomic luciferase
replicon backbone via site-directed mutagenesis and the suscep-
tibility of these constructs to DEB025 and CsA was determined
(Figure 3C). Introduction of A241P or R262Q had no effect on the
susceptibility to DEB025 or CsA. Introduction of R318W resulted
in a 62-fold change in sensitivity to DEB025 and CsA. D320E
resulted in a more pronounced effect on the antiviral phenotype
(3.92-fold for DEB025 and 6.42-fold for CsA). A genome carrying
in addition to D320E also R318W or [R318W+A241P + R262Q]
did not result in an additional reduction in sensitivity to DEB025
(3.79 and 4.33-fold WT, respectively) (Figure 3D). The D320E
mutation, when combined with R318W, either with or without
[A241P + R262Q], resulted in a phenotype that was about 10-fold
less sensitive to CsA (Figure 3E). Again, cross-resistance between
DEB025 and CsA was confirmed. The generated mutant replicons
retained WT-activity to IFN, protease (VX-950) and polymerase
(29-C-methylcytidine and HCV796) inhibitors (data not shown).
To exclude that the observed resistance is the result of generally
enhanced replication, the replication fitness of each mutant was
compared to that of WT replicon. The replicative capacity of the
single mutated replicons was slightly reduced or comparable to
that of WT, whereas replicons that carried two or more mutations
had a reduced replicative fitness compared to WT (Figure 3F).
The important role of the change from aspartic acid to glutamic
acid at position 320 (D320E) in domain II of NS5A was further
substantiated by the observation that this was the only common
mutation found in three additional independent resistance
selection assays performed in Huh 9–13 cells (two selections) and
in Huh 5–2 cells (one selection) (Table S3). The majority of
Figure 1. Characterization of DEB025resor CsAresreplicon containing cell lines. Dose-response curves for inhibition of viral subgenomic
replication by either (A) DEB025 or (B) CsA in WT (closed diamonds), DEB025res(open squares) and CsAres(grey circles) cell lines. The respective Huh 9-
13 cells were treated for 72 h with escalating concentrations of either DEB025 or CsA. HCV replicon RNA was quantified by means of RT-qPCR and is
expressed as the percentage of HCV replicon RNA of the untreated control cells. Data are mean values6standard deviations for at least three
Mechanism of Action of DEB025
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identified mutations clustered in the NS5A region. We therefore
replaced the entire NS5A gene from the WT genome with the
corresponding NS5A from the DEB025res(1stres selection)
genome. The antiviral phenotype of this construct was evaluated
in comparison with a construct into which the NS5A region
derived from WT Huh 9–13 cells (which were cultured in parallel),
was swapped. DEB025 proved 10-fold and CsA 9-fold less active
in replicons that were engineered to contain the DEB025resNS5A
sequence compared to replicons that were engineered to contain
the WT NS5A sequence (EC50: 0.3960.15 mM versus 0.046
0.01 mM for DEB025 and 2.3660.20 mM versus 0.2860.08 mM
for CsA) (Figure 4A and B).
It has been reported that CsA may select for I432V in NS5B
and that NS5B might be involved in the mechanism of action of
CsA . In our hands, I432V neither reduced the antiviral
activity of DEB025 or CsA nor enhanced the resistance observed
with the D320E mutation (Figure S1). We also introduced the
entire NS5B gene of the DEB025resreplicon in a WT background,
as well, by means of control, the NS5B region from WT Huh 9–13
cells (which were cultured in parallel). No reduction in antiviral
activity of DEB025 and CsA was observed (Figure 4A and B). The
NS5A and NS5B swapped constructs retained WT-sensitivity to
several protease and polymerase inhibitors (data not shown).
Taken together the swapping results corroborate the observation
that mutations in NS5A are responsible for the observed genome-
associated resistance. The replication fitness of all swapped
constructs was reduced compared to that of the ‘unmanipulated’
original vector (Figure 4C).
Recently we showed that JFH1-derived subgenomic replicon
(GT2a) with a ,5-fold reduced sensitivity to DEB025 carried a
mutation near the cleavage site (position 464) between NS5A and
NS5B. This mutation was demonstrated to slow down cleavage
kinetics, implying a correlation between HCV polyprotein
processing and CypA dependence of HCV replication .
Introduction of these mutations (V445A and V445L are the
corresponding mutations in the GT1b background) into the GT1b
Figure 2. Drug resistance is associated with the viral genome. Dose-response curves for inhibition of replicon RNA by either (A) DEB025 or (B)
CsA in Huh7-Lunet cells stably transfected with RNA isolated from WT (closed diamonds), DEB025res(open squares) and or CsAres(grey circles) Huh 9–
13 cell lines. HCV replicon RNA was quantified by means of RT-qPCR and is expressed as the percentage of HCV replicon RNA of the untreated control
cells. Data are mean values 6 standard deviations for at least three independent experiments.
