European Journal of Medicinal Chemistry 64 (2013) 35e41

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Short communication

Synthetic chalcones and sulfonamides as new classes of Yersinia

enterocolitica YopH tyrosine phosphatase inhibitors
Priscila Graziela Alves Martins a,1, Angela Camila Orbem Menegatti a,1,
Louise Domeneghini Chiaradia-Delatorre a, b, Kely Navakoski de Oliveira b,
Rafael Victorio Carvalho Guido c, Adriano Defini Andricopulo c, Javier Vernal a,
Rosendo Augusto Yunes b, Ricardo José Nunes b, **, Hernán Terenzi a, *
a
Centro de Biologia Molecular Estrutural (CEBIME), Departamento de Bioquímica, Universidade Federal de Santa Catarina, Campus Trindade,
88040-900 Florianópolis, SC, Brazil
b
Laboratório Estrutura e Atividade (LEAT), Departamento de Química, Universidade Federal de Santa Catarina, Campus Trindade, 88040-900 Florianópolis,
SC, Brazil
c
Laboratório de Química Medicinal e Computacional (LQMC), Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense
400, 13560-970 São Carlos, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: YopH plays a relevant role in three pathogenic species of Yersinia. Due to its importance in the prevention
Received 1 March 2013 of the inflammatory response of the host, this enzyme has become a valid target for the identification
Received in revised form and development of new inhibitors. In this work, an in-house library of 283 synthetic compounds was
7 April 2013
assayed against recombinant YopH from Yersinia enterocolitica. From these, four chalcone derivatives and
Accepted 9 April 2013
Available online 16 April 2013
one sulfonamide were identified for the first time as competitive inhibitors of YopH with binding affinity
in the low micromolar range. Molecular modeling investigations indicated that the new inhibitors
showed similar binding modes, establishing polar and hydrophobic contacts with key residues of the
Keywords:
Enzyme inhibition
YopH binding site.
Tyrosine phosphatase Ó 2013 Elsevier Masson SAS. All rights reserved.
YopH
Synthetic compounds

1. Introduction pseudotuberculosis and Yersinia pestis e are responsible for human

Protein tyrosine phosphatases (PTPs) are an important class of
enzymes that together with protein tyrosine kinases (PTKs) control
the level of phosphorylation of their protein substrate targets. It is
known that protein tyrosine phosphorylation plays a critical role in
signal transduction of many cellular events including immune
response, metabolism, growth and gene transcription [1].
Yersinia genus comprises ten species of gram-negative bacteria;
among those, three species e Yersinia enterocolitica, Yersinia
* Corresponding author. Centro de Biologia Molecular Estrutural, Universidade
Federal de Santa Catarina, Campus Trindade, 88040-900 Florianópolis, SC, Brazil.
Tel.: þ55 48 3721 6426; fax: þ55 48 3721 9672.
** Corresponding author. Departamento de Química, Universidade Federal de
Santa Catarina, Campus Trindade, 88040-900 Florianópolis, SC, Brazil. Tel.: þ55 48
3721 6844x236; fax: þ55 48 3721 6850.
E-mail addresses: nunes@qmc.ufsc.br (R.J. Nunes), hterenzi@ccb.ufsc.br,
hernan.terenzi@ufsc.br (H. Terenzi).
1
These authors contributed equally to this study.
diseases, causing different pathologies from gastrointestinal syn-
dromes to sepsis [2]. One extrachromosomal plasmid of 70 kb is
present in these three pathogenic species and is essential for their
virulence [3]. This plasmid encodes the Yop (Yersinia outer protein)
effector proteins and also the proteins forming the type III secretion
apparatus [4]. On the basis of their function, Yop proteins can be
divided in two groups: the first one, the effector group, is composed
by YopE, YopH, YopM, YopA/YopO, YopJ/P and YopT which are
injected into the lumen of the host cell, and the second group is
responsible for the formation and control of the secretion appa-
ratus [4]. The injected Yop proteins disrupt the signaling cascades of
the host cell resulting in inhibition of the innate and adaptive im-
mune responses [5].
YopH is a 51 kDa protein tyrosine phosphatase that is crucial,
and required for Yersinia pathogenesis [6,7]. The N-terminal
domain of this protein is accountable for substrate recognition [8]
while the C-terminal domain has the catalytic site which is struc-
turally similar to the eukaryotic PTPs [9]. Once inside the phago-
cytic host cell, YopH has the ability to prevent the inflammatory

