Synthesis and structure-activity relationships of phosphonic arginine mimetics as inhibitors of the M1 and M17 aminopeptidases from plasmodium falciparum

Article abstract of DOI:10.1021/jm4005972

The malaria parasite Plasmodium falciparum employs two metallo- aminopeptidases, PfA-M1 and PfA-M17, which are essential for parasite survival. Compounds that inhibit the activity of either enzyme represent leads for the development of new antimalarial drugs. Here we report the synthesis and structure-activity relationships of a small library of phosphonic acid arginine mimetics that probe the S1 pocket of both enzymes and map the necessary interactions that would be important for a dual inhibitor.

Full text of DOI:10.1021/jm4005972

Brief Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of Phosphonic
Arginine Mimetics as Inhibitors of the M1 and M17 Aminopeptidases
from Plasmodium falciparum
Komagal Kannan Sivaraman,† Alessandro Paiardini,‡ Marcin Sienczyk,§ Chiara Ruggeri,‡
́
Christine A. Oellig,† John P. Dalton,∥,⊥ Peter J. Scammells,# Marcin Drag,*,▽,○ and Sheena McGowan*,†
†Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia
‡Dipartmento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Universita di Roma, 00185 Roma, Italy
§Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland
∥Institute for the Biotechnology of Infectious Diseases (IBID), University of Technology Sydney, Ultimo, Sydney NSW 2007,
Australia
⊥Institute of Parasitology, McGill University, Sainte-Anne De Bellevue, QC H9X 3V9, Canada
#Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Australia
▽Program in Apoptosis and Cell Death Research, Sanford Burnham Medical Research Institute, La Jolla, California 92037, United
States
○Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland
S
* Supporting Information
ABSTRACT: The malaria parasite Plasmodium falciparum employs two metallo-aminopeptidases, PfA-M1 and PfA-M17, which
are essential for parasite survival. Compounds that inhibit the activity of either enzyme represent leads for the development of
new antimalarial drugs. Here we report the synthesis and structure−activity relationships of a small library of phosphonic acid
arginine mimetics that probe the S1 pocket of both enzymes and map the necessary interactions that would be important for a
dual inhibitor.
bound Zn2+ metal ion.2,8 The active site of PfA-M17 also has a
INTRODUCTION
■
carbonate ion present.5 Because chelation of the metal groups
Malaria is responsible for 2% of global mortalities. The
in either enzyme renders them inactive,2,8 much attention has
been placed on identifying chemical scaffolds that can
effectively interact with the metal ions.
A desirable quality of a potential lead antimalarial is the
discovery of new antiplasmodial compounds is necessary to
overcome the spread of malaria parasites that have become
resistant to currently available drugs. The PfA-M1 and PfA-M17
neutral aminopeptidases of the malaria parasite Plasmodium
ability to specifically inhibit both enzymes, as this would reduce
falciparum serve as key regulators of the intracellular amino acid
the prospect of drug-resistant parasites emerging. Phosphono-
pool in the blood stage of the infection within malaria cells,
peptides,2,5 peptide-based bestatin analogues,9,10 and recently
most likely by releasing amino acids from host-derived
hemoglobin.1−4 PfA-M17 was recently shown to be also
an orally bioavailable hydroxamate-containing ester, CHR-
2863, were also shown to be efficacious against murine
essential to the early life cycle of the parasite, prior to the
malaria.11 Hydroxamate derivatives have been described as
onset of hemoglobin digestion, suggesting that PfA-M17 may
inhibitors of PfA-M1.12,13
have another role outside of hemoglobin digestion.1
Optimization of lead compounds requires a detailed
biological and chemical understanding of the active site
architecture of each enzyme. Our group has described the
structure−activity relationship (SAR) data for some of these
inhibitors,1,2,5,10 and others have used our structures to model
their compounds.11,12 This body of work has identified key
architectural and chemical requirements of the active sites of
both enzymes and has revealed that the substrate binding of the
two enzymes differ dramatically. Thus, PfA-M1 has a broad
substrate tolerance, capable of efficiently hydrolyzing neutral
Both enzymes are validated and attractive targets for lead
compound discovery and optimization.2,4−6 Attempts to
genetically knock out or knock down either the PfA-M1 or
PfA-M17 genes have been unsuccessful,2,7 however, agents that
inhibit enzymatic activity in parasites have been shown to
control both laboratory and murine models of malaria.6
Similarly, the enzymes are conserved among all Plasmodium
species, indicating that future therapeutics could deliver cross-
species inhibition.5
The two enzymes are both metalloproteases but have distinct
differences in the arrangement of divalent metal cations in their
active site. PfA-M1 tightly binds a single Zn2+ metal ion,
whereas PfA-M17 contains one tightly bound and one loosely
Received: April 23, 2013
© XXXX American Chemical Society
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Journal of Medicinal Chemistry
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and basic amino acids,2,14,15 while PfA-M17 has a narrow
substrate specificity and preferentially cleaves bulky, hydro-
phobic amino acids.2,15,16
In this study, we report the synthesis and SAR of a small
library of metal-chelating phosphonic acid arginine mimetics
that have been varied to probe the S1 pocket of both enzymes.