Mechanism of Action of DEB025
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Mechanism of Action of DEB025
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subgenomic replicon resulted in a 1.28-fold change in sensitivity to
DEB025 for V445A and 1.93-fold for V445L compared to WT
(two independent experiments; Figure S2).
DEB025resreplicon is less dependent on CypA for
Several groups have identified CypA as an indispensible cellular
factor for efficient viral replication [9–11]. Since DEB025 is a
cyclophilin-binding molecule, we studied whether DEB025resHCV
replicon RNA is still able to replicate in a cell line in which CypA
was stably knocked down (KD). The efficiency of the knock down of
CypA was verified by means of Western blotting . The
replication fitness of WT and DEB025resRNA, transiently
transfected into CypA KD and control (CTRL) cells, was evaluated
(Figure 5). DEB025resreplicon proved less fit than WT replicon in
the CTRL cells (on average 6-fold lower than WT-value at 96h post
transfection). A replication-deficient replicon (the ‘GND-variant’),
was included as an internal control. Conversely DEB025resreplicon
had markedly increased replication fitness in the CypA KD cells
compared to WT (on average 8-fold WT-value at 96h post
transfection). This indicates that mutations in the DEB025res
replicon render HCV replication less dependent on CypA.
DEB025 disrupts the interaction between CypA and (WT
or DEB025res) NS5A
Since CypA is crucial for an efficient HCV replication [9,10,29]
and since direct interactions between domain II of NS5A and
CypA have recently been reported ; there may be a direct
correlation between CypA assistance to HCV replication and
CypA binding to NS5A. Based on this model, we postulated that
DEB025, as a Cyp inhibitor, would interfere with the interaction
between CypA and NS5A, thereby inhibiting HCV replication.
We therefore studied whether DEB025 and CsA are able to
disrupt the interaction between CypA and NS5A. Pull down
experiments between GST-CypA and His-tagged NS5A revealed
that DEB025 and CsA inhibit the CypA-NS5A interaction in a
dose-dependent manner (Figure 6A). DEB025 was more potent in
blocking the CypA-NS5A interaction than CsA (Figure 6B),
further supporting the earlier finding that the affinity of DEB025
to CypA is superior to that of CsA to CypA . We confirmed
earlier that GST alone does not immunoprecipitate NS5A .
We next studied whether the NS5A mutations, either alone or in
combination, have an influence on (i) the binding of NS5A to
CypA and (ii) the sensitivity of the CypA-NS5A interaction to
DEB025. All NS5A mutants studied were found to bind with the
same relative interaction to CypA as WT NS5A (Figure 6A). Also
the interactions between CypA and WT or mutated NS5A were
equally disrupted either by DEB025 or CsA.
To further quantify this observation, we prepared by chemical
synthesis two peptides (20-mers) with the sequences of the WT
domain II of NS5A (Pep-WT) or the DEB025resD320E mutant
(Pep-D320E). We centralized the 320 position in these 20-mer
peptides, while maintaining the up- and downstream proline
residues. It was previously shown that this motif in domain II of
NS5A interacts with CypA . NMR spectra of both peptides
coincided reasonably well with the spectra of the identical
sequences in the full-length domains II, underscoring the natively
unfolded character of domain II of NS5A . We then titrated
these peptides against a15N-labeled CypA sample, and derived
from the chemical shift differences a dissociation constant
(Figure 7A–D). Chemical shift perturbations were mainly
monitored for residues in the active site of CypA. The dissociation
constants derived for both peptides were weak (with KDvalues of
0.802 mM and 1.232 mM for the Pep-WT and Pep-D320E
peptide, respectively), probably reflecting the fact that we did not
use the full-length protein as substrate. Importantly, the mutation
did not dramatically alter the dissociation constants, in agreement
with the results from the pull-down experiments.
