Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target.

Abstract

Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.

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Crystal structure of Plasmodium falciparum thioredoxin reductase,
a validated drug target
Giovanna Boumisa, Giorgio Giardinaa, Francesco Angeluccic, Andrea Bellellia,b, Maurizio Brunoria,b,
Daniela Dimastrogiovannia,1, Fulvio Saccocciaa, Adriana E. Mielea,⇑
aDepartment of Biochemical Sciences and Istituto Pasteur – Fondazione Cenci Bolognetti, ‘‘Sapienza’’ University of Rome, 00185 Rome, Italy
bCNR Institute of Molecular Pathology and Biology, ‘‘Sapienza’’ University of Rome, 00185 Rome, Italy
cDepartment of Life, Health and Environmental Sciences, University of L’Aquila, 67010 L’Aquila, Italy
a r t i c l ei n f o
Article history:
Received 27 July 2012
Available online 6 August 2012
Keywords:
Malaria
Thiol-mediated redox metabolism
Thioredoxin reductase
Protein crystallography
Rational drug design
a b s t r a c t
Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the
Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug
design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the
first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its
survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this
paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at
2.9 Å and discuss its differences with respect to the human orthologue. The most important one resides in
the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking
conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites
neither in mammals, boosts its exploitability.
? 2012 Elsevier Inc. All rights reserved.
1. Introduction
Malaria, one of the major threats for human health worldwide,
is endemic in more than 100 countries, mainly in tropical and sub-
tropical areas, and causes about 1 million human deaths per year.
It represents a huge socioeconomic burden in developing coun-
tries, where limited access to therapy has also the drawback of
increasing the reservoir of infectivity [1].
Malaria is a vector borne disease caused by the unicellular Api-
complexan parasite belonging to the genus Plasmodium. There are
four main species infecting humans, and among them Plasmodium
falciparum is the most deadly and also the one with the highest rate
of drug resistance [1].
Plasmodium has a complex developmental cycle, which includes
a continuous expansion inside host erythrocytes. Therefore it is ex-
posed to high fluxes of reactive oxygen species (ROS), while main-
taining a reduced intracellular environment. Asa defense
mechanism, the parasite evolved a complex network of NADPH-
dependent redox enzymes: a complete glutathione (GSH) system
and a specialized thioredoxin (Trx) system. The first one comprises
GSH, GSH reductase, glutaredoxin (Grx) and Grx-like proteins,
GSH-S-transferase (GST),gamma-glutamylcysteine
(gamma-GCS), and a GSH-dependent glyoxalase system. The sec-
ond system includes Trx reductase (TrxR), several Trxs and Trx-like
proteins, and Trx-dependent peroxidases [2–4]. In addition Plasmo-
dium can hijack redox proteins from the host. Overall, this proved a
winning strategy for survival, despite the lack of catalase and glu-
tathione peroxidase [5].
Using genetic and chemical tools, it was demonstrated that
TrxR, the first step of the thioredoxin redox cycle, and gamma-
GCS, the rate-limiting step of glutathione synthesis, are essential
for parasite survival [3,6]. Therefore some of the enzymes involved
in this antioxidant defense pattern represent good targets for the
design of new anti-malarial active compounds [7,8]. Indeed PfTrxR
is a particularly attractive candidate because of its structural and
functional peculiarities: namely, the presence of an extension at
the N-terminus and the lack of a selenocysteine in the redox centre
at the C-terminus. This last feature is common to all the Apicom-
plexan TrxRs, where a CGGGKCG stretch replace the GCUC stretch
of mammalian TrxRs [9].
Basic to a rational drug design research project there is a thor-
ough functional and structural characterization of a selected target
that should display unique features for specificity. In this paper we
synthetase
0006-291X/$ - see front matter ? 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2012.07.156
Abbreviations: PfTrxR, Plasmodium falciparum thioredoxin reductase; HsTrxR,
homo sapiens thioredoxin reductase; Trx, thioredoxin; GSH, reduced glutathione;
NADPH, nicotinamide adenine dinucleotide phosphate; FAD, flavin adenine dinu-
cleotide; DTNB, 5,50-dithio-bis (2-nitrobenzoic acid); Tris/HCl, Tris(hydroxy-
methyl)aminomethane hydrochloride.