We have mapped the necessary interactions of these
compounds within the active sites of each enzyme and
correlated these with their binding kinetics to elucidate some
important chemistry required for a dual inhibitor.
inhibitor 4 (H-(4-Gu)PheP(OH)2). For the synthesis of 6, the
starting 4-(1H-pyrazol-1-yl)benzaldehyde, prepared from 4-
fluorobenzaldehyde and pyrazole,21 was used in amidoalkyla-
tion reaction in acetic acid. Final hydrolysis of Cbz-(4-
Pyr)PhgP(OPh)2 in refluxing HCl led to generation of 6.
PfA-M1 Inhibition and SAR. Compounds 1−8 were tested
for their ability to inhibit PfA-M1 by calculating a Ki for each
compound (Table 1). 3, 4, and 8 were found to be not
Table 1. Inhibition of PfA-M1 and PfA-M17 by 1−8
RESULTS AND DISCUSSION
■
Synthesis of Inhibitors 1−8. We have previously used
phosphonic analogues to probe the structure and mechanism of
inhibition for both PfA-M1 and -M17.15 We knew the
phosphonic acid would chelate the zinc ion(s), allowing us to
map the SAR of the arginine derivatives in the S1 pockets of
both enzymes.
The target phosphonic inhibitors (1−8) were synthesized
from parent Cbz-protected 1-aminoalkylphosphonate diphenyl
esters which were obtained via an amidoalkylation reactions
either in acetic acid17 or in dichloromethane with copper triflate
as the catalyst18 (Scheme 1). Briefly, the synthesis of 1 started
Scheme 1. General Approach for the Synthesis of
a
Phosphonic Arginine Mimetics
a
Reagents and conditions: (a) acetic acid, 80−90 °C, 2 h; (b)
Cu(OTf)2, CH2Cl2, rt, 14 h; (c) conc HCl, reflux, 12 h.
with preparation of N-phthaloyl protected 5-aminopentanol
from phthalic anhydride and 5-aminopentanol,19 which was
further oxidized to aldehyde via the Swern method. Resulting 5-
N-Phth-aminopentanal was used in amidoalkylation reaction in
acetic acid, leading to generation of side-chain Phth-protected
phosphonic analogue of lysine (Cbz-Lys(Phth)P(OPh)2). The
phthaolyl group was removed with hydrazine and the liberated
amino group was guanylated using S-ethyl-N,N′-di(Boc)-
isothiourea as the guanylating agent. Removal of Boc-protecting
groups led to generation of phosphonic homoarginine (Cbz-
hArgP(OPh)2), which was next hydrolyzed by concentrated
hydrochloric acid. Inhibitor 1 was crystallized from ethanol
after addition of propylene oxide. Inhibitors 2 and 3 were
obtained in a similar way with the exception that 4-
aminobutyrylaldehyde diethyl acetal was used as the starting
material for Cbz-OrnP(OPh)2 and Cbz-ArgP(OPh)2 syntheses.
Synthesis of Cbz-protected derivatives used for the preparation
of 5, 7, and 8 was performed as described previously,20 which
was followed by complete Cbz/ester groups deprotection as
described above. The synthesis of 4 started with the preparation
of methyl 2-(4-nitrophenyl)acetate followed by subsequent
steps such as nitro group reduction, Boc-protection, ester group
reduction to alcohol, and its oxidation to an aldehyde function.