A peptide carrying the D320E mutation has a markedly
altered cis/trans configuration than WT
Because the peptides did not show an altered affinity for the
CypA surface, we further recorded heteronuclear1H,15N HSQC
spectra at natural abundance on them. NMR is unique in the
sense that it can detect the trans and cis conformations of any prolyl
bond, even though these leave no mass signature. In the WT
peptide, we observed a major and minor conformation for several
residues preceding the Pro319 (Figure 8A and B), that we
tentatively assigned to the conformers associated with the peptide
bond preceding Pro319 in the trans and cis conformation. Upon
integration of these peaks, we obtained equilibrium values of
75.9% for the major and 24.1% for the minor form. When we
repeated the same experiment with the D320E peptide, however,
we found that the peak resonating at the minor frequency in the
WT peptide had become the major one, with populations of
70.4%. To be sure that peak positions had not shifted due to
slightly different experimental conditions, we mixed both peptides
in equimolar amounts and ran again the NMR spectrum. We now
only observed three peaks, showing unambiguously that the minor
correlation peak in the WT peptide coincides with the major one
in the D320E peptide (Figure 8C). Although further studies on the
full-length domain II of the WT and D320E NS5A proteins will be
required to elucidate the precise structural origin of this
conformational inversion, our observations suggest that CypA
resistance arises by mutations that affect directly the conformation
of domain II of the NS5A protein. The prolyl cis-trans
Figure 3. Identification and characterization of mutations conferring resistance to DEB025. (A) Schematic representation of the
selectable Con1 (GT1b) subgenomic replicon (Huh 9–13) that was used to select for DEB025 (and CsA) resistance. RNA isolated from drug resistant
and control cell cultures (passaged in parallel), was sequenced. The mutations identified in the resistant cultures are depicted above their respective
position in the polyprotein. (B) Prevalence of the variants, found in the replicon after DEB025 selection, in different genotypes in the European HCV
3del=deletion, [deletion, all of the genotype 2b and most of the genotype 2a and 4 sequences miss amino acid 262 (numbering according to
genotype 1b) in NS5A]. (C) Mutations, that had been identified in the DEB025resor CsAresreplicon, were introduced (either alone or combined) in a
WT background, after which the sensitivity to DEB025 and CsA was assessed. Data are expressed as fold reduction (based on EC50-values) in sensitivity
to the drug as compared to WT replicon. (D) Dose-response curves for inhibition of replicon replication by DEB025 or (E) CsA in Huh7-Lunet cells
transiently transfected with mutant replicon RNA (indicated on the right site of each panel). HCV replicon RNA was quantified by means of a luciferase
assay and data are expressed as percentage of untreated controls. Data are mean values 6 standard deviations for at least three independent
experiments. (F) Replication fitness of the various mutant replicons. Huh7-Lunet cells were transiently transfected with the indicated mutant replicons
and RNA replication was measured by means of a luciferase assay for 4 days post transfection. Data are normalized to the 4 h-value post
electroporation to account for differences in electroporation efficiency and expressed as a percentage of the WT-value at 96 h post electroporation.
Data are mean values 6 standard deviations for at least two independent experiments.
1Variant identified in the DEB025resreplicon.
2WT amino acid for that genotype (the amino acid with the highest prevalence).
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Figure 4. Mutations in NS5A but not NS5B contribute to resistance against DEB025. Dose-response curves for inhibition of replicon
replication by (A) DEB025 or (B) CsA in Huh7-Lunet cells transiently transfected with recombinant replicon RNA carrying either the NS5A or NS5B
region from DEB025resor control (WT) cultures. For comparative reasons also the original unmanipulated vector was included in the analysis. Cells
were treated for 72 h with escalating concentrations of DEB025 or CsA and HCV replicon RNA was quantified by means of a luciferase assay and is
expressed as percentage of the untreated controls. Data are mean values 6 standard deviations for at least three independent experiments. (C)
Replication fitness of the different recombinant replicons. Huh7-Lunet cells were transiently transfected with each of the indicated replicon
constructs and replicon RNA replication was quantified by means of a luciferase assay for 4 days post transfection. Data are normalized to the 4 h-
value post electroporation to account for differences in electroporation efficiency and is expressed as percentage of the WT-value at 96 h post
electroporation. Data are mean values 6 standard deviations for at least two independent experiments.
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isomerisation activity of CypA would thereby be not strictly
required anymore, explaining the resistance to DEB025 or CsA.
We previously reported on the potent anti-HCV activity of the
non-immunosuppressive CsA analogue DEB025  and studied
the specific in vitro characteristics of this molecule when combined
with pegIFN/RBV and several DAA inhibitors . Here we
report the in vitro characterization of DEB025resreplicon and
propose a mechanism by which DEB025, and by extension other
cyclophilin-binding agents with anti-HCV activity, inhibit viral
The selection of resistance to either DEB025 or CsA proved to
be a lengthy process, indicating that the genetic barrier towards
resistance of this class of inhibitors is high. This may be explained
by the unique mechanism of action of these inhibitors. Indeed, the
virus does not need to acquire (a) mutation(s) that prevent(s) direct
binding of the inhibitor to the viral protein, but must rather
acquire a mutation that makes it less dependent of a host factor
that is otherwise essential for viral replication. We reported that
DEB025 is highly efficient in curing hepatoma cells from their
replicon . Both in terms of drug concentration and time of
culture needed to result in clearance, DEB025 is by far more
efficient than ‘classical’ DAA inhibitors. This may be related to the
fact that drug-resistant variants are not readily selected. Moreover,
this might also be explained by the fact that DEB025 specifically
sequesters CypA and therefore prevents conformational changes
in the replication complex that are crucial for viral replication.