⇑Corresponding author. Address: Department of Biochemical Sciences, ‘‘Sapien-
za’’ University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy. Fax: +39 064440062.
E-mail address: adriana.miele@uniroma1.it (A.E. Miele).
1Present address: Department of Biochemistry, University of Cambridge,
Cambridge CB2 1GA, UK.
Biochemical and Biophysical Research Communications 425 (2012) 806–811
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Page 2

present the first crystal structure of P. falciparum thioredoxin
reductase (PfTrxR) at 2.9 Å, and discuss its properties in light of
the similarities/differences with respect to human TrxR (HsTrxR,
PDB ID: 2ZZC [10]), and to computer models of PfTrxR [11,12].
The crystallographic structure unveiled some significant peculiari-
ties of the Plasmodium enzyme, which are discussed in terms of
their exploitability for drug design.
2. Materials and methods
2.1. Expression and purification
The gene corresponding to PfTrxR (Primary accession number
Q25861) was synthesized and optimized for expression in Esche-
richia. coli cells by GeneArt (Life Technologies), and was cloned into
pGEX-4T-1 (GE Healthcare) expression vector, via BamHI and XhoI
restriction sites.
The protein fused to GST-tag was expressed in BL21(DE3) bac-
terial cells upon induction with 0.5 mM IPTG, 20 lM FAD, incubat-
ing overnight at 16 ?C. Cells were lysed by sonication in lysis buffer
[0.1 M Tris/HCl, 0.4 M NaCl, pH 7.4, 5 mM b-mercaptoethanol, 5%
glycerol, 10 lg/ml DNase, a cocktail of protease inhibitors (COM-
PLETE, Roche) and 2 mM EDTA]. The protein was purified from
the soluble fraction by affinity chromatography on Glutathione Se-
pharose (QIAGEN). Successful expression of PfTrxR was confirmed
by SDS–PAGE, highlighting a band at approximately 86 kDa (Fig-
ure not shown). The GST-tag was cleaved by thrombin (Sigma–Al-
drich) that was finally removed passing the fractions on a 1 ml
benzamidine FF (HS) column (GE Healthcare). The expression gave
a yield of 7 mg/L culture, with a good FAD incorporation, according
to the Abs280/Abs340ratio (data not shown).
Purified PfTrxR was exchanged into crystallization buffer
(25 mM Tris/HCl, 0.2 M NaCl, pH 7.4, 5 mM b-mercaptoethanol),
and concentrated to 3.5 mg/mL by ultrafiltration (Millipore), ali-
quoted, and stored at ?20 ?C.
2.2. Crystallization
Crystallization conditions were initially screened by robot
(Phoenix, ArtRobbins) and then optimized by standard hanging
drop methods. Crystals of PfTrxR grew over 1 month in a drop com-
posed by 1 ll protein (3.5 mg/mL) and 1 ll well solution (16% (w/
v) PEG 4000, 0.1 M Tris/HCl, pH 8.5, 0.2 M sodium acetate).
2.3. Data collection, processing, and refinement
Diffraction data have been collected on BL14.1 (HZB BESSY II
electron storage ring of Berlin-Adlershof, Germany [13]) from a
crystal diffracting up to 2.9 Å. Data were indexed and processed
with XDS [14]. Phases were determined by molecular replacement
using Phaser [15] within the CCP4 Suite [16,17], taking mitochon-
drial mouse TrxR as a model (PDB entry: 1ZKQ, 46% identity [18]).
The structure was refined using REFMAC5 [19] and fitted to gen-
erated electron density maps by Coot [20]. MolProbity [21] and Pro-
Check [22] were used to assess the quality of the final model. Data
collection and refinement statistics are summarized in Table 1.
Figures were prepared with CCP4MG [23]. All the sequence
alignments were made with ClustalW2 [24] available on the EBI
website (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
2.4. Functional assays
2.4.1. DTNB reduction assay
5,50-dithio-bis (2-nitrobenzoic acid) (DTNB) reduction assay
was performed by adding PfTrxR (5–500 nM) to a mixture of
100 mM potassium phosphate pH 7.0, 2 mM EDTA, 0.2–5.0 mM
DTNB, and 300 lM NADPH at 20 ?C. The increase in absorbance
at 412 nm was monitored (e412= 13.6 mM?1cm?1) [25].