0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.018
36 P.G.A. Martins et al. / European Journal of Medicinal Chemistry 64 (2013) 35e41
response by different processes: inhibition of phagocytosis, sup-
pression of reactive oxygen intermediates production by macro-
phages and neutrophil polymorphonuclear leukocytes (PMNs) and
disruption of focal adhesion by dephosphorylation of FAK and
p130cas [10,11]. YopH can also avoid the adaptive immune response
by impairing T and B lymphocyte activation [12].
Due to the fact that the pathogenicity of Y. enterocolitica,
Y. pseudotuberculosis and Y. pestis is closely related to the activity of
YopH, this enzyme is a promising target for therapeutic in-
terventions against diseases provoked by these bacteria. In the last
years, several compounds have been reported as effective YopH
inhibitors, as a-ketocarboxylic acid [13], aurintricarboxylic acid
[14], tripeptides [15,16], furanyl-salicylate derivatives [17], oxalyl
derivatives [18], salicylic acid derivatives [19], phosphonate de-
rivatives of calixarenes [20] and oximes [21,22].
So, in this work, we screened an in-house library of 283 syn-
thetic compounds, in order to identify new inhibitors of YopH. The
results led to the determination of potency and investigation of the
structureeactivity relationships (SAR), mechanism of inhibition,
and mode of interaction of the most potent compounds.
2. Results and discussion
2.1. Biochemical evaluation and SAR studies
Having an in-house library of 283 synthetic compounds,
including 204 chalcones, 37 hydrazones, 10 oxadiazoles, 26 sulfonyl-
hydrazones and 6 sulfonamides, the inhibitory activity of them was
evaluated in vitro against recombinant YopH from Y. enterocolitica,
using p-nitrophenyl-phosphate (pNPP) as substrate. The percentage
of inhibition was assessed spectrophotometrically at a single con-
centration of 25 mM for each compound (data not shown). On the
basis of the initial screening, twelve compounds showed significant
YopH inhibition (>25%), when compared to the control (Table 1). The
bioactive compounds belong to three chemical classes, including
chalcone derivatives (1e9), sulfonamide derivatives (10 and 11) and
a sulfonyl-hydrazone derivative (12).
Chalcones are intermediate compounds in flavonoid biosyn-
thesis and exhibit a wide range of biological activities [23e25].
Sulfonyl-hydrazones and sulfonamides are known bioactive com-
pounds that show antibacterial activity against Escherichia coli and
Staphylococcus aureus [26,27]. Recently, chalcones and sulfonyl-
hydrazones were able to inhibit the activity of Mycobacterium
tuberculosis protein tyrosine phosphatases A and B (PtpA and PtpB,
respectively) [28e31].
In order to better understand and assess the molecular aspects
involved in YopH inhibition, IC50 values were determined using, at
least, five different concentrations of each compound and pNPP as
substrate (Table 1). Compounds 1e3, 5, 10 and 11 showed sub-
stantial inhibition of YopH with IC50 values <50 mM. Compounds 4,
8, and 12 were moderated inhibitors (IC50 ¼ 50e150 mM), while
compounds 6, 7 and 9 showed low inhibition of YopH
(IC50 > 150 mM) (Table 1). The most potent YopH inhibitors were
compounds 1 (chalcone derivative, IC50 ¼ 9.9  2.2 mM) and 10
(sulfonamide derivative, IC50 ¼ 9.0  1.4 mM).
Among the chalcone derivatives, inhibitor 1 (IC50 ¼ 9.9 
2.2 mM) exhibits a 3-nitrophenyl group as A-ring and a 3,4-
methylenedioxy-phenyl group as B-ring. The SAR studies con-
ducted on these derivatives indicated that the nitro group is well
tolerated in the A-ring by the putative binding site (compound 2,
IC50 ¼ 15.1  2.8 mM). Switching the A-ring nitrophenyl group for a
3,4-methylenedioxy-phenyl decreased 4- and 12-fold the inhibi-
tory activity of the compounds (compounds 3 and 4, respectively).
The chalcone derivative 5 (IC50 ¼ 20.3  1.1 mM) exhibits the 2-
naphthyl group as A- and B-rings. Interestingly, the substitution