Altering the conformation of replication complex components may
result in an irreversible modification (which is not the case with
DAA inhibitors) and thus efficient clearance. Earlier clinical
studies have shown that resistance mutations to protease inhibitors
emerged in patients within days of treatment , leading to
treatment failure. The low barrier to resistance to protease
inhibitors and other DAAs therefore requires combination
therapy. Since DEB025 is fully active against resistant replicons
selected by other DAAs  and DAA inhibitors retained WT-
Figure 5. RNA replication of DEB025 resistant replicon is less dependent on CypA. Replication competence of replicon RNA isolated from
DEB025resor WT Huh 9–13 cultures in Huh7-Lunet cells (A) transduced with a lentiviral control vector (CTRL) or with (B) a stable knock down of CypA
(KD). Replication was quantified at various time points post electroporation by means of RT-qPCR. A replication-deficient construct (GND) was
included as a negative control. Data are expressed relative to the 4 h-value to normalize for differences in electroporation efficiency. Means of a
representative experiment are shown.
Mechanism of Action of DEB025
PLoS ONE | www.plosone.org8 October 2010 | Volume 5 | Issue 10 | e13687
activity against these DEB025resor CsAresreplicons, the high
resistance barrier and favorable safety profile of DEB025
potentially allow it to be the backbone of future combination
therapy as well as an essential component of rescuing regimen for
patients who failed protease inhibitors or other DAAs treatment.
In this study, transfection of RNA isolated from these
DEB025resor CsArescultures into naı ¨ve hepatoma cells transferred
part of the originally observed resistance, indicating that resistance
can be conferred by the viral genome. Four independent resistance
selection assays were carried out; with NS5A D320E being the
only mutation identified in all resistant replicons. A number of
other NS5A mutations at relatively conserved sites were also seen
in one of the four resistant lines; these are not known as adaptive
mutations that generally enhance replication. However, reintro-
duction of single as well as combinations of mutations into a WT
background identified D320E as the single mutation conferring
part of the observed genome-associated resistance. The region
around the D320 position is well-conserved among a diverse set of
HCV genotypes. This suggests that it might play an important role
in some aspect of the HCV life cycle . In addition, other
groups recently reported that D320E and the near variant Y321N
are involved in resistance to cyclosporins in vitro [13,14,17].
However the full extent of genome-associated resistance was not
restored by the single mutation D320E. Exchanging the NS5A gene
from WT replicons by the corresponding NS5A from DEB025res
replicon resulted in a yet more pronounced resistance. A number of
other mutations could be found in subgenomic replicons (relative to
the original Con1 sequence), which generally enhance subgenomic
replication. Although these mutations alone were unable to confer
resistance to DEB025 or CsA, they did contribute, in concert, to the
elevated resistancelevelaswasevident intheswappingexperiments.
It was demonstrated that CypA binds to NS5B and that the
enzymatic pocket of CypA is critical for this interaction. DEB025
was reported to prevent the CypA-NS5B interaction . Moreover,
it has been suggested that NS5B is involved in the mechanism of
action of anti-HCV activity of Cyp inhibitors . However our
observations argue against this hypothesis (at least in the context of
GT1b), as introducing the NS5B gene derived from DEB025res
replicon into a WT background did not result in transfer of
resistance. Also I432V in NS5B, a variant previously reported as
being involved in CsA resistance , was not able to confer CsA or
DEB025 resistance in our hands.
The HCV NS5A protein has no known intrinsic enzymatic
activity, but likely exerts its functions through interactions with
Figure 6. DEB025 disrupts the CypA-NS5A interaction in a dose-dependent manner. Recombinant GST-CypA (100 ng) was incubated with
recombinant NS5A Con1-His (10 ng) together with increasing concentrations of DEB025 (A) or CsA (B) for 3 h at 4uC. Glutathione beads were added
to the GST-CypA/NS5A-His mixture for 30 min at 4uC and washed. Bound material was eluted and analyzed by Western blotting using anti-His and
anti-CypA antibodies. (C) The percentage of NS5A pulled down in the presence of increasing concentrations of DEB025, relative to the amount of
NS5A pulled down in the absence of the drug.