Resulting 4-(N-Boc-amino)phenylacetaldehyde was used for
the copper triflate-catalyzed amidoalkylation reaction under
mild conditions. After Boc-deprotection, the obtained Cbz-(4-
NH2)PheP(OPh)2 was guanylated and all protective groups
were hydrolyzed in refluxing hydrochloric acid, leading to final
inhibitory for the enzyme. The most potent inhibitor was 1
with a Ki in the low micromolar range, while the least inhibitory
compound was 7 with a Ki at 345 μM (Table 1).
We determined the high resolution X-ray crystal structures of
each of the inhibitory compounds bound to PfA-M1 (see
Supporting Information (SI) Table 1 for crystallization and
refinement details, Figure 1). The PfA-M1 enzyme has a
canonical alanyl-aminopeptidase fold with four domains. The
catalytic domain contains an essential zinc ion that is well
coordinated by a catalytic triad of residues His496, His500, and
Glu519. Each of the compounds showed the same interactions
with the zinc ion with the inhibitors primarily interacting via
metallo-bonds to the O1 and O3 oxygen of the phosphate
group. A further H-bond common to all inhibitors was noted
between the O1 atom in the phosphate group and the −OH of
Tyr580. With the exception of 7, water molecules contributed
two further interactions with the phosphate group through H-
bonds with the O2 atom. The inhibitor’s amine group (N4)
formed 3 H-bonds with the OE oxygens of Glu319, 463, and
519. 7 was again missing a H-bond, forming interactions with
only Glu319 and 519.
The most potent inhibitor was homoarginine derivative 1
with the 1-amino-5-guanidinopentyl group in a side-chain. The
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Journal of Medicinal Chemistry
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H-bonds with Glu572 and Glu319, as well as two water
molecules (PDB 4K5N). 6 only forms a H-bond with the O-
atom of Glu319 and one water molecule. Moreover, the
pyrazole moiety of 6 makes hydrophobic contacts with the side-
chain of Met1034, completely filling the cleft (PDB 4K5O).
Although 7 forms a similar H-bond with Glu319, this is not
stabilized by any further interactions with the protein or water
and is the likely reason for its reduced potency (PDB 4K5P).
PfA-M17 Inhibition and SAR. Arginine is not a substrate
for PfA-M17,15 and we were interested to see if the Arg
mimetics were inhibitory against the aminopeptidase. 1−8 were
tested for their ability to inhibit the PfA-M17 aminopeptidase
by calculating a Ki for each compound (Table 1). Compounds
with smaller groups, 2, 3, and 7, were not inhibitory. The most
potent inhibitor was 6 with a Ki of 11 nM, while the least
inhibitory compound was 1 with a Ki at 268 μM (Table 1).
With the exception of 1, the inhibitors all displayed a Ki in the
lower μM to nM range. This immediately told us that although
PfA-M17 had a requirement for a hydrophobic arm to satisfy
the hydrophobic nature of S1 pocket in PfA-M17, that
inhibition of the enzyme was possible with a polar compound.
To investigate how the guanidino groups were being
accommodated within the active site and S1 pocket, we
attempted to determine the X-ray crystal structures of each of
the compounds bound to the PfA-M17. Despite extensive
efforts, the only compound that was successfully crystallized
with PfA-M17 was 6 (see SI Table 1 for crystallization and
refinement details, Figure 2, PDB 4K3N). 6 showed extensive
Figure 1. Diagram of inhibitors binding to active site of PfA-M1.
Carbon atoms of PfA-M1 residues are colored in gray. Zinc is shown as
black sphere, water as blue sphere. 1−8 are colored by carbon atom, 1
(red, 4K5L), 2 (cyan, 4K5M), 5 (green, 4K5N), 6 (yellow, 4K5O),
and 7 (pink, 4K5P).