2.4.2. Insulin reduction assay
The classical turbidimetric assay [26] was performed by follow-
ing the precipitation of insulin at 650 nm after addition of 300 nM
PfTrxR to a reaction mixture composed of 100 mM potassium
phosphate pH 7.0, 2 mM EDTA, 300 lM NADPH, 200 lM insulin,
and 10 lM thioredoxin from Schistosoma mansoni. Stock solutions
of insulin were prepared according to [26]. The parameters used
to express the enzymatic activity were the starting time of precip-
itation and the precipitation rate (DAbs650min?1).
3. Results
3.1. The crystal structure
PfTrxR crystals belong to space group P212121, with two mono-
mers per asymmetric unit, in agreement with the dimeric active
form of the enzyme [5]. Crystal diffraction at 2.9 Å allowed the res-
olution of the overall fold, which is superimposable to that of other
members of the class-I pyridine nucleotide-disulfide oxidoreduc-
tase family. PfTrxR is a homodimeric enzyme, whose monomer
has three domains: a NADPH binding domain, a FAD binding do-
main, and a monomer–monomer interface. It possesses three redox
centers: (i) the non covalently bound FAD, which takes up electron
from NADPH; (ii) the first di-thiol couple Cys88–Cys93, which
takes up electrons from the isoalloxazine ring of FAD; (iii) the sec-
ond di-thiol couple Cys535–Cys540 at the end of the C-terminal
arm. The enzyme is an obligate dimer since the C-term Cys couple
of one monomer is in close contact with the FAD Cys couple of the
adjacent one, from which it takes up electrons to subsequently re-
duce the macromolecular substrate Trx.
All the secondary structure elements, the active site in the prox-
imity of FAD (Supplementary material Fig. 1), the NADPH binding
Table 1
Summary of data collection and refinement statistics for 4B1B.
Data collection
Space group
Cell dimensions: a, b, c (Å)
Resolution range (Å) (last shell)
Rmerge(last shell)
I/rI (last shell)
Completeness (%)
Multiplicity
No. total reflections
No. unique reflections
P212121
63.12, 109.18, 182.39
15.7–2.9 (3.07–2.90)
0.19 (0.76)
9.23 (2.02)
96.7 (92.7)
5.1 (4.4)
137765
27908
Refinement
Resolution (Å)
No. reflections used
R factor
Rfree
No. atoms
Protein
Ligand
B-Wilson (Å2)
B-factor (Å2)
15.7–2.9
26474
0.26
0.29
6848
6739
109
52.2
34.25
Model quality
R.m.s deviations
Bond lengths (Å)
Bond angles (?)
Chiral volume (Å2)
Ramachandran plot (%)
Favored
Generously allowed
Disallowed
0.002
0.39
0.028
99.7
0.3
0
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pocket and the interface domain are clearly visible in the electron
density map (Fig. 1).
The FAD active site is conserved, its binding mode being essen-
tially identical to that of the mammalian enzymes. The only nota-
ble peculiarity is around the adenosine ring next to the surface,
where Lys74, Leu160 and Asp319 in PfTrxR correspond, respec-
tively, to Thr45, Tyr131 and Cys296 in HsTrxR.
Although PfTrxR was crystallized without NADPH, its binding
pocket, formed by segments Gly229–Val233, Arg253–Phe260 and
Ile314–Arg316, is well structured, being a canonical Rossman’s
fold; thus a molecule of NADPH, docked for a better localization
of this domain in Fig. 1C, fits very well.
The delay in obtaining an experimental structure of such a key
enzyme points to the difficulty in growing well diffracting crystals.