Mechanism of Action of DEB025
PLoS ONE | www.plosone.org9 October 2010 | Volume 5 | Issue 10 | e13687
viral and cellular factors. It is a pleiotropic protein which plays an
essential role in the HCV viral life cycle for example by supporting
viral RNA replication as well as by modulating the physiology of
the host cell to favour viral replication . It occurs in a basally-
and hyperphosphorylated form, with different putative functions
during HCV replication . NS5A consists of three domains,
separated by low-complexity sequences . Whereas domain I
appears to be involved in RNA-binding [36–38] and domain III is
essential for infectious particle assembly [39–41], the specific role
of domain II, which is natively unfolded [42,43], still remains to be
elucidated. Direct molecular interactions between domain II and
both CypA and CypB have been observed . Surprisingly, we
observed that the interaction of CypA with DEB025resNS5A was
as sensitive to inhibition by DEB025 and CsA as the interaction
between CypA and WT NS5A. This result implies that DEB025res
NS5A does not depend on CypA-binding to efficiently contribute
to RNA replication. Small molecule inhibitors of HCV that target
NS5A have been shown to reduce the hyperphosphorylated form
of NS5A . Since DEB025resreplicons carry mutations in
NS5A, we studied by means of Western blot and phosphatase
treatment experiments whether NS5A hyperphosphorylation was
different in either WT, CsA or DEB025rescultures. No such
differences were however observed (data not shown). Mutations
conferring resistance to these NS5A inhibitors reside in domain I
of NS5A, so cross-resistance between DEB025 and this type of
inhibitors is unlikely to occur .
Replication of the DEB025resgenome proved, unlike replication
of the WT genome, to be efficient in CypA KD cells, indicating
that the drug resistant genome is less dependent on CypA (and
thus likely on the cis-trans isomerase activity) for efficient
replication. Given the fact that domain II of NS5A contains
many proline residues that form potential valid substrates for the
enzymatic peptidyl-prolyl cis-trans isomerase activity of cyclophi-
lins, we studied by means of NMR the conformation of a 20-mer
peptide carrying the E320 in the middle and compared this to that
of a corresponding WT peptide. The residues preceding Pro319 in
the WT peptide occurred on average for 24.1% in a minor
conformation that we tentatively assigned to the Pro319 cis form.
Based on the chemical shift identity, this same minor conformer in
the WT-peptide becomes the dominant one in the D320E-peptide,
with a relative population of 70.4%. We therefore assume that the
DEB025resgenome lowers CypA dependence by acquiring a
conformational inversion mediated by the D320E mutation, that
otherwise is supported by CypA. The data generated with these
peptides may not necessarily reflect the situation of the full length
NS5A protein in the host cell. However, based on the dataset
obtained, it can be hypothesized that the change in conformation
is in some way crucial for HCV replication. Blocking the
Figure 7. NMR titration experiments between CypA and NS5A-derived peptides, Pep-WT or Pep-D320E. Panels (A) and (C) correspond to
the superimposition of the1H,15N HSQC spectra of15N-CypA acquired in the presence of increasing amounts of unlabeled peptides; Pep-WT in (A) and
Pep-D320E in (C). Whereas both peptides correspond to residues 308–327 of HCV NS5A protein (GT1b, Con1), Pep-WT correspond to the WT sequence
(308-KFPRAMPIWARPDYNPPLLE-327) and Pep-D320E carries the D320E mutation (308-KFPRAMPIWARPEYNPPLLE-327). M100 in CypA is a residue that is
located at the periphery of the PPIase active site. (B) and (D), titration curves corresponding to experiments in (A) and (C) respectively. The1H,15N
combined chemical shift perturbations dD (in ppm) (dD=|d(1HN)| + 0.2x|d(15N)|) were plotted as a function of the Peptide:CypA molar ratios. The
dissociation constants (KD) were obtained by fitting the experimental data with the following equation: KD=[CypAfree]x[Peptidefree]/[CypA:Peptide].
Mechanism of Action of DEB025
PLoS ONE | www.plosone.org10October 2010 | Volume 5 | Issue 10 | e13687
interaction between CypA and NS5A by a cyclophilin-binding
compound such as DEB025, results therefore in inhibition of HCV
replication. It has been reported that NS5A interacts with NS5B
through two independent regions (of which one includes the D320
position) and that NS5A modulates the activity of NS5B RdRp
through this interaction . Possibly the conformational changes
in NS5A catalyzed by CypA contribute to these crucial
interactions with NS5B.
Taken together, our data point to an entirely novel and exciting
mechanism by which HCV (GT1b) replication can be blocked by
CypA inhibitors. Resistance mutations may trigger a particular
conformation in NS5A that is (directly or indirectly) required for
efficient viral replication. The fact that the virus needs to develop a
strategy to efficiently replicate largely independent from a host
factor may explain the high genetic barrier that has to be passed to
develop resistance against cyclophilin inhibitors.