5-carbon chain showed a 10-fold increase in its Ki compared to
arginine derivative 2 (Table 1). A comparison of the bound
structures of these two compounds demonstrates that 1 is long
enough to place the guanidino group in reach of Glu572, a key
residue in the formation of the S1 pocket of PfA-M1.10 Indeed,
in the crystal structure of PfA-M1-1 (PDB 4K5L), a H-bond is
present between N2 and the backbone oxygen of Glu572.2 In
this structure, the side-chain of Glu572 remains unresolved, a
feature common to the crystal structures of PfA-M1 (SI Figure
1A).10 The S1 pocket of PfA-M1 has been shown to possess a
plasticity that allows for substantial inhibitor-induced rearrange-
ment of Glu572.10 Neither 1 nor 2 PDB in this study altered
the c−α position of Glu572. The crystal structure of PfA-M1-2
(PDB 4K5M) shows that 2 can occupy two alternative
positions in the active site, identifying the reason for the
reduced potency observed with this inhibitor (SI Figure 1B).
The electron density clearly demonstrated that the compound
branched at the C3 position, with one position occupying the
same position as 1 and the second reaching away to form a H-
bond with the backbone O of Val459 (SI Figure 1B). The
shorter carbon chain fails to reach the Glu572 residue. Both of
these structures show an alternative position for the Val459
residue in comparison to the unbound PfA-M1 (PDB 3EBG)
and other inhibitor bound structures.1,2,10 The side-chain
position in these structures points away from the active site
and results in minor changes in the c−α backbone of the
pocket (SI Figure 1C). This moves the backbone oxygen into a
position where it can form the H-bond with 2 (SI Figure 1B,C).
The amino group of the 4-carbon chain noninhibitory
compound 3 simply lacks the ability to interact with the S1
pocket.
Figure 2. Diagram of 6 binding to PfA-M17 (4K3N). Colors as per
Figure 1.
interactions of its phosphate group with the two metal ions and
carbonate ion present in each active site of PfA-M17 (Figure 2).
Both zinc ions and the carbonate ion formed a metal bond with
O1, while O2 formed a metal bond with only one of the zinc
ions but also engaged the side-chain of Lys386. The amine
group of the compound formed H-bonds with Asp379 and 399.
The functional group in 6 formed no further interactions with
the enzyme. The 4-(1H-pyrazol-1-yl)phenyl group fitted neatly
into the hydrophobic S1 pocket, efficiently filling the cavity
(Figure 2) but forming no H-bonds, only hydrophobic packing
interactions.
Compounds 5−8 all contain a phenyl group. 4 and 8 were
noninhibitory, and both contained a rigid phenyl group with a
2-(4-guanidinophenyl) (4) or a 3-guanidinophenyl (8). This
confirms the preference of PfA-M1 for a straight chain P1
residue or functional group. 5 and 6 contained a 4-
guanidiophenyl and a (1H-pyrazol-1yl) phenyl, respectively,
but had equivalent Ki values. The guanidino nitrogens of 5 form
C
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Journal of Medicinal Chemistry
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To examine the interactions of the remaining compounds,
we used our atomic data from the cocrystal structure of PfA-
M17-6 to perform template-based molecular docking of the
remaining compounds.22 Template docking can be used when
knowledge about the 3D conformation of a ligand is already
available. Features expected to be relevant for the binding of the
known ligand are extracted and used as energy constraints
during the docking procedure. This allows the docking engine
to focus the search on poses similar to the docking template,
taking at the same time the ligand’s flexibility into account. By
utilizing active site interactions (zinc, carbonate, and PfA-M17
residues) of 6 that are plausibly shared with the other
compounds (phosphate and amino groups), we were able to
generate models for each of them. With the exception of 1, the
compounds were docked into a single conformation within the
active site. 1 was seen to have two equivalent conformations
and both were subsequently analyzed.