In fact the prediction server XtalPred [27] highlighted an intrinsic
low propensity to crystallize. We can now infer that this is due
to the presence of three natively unstructured regions, which are
not visible in the electron density map, namely (i) the N-terminal
arm residues 1–37, (ii) the loop 428–456, and (iii) the C-terminal
residues 506–541. The N-terminal arm, which was predicted to
be unstructured by Psipred server [28] (Supplementary material
Fig. 2) is characteristic of TrxR from other Plasmodium and Apicom-
plexa species (though not conserved) but is absent in mammalian
TrxR (Supplementary material Fig. 3). At the C-terminus, the elec-
tron density map is well defined only up to Cys505, after which the
polypeptide chain is disordered and thus non visible. This is not
entirely unexpected, since the role of this C-terminal loop is to
shuttle electrons from Cys88–Cys93 facing FAD to the macromo-
lecular acceptor Trx via the active couple Cys535–Cys540. The
topologically corresponding region, visible in the human enzyme,
is shown for comparison in Fig. 2.
The long insertion loop 428–456, preceded and followed by b-
strands and predicted to contain two short helices and coiled seg-
ments (Supplementary material Fig. 2), is 14 residues longer than
the corresponding one in the mammalian enzymes (Supplemen-
tary material Fig. 3). Unlike the N- and C-termini, whose electron
density is completely missing, in this region small volumes of elec-
tron density are present, although it is not possible to unequivo-
cally assign them either to the backbone or to side chains. Hence
this highly flexible loop can assume an ensemble of different con-
formations, possibly originating the spots of electron density.
Therefore residues 428–456 were not included in the final model.
3.2. Functional studies
Functionality of recombinant PfTrxR was assessed by using (i)
the classical DTNB assay at 20 ?C, to monitor electron transfer from
NADPH to the Cys88–Cys93 couple via the FAD, and (ii) the insulin
precipitation assay, to monitor electron transfer to Trx, the macro-
molecular substrate.
Fig. 1. Ribbon representation of the overall structure of PfTrxR. Subunit A is in blue and subunit B in light blue. In stick representation are indicated FAD (green) and Cys88–
Cys93 couple of the active site (yellow). N-term and C-term of the two monomers A and B (as per crystal structure) are also labelled. The final model contains a total of 437
residues, from 38 to 174, from 177 to 427 and from 457 to 505 in each subunit. Panel A: Front view. Panel B: Top view, in which NADPH, in red, was inserted after
superposition with the human enzyme (PDB ID 2ZZC [10]) and after having swung out the active site Tyr-232. Panel C: Bottom view. Panel D: Front view centered on the 2-
fold axis (i.e. rotated by 30? towards the right with respect to panel A), for a better view of the central cavity. The 2-fold axis is represented either as a black stick or as an oval.
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InthefirstassayKmwas240 lMandkcatwas14.7 s?1;inthesec-
ond one the starting time was 150 s and the rate 0.68 DAbs min?1.
Bothsetsof dataareinagreementwithpreviouslypublishedresults
on the wild type enzyme [9,29].
To investigate the role of the N-terminal extension, we pro-
duced a truncated recombinant enzyme lacking the first 34 resi-
dues, in an attempt to produce crystal-prone boundaries and
improve protein stability. This truncated form displayed the same
enzymatic activity against DTNB and Trx as the full length enzyme
(data not shown); therefore the functional role of this N-terminal
domain remains unclear; unfortunately no improvement in crystal
quality was observed.
4. Discussion
Thioredoxin reductase (PfTrxR) is a vital enzyme for the redox
metabolism of P. falciparum, particularly during the erythrocytic
stage of the parasite life cycle. Although the 3D structures of sev-
eral mammalian proteins belonging to the same class are avail-
able, up to date Glutathione reductase is the only redox enzyme
from P. falciparum whose structure has been solved [30]. Here
we report the first crystallographic structure of PfTrxR in the
oxidized form, and discuss its peculiar features by reference to
human TrxR [10] and to the in silico models of PfTrxR recently
published [11,12].
The experimental structure of PfTrxR displays several features
unique to the parasite that will be useful to increase our under-
standing of the mechanism of binding of specific antimalarial
drugs, and to unveil the dynamics of a protein belonging to Api-
complexa parasites, which spend a significant part of their lifecycle
inside the red blood cell.