The distinctive resistance profile of DEB025 provides a unique
option in treating chronic HCV infection, both as the backbone of
future combination therapy with other compounds in treatment-
naı ¨ve patients and as rescue therapy for patients harbouring
resistance mutations to other classes of anti-HCV agents.
Materials and Methods
The compounds used were described previously .
Huh7 cells containing subgenomic HCV genotype 1b repli-
con I377/NS3-39/wt (Huh 9–13) [47,48] and I389luc-ubi-neo/
NS3-39/5.1 (Huh 5–2) were used to select for resistance.
Transfection experiments were performed in Huh7-Lunet cells
 supporting high level of viral RNA replication. The
generation of CypA knock down (KD) cells has been described
before . Stable Cyp KD cell lines were obtained under
puromycin (1 mg/ml) selection and CypA KD was verified by
Western blotting as described previously . Cells were grown in
Dulbecco’s modified Eagle’s Medium (DMEM; Gibco) supple-
mented with 10% heat-inactivated fetal bovine serum (FCS)
(Integro), 1x non-essential amino acids (Gibco), 100 IU/ml
penicillin (Gibco), 100 mg/ml streptomycin (Gibco), 250 mg/ml
G418 for Huh 5-2 and 1000 mg/ml G418 for Huh 9–13 cells or
puromycin (1 mg/ml) for Cyp KD cells.
Selection of drug resistant replicon cell lines
Drug resistant replicons were generated by passaging HCV
subgenomic replicon containing cells (Huh 9–13 cells) under G418
selection (1 mg/ml G418) in the presence of gradually increasing
concentrations of DEB025 or CsA. When cells suffered from
compound pressure, G418 pressure was removed till cells
recovered. Thereafter, cells were re-cultured with either CsA or
DEB025 and G418 pressure. Four independent resistance
selection experiments were carried out, three with Huh 9–13
replicons and one with Huh 5–2 replicons.
Antiviral assays and RT-qPCR
HCV replication assays were performed and analysed using
published procedures .
Figure 8. NMR analysis of the conformational consequences of the D320E mutation.1H,15N HSQC spectra were acquired at natural
abundance on Pep-WT (A) and Pep-D320E (B). The NMR backbone proton amide resonances corresponding to each residue in the 316-WARP(D/E)-
320 motif are shown in the different inserts. The resonances indicated by an arrow correspond to the major form of the residue whereas the ones
marked with an asterisk correspond to the minor form. The population of the minor form is given as a percentage of the total peptide population. (C)
1H,15N HSQC spectrum acquired at natural abundance on a mixture of Pep-WT and Pep-D320E peptides at equimolar ratio (3 mM each).
Mechanism of Action of DEB025
PLoS ONE | www.plosone.org11 October 2010 | Volume 5 | Issue 10 | e13687
Total RNA from pooled drug resistant or control (non-treated)
cultures was isolated (RNeasy mini kit, Qiagen), according to the
manufacturer’s instructions, and amplified by RT-PCRs to
generate overlapping fragments that covered the full length of
the replicon. The sequence of the entire region encoding HCV
non-structural proteins was determined. Alternatively, the RT-
PCR fragments from the NS5A region were cloned into a pCRII-
TOPO TA vector (Invitrogen) and plasmid DNA from 10
bacterial colonies was sequenced (Big DyeH Terminator v3.1,
Applied Biosystems). The sequences were aligned and analysed
using Vector NTI advance software (Invitrogen).
Plasmid pFKI389Lucibineo EI NS3-39ET  was used for the
reintroduction of identified mutations. Initially, a NotI and MluI
fragment containing the NS3, NS4A, NS4B and part of the NS5A
coding region was subcloned into the pCRII-TOPO vector
(Invitrogen) for reintroduction of the A241P mutation in NS3. A
MluI and XhoI fragment that contains a part of the NS5A coding
region was subcloned into a pCRII-TOPO vector in which a part
of the yellow fever virus was inserted to provide the vector with the
correct restriction sites. This vector was used to reintroduce the
R262Q mutation in NS5A. An XhoI and SpeI fragment
containing part of the NS5A and the NS5B coding region was
subcloned into the pCRII-TOPO vector for reintroduction of the
R318W and D320E mutations in NS5A. Site-directed mutagenesis
was performed using the ‘‘QuickChange Site Directed Mutagen-
esis Kit’’ (Stratagene) according to manufacturer’s instructions.