The docking experiments revealed that positioning of the
ligand with respect to the hydrophobic surface of the S1 cavity
is key for potency. 7 was noninhibitory, and its carbaminidoyl-
phenyl was shown to pack directly against the hydrophobic
residues of the cavity. The positively charged guanidino group
in 4 is in close proximity with the hydrophobic residues
Met392, Leu492, and Phe583. Although it is likely that subtle
structural rearrangements would occur to accommodate the
side-chain of 4 into the hydrophobic cleft, this scenario
presumably accounts for the observed reduced potency of the
latter. Compared to 4, 5 is able to shift the position of the
positively charged group, moving the head moiety away from
Phe583 and placing the guanidine group at favorable hydrogen-
bonding distance (∼2.8 Å) with the O atom of Ala577. Finally,
the meta- substitution of 8 pushes its guanidino headgroup
aside from the hydrophobic cleft formed by Met392, Leu492,
and Phe583 and places it in a similar pose to that of 1 (position
2, see below), where the N3/N4 can form H-bonds with the
backbone O of Gly390. This favorable interaction probably
accounts for the observed increase inhibitory property, Ki =
0.16 μM, of the latter.
The straight carbon backbones proved to be relatively
ineffective at inhibiting PfA-M17. Only the 5-carbon chain in 1
was found to be moderately inhibitory. The two positions
within the active site of PfA-M17, identified by molecular
docking, show that the guanidino group reaches out toward the
center of the active site cavity. Position 1 (SI Figure 1) shows
that the ligand can occupy a similar space to 6, sitting effectively
in the middle of the S1 cavity (SI Figure 1B). In this position,
however, the guanidino group is close to the top of the S1
pocket, in particular to Ala577. Position 2 indicates that the
compound could also reach away from the highly hydrophobic
surface at the back of the pocket, forming a H-bond with the
backbone O of Ser391 (SI Figure 2B). Analysis of docked
models of the noninhibitory 2 and 3 show that the shorter
carbon chains position the respective guanidino and amino
groups in close proximity to the hydrophobic surface of the
cavity, in particular to Phe398 and Phe583.
responsible for binding to the zinc ion(s) in the enzyme’s active
site, while the side-chain binds specifically to S1 pocket of the
aminopeptidase. In our work, we have demonstrated a series of
aminophosphonic acid derivatives have been prepared and
evaluated as inhibitors of the PfA-M1 and PfA-M17 amino-
peptidase enzymes of P. falciparum. 1-Amino-5-guanidinopen-
tylphosphonic acid (1) proved to be the most potent inhibitor
of PfA-M1, with a Ki value of 11 μM. Reduction of the length of
the carbon backbone or rigidification via the incorporation of a
phenyl group significantly reduced PfA-M1 inhibitory activity.
Similarly, retention of the guanidino groups also proved to be
important for PfA-M1 inhibitory activity. In contrast, 4-
(pyrazol-1-yl)phenyl)(amino)methylphosphonic acid (6) was
found to be the most potent inhibitor of PfA-M17, with a Ki
value in the low nanomolar range (11 nM). Encouragingly, this
compound also possessed modest inhibitory activity against
PfA-M1 (Ki 104 μM), forming few interactions with the large
S1 pocket of this enzyme. This compound supports the
feasibility of developing dual PfA-M1/M17 inhibitors. The
scaffold of 6 has considerable scope for further optimization,
both in terms of enhancing its PfA-M1 inhibitory activity and its
physicochemical properties for parasite killing.
EXPERIMENTAL SECTION
■
Chemistry. A full description of materials and details of synthetic
protocol as well as spectroscopic analysis for every compound can be
found in SI. A representative example of target compound 6 synthesis
is described below. The purity of final compounds was examined by
HPLC (Dionex, Ultimate 3000, C8 column, SunFire Prep C8 column,
10 mm × 250 mm, 5 μm, Supelco, Poland) and was greater than 95%.
The synthesis of 6 started with preparation of 4-(1H-pyrazol-1-
yl)benzaldehyde.21 Briefly, anhydrous K2CO3 (22 mmol) was
suspended in DMSO (20 mL) and placed in an ultrasonic bath for
30 min. Next, 4-fluorobenzaldehyde (22 mmol) and pyrazole (20
mmol) were added and kept in an ultrasonic bath until the
temperature dropped to 80 °C (1−2 h) and then was heated at 100
°C for 3 h. The reaction mixture was then extracted with diethyl ether
(25 mL) and chloroform (3 × 100 mL). The ether fraction was
discarded, and the combined chloroform fractions were washed with
water and brine, dried over MgSO4, filtered, and evaporated to
dryness. The product was crystallized from hexane as a pale-yellow
solid (56%). Next, the 4-(1H-pyrazol-1-yl)benzaldehyde (11 mmol),
triphenyl phosphite (10 mmol), and benzyl carbamate (11 mmol)
were dissolved in glacial acetic acid (25 mL) and heated at 80−90 °C
for 2 h.17 The solvent was evaporated, and the resulting oil was
dissolved in methanol (100 mL) and left at −20 °C for crystallization.