of 1-naphthyl for 2-naphthyl as B-ring led to a 10-fold less inhib-
itory activity (compound 6, IC50 ¼ 193.1  2.8 mM), suggesting
some steric constraints for the putative binding site or some very
specific interaction of the 1-naphthyl. In line with this, the
replacement of 1-naphthyl for a phenyl derivative substantially
decreased the binding potency of the inhibitor (compound 7,
IC50 ¼ 245.6  5.6 mM). The substitution of the 1-naphthyl group
for a nitrophenyl as B-ring partially restores the inhibitory activity
(compound 8, IC50 ¼ 122.2  1.7 mM), however, further modifica-
tion in the B-ring was detrimental for the binding potency (com-
pound 9, IC50 ¼ 268.1  9.0 mM).
The SAR for the sulfonamide derivatives were based on the
biological data of the potent inhibitor 10 (IC50 ¼ 9.0  1.4 mM), a
compound obtained from the condensation of 4-(3,5-dioxo-10-
oxa-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)benzenesulfonyl chlo-
ride, a phenylmaleimide derivative, with a pyrrolidine ring [32].
The data indicated that replacement of the pyrrolidine substituent
for morpholine and the presence of bulky groups at the 4-(phe-
nylsulfonyl) substituent is tolerated by the YopH binding site
(compound 11, IC50 ¼ 15.5  3.7 mM), suggesting that polar and
hydrophobic interactions are important to the inhibitory activity
underlying this class. Moreover, the related sulfonyl-hydrazone
derivative (compound 12, IC50 ¼ 62.2  3.3 mM) also showed
moderate inhibitory activity.
2.2. Kinetics measurement and mechanism of inhibition
On the basis of the collected IC50 values, we investigated the
mechanism of YopH inhibition of the six most potent compounds
(1e3, 5, 10 and 11). Kinetic analysis revealed that compounds 1e3, 5
and 10 are competitive inhibitors. The LineweavereBurk double-
reciprocal plots (Fig. 1) were obtained with three different inhibi-
tor concentrations: all lines converged at the y-axis (1/Vmax),
whereas the slope (KMapp/Vmax) and x-axis interception (1/KMapp)
vary according to the inhibitor concentration. The constant value of
Vmax and the increased values of KMapp (proportional to increasing
inhibitor concentrations) are consistent with a mutually exclusive
binding mode, resulting in a competition for the enzyme active site
by the bioactive molecules and the substrate [29e31,33,34]. The
evaluated Ki values range from 4.4 to 14.1 mM indicating the affinity
of these compounds for the YopH enzyme (Table 2). Conversely, the
graphical analysis for compound 11 (Fig. 1), indicated that all lines
converge at the x-axis (1/KMapp), and the y-axis interception (1/
Vmax) varied as a function of the inhibitor concentration. The con-
stant value of KMapp and the increased values of Vmax indicated that
this compound is a noncompetitive inhibitor with equivalent af-
finity for both the free enzyme and enzymeesubstrate complex
(Ki ¼ 36.0  2.5 mM) [35]. This finding is in agreement with the
previous observation that YopH possesses a second phosphate
binding site that allows simultaneous interactions of an inhibitor at
both catalytic site and an adjacent peripheral site [18,36].
2.3. Molecular modeling
In an effort to understand the interactions of the chalcone and
sulfonamide derivates with YopH at the molecular level, modeling
studies were conducted based on the crystal structure of p-nitro-
catechol sulfate (pNCS) inhibitor bound to YopH [37e39]. Firstly,
with the aim of validating the molecular docking protocol, the
ligand pNCS was re-docked to the binding site of the protein using
the genetic algorithm implemented in GOLD [40]. The docked
conformation corresponding to the top ranked pose showed a
RMSD value of 0.3  A, indicating a high docking reliability of GOLD in
reproducing the experimentally observed binding mode for YopH
inhibitors. Subsequently, the competitive inhibitors 1, 2 and 10
 P.G.A. Martins et al. / European Journal of Medicinal Chemistry 64 (2013) 35e41 37