The primers used for mutagenesis are shown in Table S1. The
mutated fragments were cloned back into the pFKI389Lucubineo
EI NS3-39ET plasmid. DNA sequencing confirmed the presence
of the introduced mutations.
RNA was isolated from either DEB025resor WT HCV
subgenomic replicon cells using the RNeasy mini kit (Qiagen).
First-strand cDNA was synthesized using SuperScript II RT
(Invitrogen). The NS5A or NS5B-containing regions were
amplified in an AccuPrime Pfx DNA polymerase reaction
(Invitrogen) for subsequent cloning into the WT Con1 plasmid.
All constructs were sequenced to verify the correct manipulation.
In vitro transcription
Five mg of mutated or WT plasmids were linearized with ScaI
(Promega) and AseI (New England Biolabs) (only for pFKI389
Lucibineo EI NS3-39ET plasmid). Thereafter, linearized plasmid
was phenol/chloroform extracted, ethanol precipitated and
dissolved in RNase free water. HCV replicon RNAs were
generated using the RiboMAXTMLarge scale RNA production
system-T7 (Promega). RNA was purified and collected by using
the RNeasy mini kit (Qiagen). The concentration and purity of
RNA were spectrophotometrically measured.
Replicon RNA was delivered to Huh7-Lunet cells by electro-
poration or liposome-mediated transfection (for the generation of
stable cell lines).
One day before lipofection, Huh7-Lunet cured
cells were seeded in a 25 cm2flasks at a cell density of 5.266105
cells. Just before transfection, the cells were washed once with opti-
MEMH I (Invitrogen) and incubated for 4 h at 37uC with
transfection medium containing: 26.3 mg of RNA, DMRIE-C
Reagent (Invitrogen) and opti-MEM I serum free medium. Four
hours post transfection, medium was replaced by culture medium
without G418. Depending on the proliferation of the cells, G418
was gradually added to the cultures (to a final concentration of
1 mg/ml) to generate stable cell lines.
Huh7-Lunet single-cell suspensions were
prepared by trypsinization, washed twice with Opti-MEMH I
(Invitrogen) and then resuspended at 1610‘7 cells per ml in
Cytomix  containing 2 mM ATP and 5 mM glutathione.
Ten mg of in vitro transcribed RNA was mixed with 400 ml cell
suspension in a cuvette with a gap width of 4 mm (VWR
International) and an ECM 830 Electro Square PoratorTM(BTX
Harvard Apparatus) was used to deliver 5 pulses at 900 V, 99 msec.
Cells were immediately transferred to 20 ml complete DMEM
and seeded as required for the assay. Briefly, 100 or 700 ml
aliquots of the cell suspension were seeded in a 96- or 24-well plate
(Iwaki), respectively, filled with serial dilutions of the antiviral
molecule in complete DMEM. Cells were allowed to proliferate
for 4 days at 37uC, after which the luciferase signal was
(Promega) in a Luminoskan Ascent (Thermo).
Transfections were performed in Huh7-Lunet cells as described
above with the exception that cells were transfected with 5 mg
RNA and 5 mg tRNA as a carrier (Sigma Aldrich). Tranfected cells
were immediately transferred to 24 ml of complete DMEM and a
1.5 ml aliquot of the cell suspension was added to a 6 well plate
(Iwaki). Cells were collected at 4 h (normalization point) and 1, 2,
3, 4 days post transfection to compare mutant luciferase values
with WT values. Cells only electroporated with tRNA were
analyzed at the indicated time points for luciferase activity and
used as background activity from the residual input RNA. A GND
replicon (a replication-deficient subgenomic replicon encoding a
GDD to GND mutation in NS5B ) served as a negative
Production of recombinant CypA and NS5A proteins
Recombinant GST (glutathione S transferase)-CypA was
produced and purified as we described previously , as well
as full-length NS5A GT1b (pET-Ub-NS5A Con1-His) was
expressed as described previously . NS5A mutants were
created by site-directed mutagenesis.
CypA-NS5A pull-down studies
Pull-down experiments were conducted as described previously
. Briefly, GST-CypA (100 ng) was mixed with 10 ng of NS5A-
His in a total volume of 200 ml for 3 h at 4uC on a rotating wheel.
Glutathione beads (25 ml) were added to the GST-CypA/NS5A-
His mixture for 30 min at 4uC and samples were washed 3 times
thereafter. Beads were pelleted for 30 sec at 2000 g in a
microcentrifuge and bound material was eluted with 25 ml of 2x
SDS sample buffer, heated for 5 min, and frozen at 220uC.