Despite the non-atomic resolution of the structure (2.9 Å) and
the lack of electron density over three small regions of the pro-
tein (see above), we shall discuss the peculiarities of the crystal-
lographic data since they are crucial for a rational analysis of the
action of novel antimalarial drugs. This is of some immediate
interest given the recent discovery of a new class of PfTrxR inhib-
itors [8], which are presumed to bind in the cavity next to the di-
mer interface. The specific features of this cavity in PfTrxR,
relative to that observed in human TrxR [8,30], will be discussed
in some detail.
Fig. 2. Superposition of PfTrxR (blue) with HsTrxR (green). Panel A: Overall superposition in ribbon representation of the two enzymes along the dimer interface (same
orientation as Fig. 1D). The red oval highlights the interface region enlarged in the next panels.⁄denotes the position of the C-terminal in HsTrxR;⁄⁄marks the interface loop
from residue 407 to 421 of HsTrxR. Panel B: Bottom view, showing the H-bond zipper stabilizing the PfTrxR antiparallel loop 118–125 from the two monomers. Main chain
atoms and Asp121 and Asn122 side chains are shown in yellow stick. Distances are in Å. Panel C: Bottom view, after clipping the antiparallel loop and highlighting helices a3
belonging to the two monomers. In stick representation are shown: Met105 and Phe 109 of PfTrxR (red), Leu76 and Leu80 of HsTrxR (green). Panel D: Front view highlighting
the hydrophobic cluster between helix a3 and loop 118–125 of PfTrxR.
G. Boumis et al./Biochemical and Biophysical Research Communications 425 (2012) 806–811
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4.1. The interface domain
As for the other TrxR enzymes, the homodimer is stabilized
essentially by two interface contact regions (Figs. 1 and 2): the first
comprising the last two a-helices of the C-terminal domain (the
long a14 and the short a15), and the second being constituted
by residues belonging to the last part of a3 and by its downstream
loop (residues 118–125). It is of significance that the unstructured
C-terminal arm is located in the cleft arising from these two inter-
face regions and the FAD domain, with each C-terminal contacting
the FAD domain of the partner subunit.
While the first interface region is conserved and very similar to
that observed for mammalian and Apicomplexan TrxRs (Supple-
mentary material Fig. 3), the second one is substantially different
and quite peculiar. In PfTrxR helix a3 is bent due to the presence
of Met105 and Phe109 (Fig. 2C), which are conserved only in the
Plasmodium species (Supplementary material Fig. 3). These two
bulky residues push the two interface helices apart, inducing a
bending that triggers a conformational reorganization of the down-
stream loop, also part of the same interface. While in the mamma-
lian structures these loops are open, in PfTrxR they assume an anti-
parallel conformation, forming an extended and well defined H-
bonding network centered on Asp121 and Asn122, which further
stabilizes the interface (Fig. 2B).
In the region between a3 and the anti-parallel loops, a dense
cluster of aromatic and hydrophobic residues is observed, compris-
ing not only the above mentioned Met105 and Phe109, but also
Ile108, Trp118, Phe120 and Leu123 (Fig. 2D). Overall, even though
the buried surface area of the entire interface is similar to that re-
ported for other enzymes, in PfTrxR it is stabilized by more polar
contacts, yielding a dissociation energy of 31 kcal/mol versus
19 kcal/mol calculated for the human enzyme after removal of
the C-terminal arm (residues 468–493), using PISA [31] at EBI
(http://www.ebi.ac.uk/pdbe/prot_int/pistart.html).
agreement with the hypothesis that in PfTrxR the mobile C-termi-
nal arm only marginally contributes to the dimer stability.
Thisisin
4.2. The interface cavity
The interface cavity between the two interface contact regions
has often been invoked as a potential inhibitor binding site. In fact
in PfTrxR it is peculiarly narrower than in HsTrxR due to the pres-
ence of Tyr101 and His104. Moreover, these residues modify the
stereo-chemical properties of the interface surface, given that their
counterparts in the human enzyme are, respectively, Gln and Leu
(Fig. 3 and Supplementary material Fig. 3). Furthermore Tyr101
of one monomer is engaged in H-bond with Asp112 on a3 of the
other monomer. Therefore both the size of this cavity and its nat-
ure are exploitable properties of the plasmodial enzymes, since
they could presumably host smaller and slightly more amphipathic
molecules than the human enzyme. These features might be cru-
cial to obtain a good pharmacophoric map and achieve better
selectivity.