Table 1
Biological activity of the chalcone (1e9), sulfonamide (10 and 11) and sulfonyl-hydrazone (12) derivatives; data are the averages  SD of at least three independent
experiments.

Compound Structure % YopH inhibition (compound at 25 mM) IC50 value (mM)

1 31.3  1.7 9.9  2.2

2 65.7  1.2 15.1  1.1

3 33.8  7.4 38.6  0.6

4 44.2  4.3 116.9  4.6

5 66.9  2.0 20.3  1.1

6 26.5  2.7 193.1  2.8

7 30.9  4.24 245.6  5.6

8 34.7  0.9 122.2  1.7

9 34.6  4.9 268.1  9.0

10 76.4  5.9 9.0  1.4

11 82.1  1.2 15.5  3.7

12 45.6  3.3 62.2  3.3

38 P.G.A. Martins et al. / European Journal of Medicinal Chemistry 64 (2013) 35e41

Fig. 1. Inhibitory profile of compounds 1e3, 5, 10 and 11 against YopH from Yersinia enterocolitica. Kinetic experiments were conducted in the presence of increasing concentrations
of inhibitors; pNPP was used as substrate in all experiments. Assays were done in duplicate in at least four independent experiments.

were docked into the catalytic site (Fig. 2A) [39]. According to the (A-ring) is engaged in extensive hydrophobic interactions with the
model, the chalcone derivatives 1 (Ki ¼ 4.5  0.7 mM) and 2 active site flanked by Ile232, Arg242, Ala405 and Val407 (Fig. 2B
(Ki ¼ 4.4  1.6 mM) orient the 3- and 4-nitrophenyl substituent, and C). The central propenone moiety of the inhibitors 1 and 2 is in
respectively, toward the YopH catalytic P-loop (residues 403e410) van der Waals contact with the side-chain of Phe229, whilst the
(Fig. 2B and C). The nitro group is coordinated by ionic interactions 3,4-methylenedioxy-phenyl substituent of both inhibitors is
with the side-chain of the conserved Arg409 residue and the exposed to the solvent. Therefore, the predicted interactions of
crystallographic water W810, which acts as a hydrogen bond donor. YopH catalytic residues with the high affinity inhibitors 1 and 2 are
Furthermore, the electronegative nitro group is complementary to consistent with the SAR data and suggest the structural basis un-
the helix dipole, thereby assisting the stabilization of the binary derlying the tolerance observed for the substitution pattern of the
complex. In addition to the polar interaction between the nitro chalcone derivatives.
group and the side-chain of Arg409, there are key van der Waals The second class of competitive inhibitors is represented by
and hydrophobic contacts between YopH and the chalcone in- compound 10 (Ki ¼ 4.4  0.4 mM), a sulfonamide derivative that
hibitors. The phenyl ring of the 3- and 4-nitrophenyl substituents exhibits low micromolar affinity for YopH. The predicted binding
mode of compound 10 indicated that the 7-oxabicyclo[2.2.1]hep-
Table 2 tene group fits into the catalytic P-loop (Fig. 2D). Additionally, the
Mode of inhibition and Ki values for selected YopH inhibitors. imido moiety is in favorable orientation to accept two hydrogen
Compound Ki Mode of inhibition bonds from the side-chains of Gln446 and Arg242, the central
phenyl substituent makes hydrophobic interactions with Phe229
1 4.5  0.7 Competitive
2 4.4  1.6 Competitive and the sulfonyl-pyrrolidine group is exposed to the solvent
3 11.1  0.9 Competitive (Fig. 2D). The extensive polar and hydrophobic contacts of com-
5 14.1  0.8 Competitive pound 10 to the binding cavity of YopH are similar to those
10 4.4  0.4 Competitive observed for the chalcone derivatives, which may explain the
11 36.0  2.5 Noncompetitive
comparable binding affinities among the inhibitors.
 P.G.A. Martins et al. / European Journal of Medicinal Chemistry 64 (2013) 35e41 39

Fig. 2. Superimposed binding mode of competitive inhibitors 1, 2 and 10 into YopH catalytic cavity (A). Binding mode of inhibitors 1 (B), 2 (C) and 10 (D). Key residues and water
molecule involved in ligand binding are indicated as green stick and red sphere, respectively. Hydrogen bonds are indicated as blue dashed lines. (For interpretation of the ref-
erences to color in this figure legend, the reader is referred to the web version of this article.)