Bound material was then analyzed by Western blotting using anti-
CypA and anti-His antibodies as described previously . CsA or
DEB025 was added together with NS5A and CypA. To calculate
the percent of NS5A pulled down relative to the amount of NS5A
pulled down in the absence of the drug scans of ECL-developed
films were analyzed by Bio-Rad densitometer model 620 and
quantified by Bio-Rad Program Manager software (Bio-Rad
Laboratories, Inc., Hercules, CA). Data are expressed in
percentage of pulled down NS5A in the presence of increasing
concentrations of DEB025 by arbitrarily fixing the percentage
Mechanism of Action of DEB025
PLoS ONE | www.plosone.org 12 October 2010 | Volume 5 | Issue 10 | e13687
(band intensity) of each pulled down NS5A protein in the absence
of the drug at 100.
NS5A-derived peptides NMR analysis
Purified synthetic peptides, Pep-WT (308-KFPRAMPIWARP-
PEYNPPLLE-327), corresponding to residues 308–327 of NS5A
from HCV GT1b (Con1) were obtained from Neosystems
(Strasbourg, France). The purity of the peptide was verified by
high pressure liquid chromatography and mass spectrometry as
greater than 95%. Recombinant15N-labelled CypA was produced
and purified as described previously . Spectra were acquired
on a Bruker Avance 600 MHz equipped with a cryogenic triple
resonance probe head (Bruker, Karlsruhe, Germany). The proton
chemical shifts were referenced using the methyl signal of TMSP
(sodium 3-trimethylsill-[2,2_,3,3_-d4]propionate) at 0 ppm. Spec-
tra were processed and analyzed with the Bruker TopSpin
software package 2.1. To study the interaction between CypA
and peptide Pep-WT or Pep-D320E,15N-labelled CypA (100 mM)
was mixed with increasing concentrations of non labelled peptide
(from 0 to 2.5 mM) and a1H,15N HSQC spectrum was recorded
at each step. The combined chemical shift perturbations (dD, in
ppm) following peptide addition were calculated as dD=|d(1HN)|
+ 0.2x|d(15N)| whereby d(1HN) and d(15N) are the chemical shift
perturbations in the
study the solution behaviour of Pep-WT and Pep-D320E, each
peptide (10mM) was analyzed by NMR at natural isotopic
abundance. Proton amide resonances were assigned by classical
procedure using homonuclear
NOESY experiments. To obtain the population of the major
and minor form of the peptides we measured the integrals of the
proton amide resonances on a1H,15N HSQC spectrum. In order
to exclude sample variations as a reason for the small shifts
between resonances, a mixture of both peptides (3mM each) was
analyzed in a similar manner.
15N dimensions, respectively. To
1H,1H TOCSY and
Found at: doi:10.1371/journal.pone.0013687.s001 (2.72 MB TIF)
Primers used for site-directed mutagenesis.
DEB025 selection in different genotypes in the Los Alamos
Found at: doi:10.1371/journal.pone.0013687.s002 (1.80 MB TIF)
Prevalence of the variants found in the replicon after
DEB025 or CsA resistant replicons.
Found at: doi:10.1371/journal.pone.0013687.s003 (1.53 MB TIF)
Mutationsidentified in independently selected
replication by DEB025 (A) or CsA (B) in Huh7-Lunet cells
transiently transfected with mutant replicon RNA (indicated on
the right site of each panel). HCV replicon RNA was quantified by
means of a luciferase assay and data are expressed as percentage of
untreated controls. Data are mean values 6 standard deviations
for at least two independent experiments.
Found at: doi:10.1371/journal.pone.0013687.s004 (0.94 MB TIF)
Dose-response curves for inhibition of replicon
replication by DEB025 in Huh7-Lunet cells transiently transfected
with mutant replicon RNA (indicated on the right site of each
panel). HCV replicon RNA was quantified by means of a
luciferase assay and data are expressed as percentage of untreated
controls. Data are mean values 6 standard deviations for at least
two independent experiments.
Found at: doi:10.1371/journal.pone.0013687.s005 (0.59 MB TIF)
Dose-response curves for inhibition of replicon
We thank Lien Kerremans, Ine Jaspers and Katrien Geerts for excellent
We thank Prof. C. Cameron for providing us with the bacterial
expression NS5A plasmid.
Conceived and designed the experiments: LC XH UC CB MB JP GV RB
PG GL JN. Performed the experiments: LC XH UC CB MB PL GL.
Analyzed the data: LC XH UC CB MB PL GL. Contributed reagents/
materials/analysis tools: UC JS MB IV JP AMV RB PG. Wrote the paper:
LC XH PG GL JN.
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Mechanism of Action of DEB025
PLoS ONE | www.plosone.org14 October 2010 | Volume 5 | Issue 10 | e13687