The product (Cbz-(4-Pyr)PhgP(OPh)2) was obtained as a white solid
(53%). Finally, acid hydrolysis of Cbz-(4-Pyr)PhgP(OPh)2 (1.26
mmol, 1 g) led to generation of 6 as a white solid (29%), mp 278 °C.
Biochemistry. Recombinant PfA-M1 and PfA-M17 were produced
as described previously.2,5 Aminopeptidase activity of both enzymes
and calculations of Ki are described in the SI. Co-crystallization of
enzymes was achieved by the addition of 1 mM compound. Data
collection and refinement statistics are available in the SI. Raw data is
available from TARDIS (www.tardis.edu.au).24 Molecular docking
methods are described in the SI.
ASSOCIATED CONTENT
* Supporting Information
■
S
CONCLUSION
Experimental procedures for synthesized compounds and
protein crystallography including data collection and refine-
ment statistics. Figures detailing interactions of each compound
for each model generated. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
Aminophosphonates have been identified as potent inhibitors
of the aminopeptidases for more than two decades.23 They are
simple analogues of amino acids, in which the carboxy group is
exchanged for phosphonate, mimicking the transition state of
the peptide bond hydrolysis. Aminophosphonates are consid-
ered as dual inhibitors, in which phosphonate fragment is
Accession Codes
PDB IDs: 4K5L, 4K5M, 4K5N, 4K5O, 4K5P, 4K3N.
D
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Journal of Medicinal Chemistry
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1666.
(11) Skinner-Adams, T. S.; Peatey, C. L.; Anderson, K.; Trenholme,
K. R.; Krige, D.; Brown, C. L.; Stack, C.; Nsangou, D. M.; Mathews, R.
T.; Thivierge, K.; Dalton, J. P.; Gardiner, D. L. The aminopeptidase
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malaria. Antimicrob. Agents Chemother. 2012, 56, 3244−3249.
(12) Deprez-Poulain, R.; Flipo, M.; Piveteau, C.; Leroux, F.;
Dassonneville, S.; Florent, I.; Maes, L.; Cos, P.; Deprez, B.
Structure−activity relationships and blood distribution of antiplasmo-
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(13) Flipo, M.; Beghyn, T.; Leroux, V.; Florent, I.; Deprez, B. P.;
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aminopeptidase PfA-M1 as potential antimalarial agents. J. Med. Chem.
2007, 50, 1322−1334.
AUTHOR INFORMATION
■
Corresponding Author
*For M.D.: phone, +48 71 3204526; fax, +48 71 3202427; E-
mail, marcin.drag@pwr.wroc.pl. For S.M.: phone, +613
99029309; fax, +613 99029500; E-mail, Sheena.McGowan@
monash.edu.
Author Contributions
All authors have given approval to the final version of the
manuscript. K.K.S. and A.P. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.D. is grateful to the Foundation for Polish Science for
financial support. S.M. is an Australian Research Council Future
Fellow. We thank the NHMRC and the ARC for funding
support. We thank the Australian Synchrotron (MX-1 and MX-
2) and the beamline scientists for beamtime and for technical
assistance. We thank the Monash Platforms (Protein
Production and Crystallization) for technical assistance.
(14) Allary, M.; Schrevel, J.; Florent, I. Properties, stage-dependent
expression and localization of Plasmodium falciparum M1 family zinc-
aminopeptidase. Parasitology 2002, 125, 1−10.
(15) Poreba, M.; McGowan, S.; Skinner-Adams, T.; Trenholme, K.
R.; Gardiner, D. L.; Whisstock, J. C.; To, , J.; Salvesen, G. S.; Drag, M.;
Dalton, J. P. Fingerprinting the substrate specificity of M1 and M17
neutral aminopeptidases of human malaria, Plasmodium falciparum.
PloS One 2012, 2, e31938.
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