3. Conclusions Department, Universidade Federal de Santa Catarina, Florianópolis-

In sum, an in-house library of 283 synthetic compounds was
assayed against YopH, and off these, four chalcone and two sul-
fonamide derivatives exhibited potent inhibitory activity (com-
pounds 1e3, 5, 10 and 11). Five out of the six bioactive compounds
exhibited competitive inhibition with respect to the substrate pNPP,
with Ki values in low micromolar range. In addition, structure-
based molecular modeling investigations were performed, indi-
cating that the competitive inhibitors present similar binding
modes through key polar and hydrophobic contacts with the cat-
alytic pocket residues of the protein. So, the chalcone and sulfon-
amide derivatives described herein represent new lead candidates
for the development of therapeutic agents against pathogenic
Yersinia species.
4. Methods
4.1. Recombinant protein expression and purification
The expression and purification of recombinant YopH were
performed as previously described [41]. After purification, the
protein was concentrated and the original buffer was changed to
buffer C (20 mM Bis-Tris propane pH 6.5, 100 mM sodium chloride,
1 mM DTT, 1 mM EDTA) by ultrafiltration with a 10 kDa pore
membrane (Amicon Ultra-15 Millipore). The final protein concen-
tration was determined by Bradford assay [42].
SC, Brazil). The active compounds (chalcones 1e9, sulfonamides
10e11 and sulfonyl-hydrazone 12) were prepared as previously
described [28e30,32,43,44].
4.3. Biochemical evaluation
The phosphatase activity of YopH was assayed using pNPP as
substrate. The recombinant protein (14 nM) was incubated at 37  C
for 10 min in 20 mM Imidazole buffer pH 7.0, 25 mM of each com-
pound in DMSO 100% and MilliQ water. After adding 20 mM pNPP,
the quantity of p-nitrophenol produced was monitored spectro-
photometrically at 410 nm for 10 min. The screening was carried
out in 96-well plates and the absorbance was measured on an ELISA
plate spectrophotometer (TECAN Infinite M200). It was considered
as positive control the enzyme with DMSO in place of the com-
pound and as negative control the absence of enzyme. The enzy-
matic activity was expressed in % of phosphatase residual activity
compared with wells without inhibitor. All assays were done in
triplicate in at least three independent experiments.
The IC50 values were determined with increasing concentrations
of inhibitor (1e100 mM) versus % of inhibition, in triplicate in at least
three independent experiments. The experimental data was
analyzed with GraphPad Prism 5.0 and the IC50 values determined
by linear regression.
4.4. Kinetic measurements

4.2. Synthetic compounds To determine the kinetic parameters, three concentration of

each inhibitor and seven concentration of pNPP were used (0.2, 0.4,
The library of 283 synthetic compounds was obtained from our 0.8, 1.6, 3.2, 6.4 and 12.8 mM). The absorbance was converted
research group (Structure-Activity Laboratory of the Chemistry using p-nitrophenol molar absorptivity: 2249.6 M1 cm1 [45]. The
40 P.G.A. Martins et al. / European Journal of Medicinal Chemistry 64 (2013) 35e41
p-nitrophenol production was quantified and analyzed as a Line-
weavereBurk plot (1/V  1/[S]). The linear regression analysis was
used to determine Ki, Vmax and KMapp for each compound. The Ki
values were determined by plotting the KMapp  [I], considering Ki
the curve interception in x-axis. All assays were done in duplicate in
at least four independent experiments.
4.5. Molecular modeling
The 3D structures of the inhibitors were constructed using
standard geometric parameters of the molecular modeling soft-
ware package SYBYL 8.0. Each single optimized conformation of
each molecule in the data set was energetically minimized
employing the Tripos force field and the Powell conjugate gradient
algorithm [37] with a convergence criterion of 0.05 kcal/mol 
A and
GasteigereHückel charges [38]. Molecular docking and scoring
protocols as implemented in GOLD [40] were used to investigate
the possible binding conformations of the ligands within the YopH
binding pocket. The X-ray crystallographic data for YopH deter-
mined at 2 A (PDB ID 1PA9) [39] used in the docking simulations
were retrieved from the Protein Data Bank (PDB). Hydrogen atoms
were added in standard geometry using the Biopolymer module
implemented in SYBYL 8.0. Histidines, glutamines, and asparagines
residues within the binding site were manually checked for
possible flipped orientation, protonation, and tautomeric states
with Pymol 1.2 (DeLano Scientific, San Carlos, USA) side chain
wizard script. The binding site was defined as all the amino acid
residues encompassed within a 20 
A radius sphere centered on Cb
atom of the Ile281 residue, including the crystallographic water
molecule W810, which was considered part of the binding site
during the docking simulations. The docking procedures were
repeated 30 times for each inhibitor. The implemented GOLDscore
scoring function and visual inspection were employed to select the
representative conformation for each inhibitor.
Acknowledgments
We thank Dr. Tiago A.S. Brandão (Universidade Federal de Minas
Gerais) for the plasmid p7-7 (YopH51*D162) and CNPq, CAPES,
FAPESC, MCT and FINEP for financial support and fellowships.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.04.018.
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