Both published models of PfTrxR, based either on the human
enzyme [11] or on the mouse enzyme [12] did not spot down these
features, or they were mis-regarded by the authors. Therefore, in
silico models based on a C-terminal arm that strengthens the inter-
face region should be regarded as highly suspicious.
4.3. Exploitability
PfTrxR crystal structure highlighted the conservation of the
binding site for Trx, which is located at the bottom of a4. Since
the structure of HsTrxR in complex with HsTrx is available [32],
we have superimposed this complex (PDB ID: 3QFB) to PfTrxR
and indeed the contact surface area between PfTrxR and Trx is
nearly identical (Figure not shown), the only substitution being
Arg139, conserved in all plasmodial TrxR, that replaces Gly260 of
Fig. 3. The cavity of PfTrxR (blue) in comparison with HsTrxR (green). Panel A:
Enlarged view of the central cavity, highlighting Tyr101 and His104 from both
monomers of PfTrxR, and the corresponding Gln72 and Leu75 of HsTrxR. Panel B:
Electrostatic surface of the cavity of PfTrxR. Panel C: Electrostatic surface of the
cavity of HsTrxR, showed after having sliced out the loop from residue 407 to 421.
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the human enzyme. This mutation is counteracted by the substitu-
tion of Thr30 of HsTrx with Glu28 of PfTrx, thus changing the inter-
action between the two proteins from H-bond to salt bridge. The
functional conservation has been indirectly confirmed by the func-
tional assays, where Trx from an unrelated parasite (Schistosoma
mansoni), which has Thr as HsTrx, has been used instead of PfTrx
as an electron acceptor.
Therefore we might speculate inhibitors binding in this region
to be highly toxic, since they would possibly interfere with both
HsTrxR and PfTrxR binding to Trx. Indeed it has been reported that
an anticancer drug Terpyridine Platinum(II) inhibits the complex
formation between HsTrxR and HsTrx, on top of being a topoiso-
merase inhibitor [10]. We can speculate that this class of com-
pounds would not be useful as antiplasmodial agents since they
would be too cytotoxic.
Very recently a paper has been published presenting two new
classes of potent electrophilic non competitive inhibitors of PfTrxR
[8]. The authors hypothesize that these compounds might bind in
the interface domain. In light of our results, indeed the differences
in the interface cavity between the parasite and human enzyme
could be exploited for selecting specific drug compounds.
In conclusion, PfTrxR structure has revealed unique unexpected
features on top of a conserved 3D-fold. This enzyme displays a
more stable monomer–monomer interface and an extended and
well defined H-bonding network along the loop 118–125 of both
subunits, both features being absent in the human enzyme. The
greater stability of the dimer in the parasite could partially com-
pensate for the more extended structural flexibility, largely due
to the loop 429–458 and the C-terminal arm. Relevant questions
remain presently unanswered such as: why the Plasmodium spe-
cies evolved such a diverse interface and what the evolutionary
advantage of these flexible insertions might be.
5. Accession numbers
Coordinates and structure factors have been deposited in the
Protein Data Bank with accession number 4B1B.
Acknowledgments
We would like to thank Prof. Robert Hondal and Dr. Gregg Sni-
der (University of Vermont, USA) for helpful discussion. The re-
search leading to these results has received funding from:
Fondazione Roma under Grant ‘‘Rational approach to the specific
inhibition of Plasmodium falciparum and Schistosoma mansoni’’ to
MB; MIUR of Italy under Grant FIRB RBRN07BMCT_007 to MB
and International FIRB RBIN06E9Z8 to AB; ‘‘Sapienza’’ University
of Rome under Grants ‘‘Progetti di Università’’ 2010 and 2011 to
AB; the European Community’s Seventh Framework Programme
(FP7/2007-2013) under Grant agreement no.226716 to AEM. GG
is a supported fellow of Fondazione Roma.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bbrc.2012.07.156.
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