A chalcone derivative binds a putative allosteric site of YopH: Inhibition of a virulence factor of Yersinia

Article abstract of DOI:10.1016/j.bmcl.2020.127350

Identification of allosteric inhibitors of PTPs has attracted great interest as a new strategy to overcome the challenge of discover potent and selective molecules for therapeutic intervention. YopH is a virulence factor of the genus Yersinia, validated a

Full text of DOI:10.1016/j.bmcl.2020.127350

Bioorganic&MedicinalChemistryLetters30(2020)127350
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A chalcone derivative binds a putative allosteric site of YopH: Inhibition of a
virulence factor of Yersinia
Ana C.A. de Souzaa, Mattia Morib, Larissa Sensc, Ruth F. Rochaa, Tiago Tizzianic,
Luiz F.S. de Souzac, Louise Domeneghini Chiaradia-Delatorrec, Maurizio Bottab,1
,
⁎
⁎
Ricardo J. Nunesc, Hernán Terenzia, , Angela C.O. Menegattia,d,
a Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Campus Trindade, Florianópolis, SC 88040-900, Brazil
b Department of Biotechnology, Chemistry and Pharmacy, “Department of Excellence 2018-2022”, University of Siena, via Aldo Moro 2, Siena 53100, Italy
c Laboratório Estrutura e Atividade, Departamento de Química, Universidade Federal de Santa Catarina, Campus Trindade, Florianópolis, SC 88040-900, Brazil
d Universidade Federal do Piauí, CPCE, Bom Jesus, PI 64900-000, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Identification of allosteric inhibitors of PTPs has attracted great interest as a new strategy to overcome the
challenge of discover potent and selective molecules for therapeutic intervention. YopH is a virulence factor of
the genus Yersinia, validated as an antimicrobial target. The finding of a second substrate binding site in YopH
has revealed a putative allosteric site that could be further exploited. Novel chalcone compounds that inhibit
PTPs activity were designed and synthesized. Compound 3j was the most potent inhibitor, interestingly, with
different mechanisms of inhibition for the panel of enzymes evaluated. Further, our results showed that com-
pound 3j is an irreversible non-competitive inhibitor of YopH that binds to a site different than the catalytic site,
but close to the well-known second binding site of YopH.
Non-competitive inhibitor
Yersinia
PTP
Allosteric site
Chicoric acid
Protein tyrosine phosphatases (PTPs) are an important topic in
current biomedical research. Since perturbation of the delicate balance
between the opposing activity of protein-tyrosine kinases (PTK) and
PTP results in abnormal tyrosine phosphorylation, these enzymes have
been associated with the etiology of many human diseases, including
tuberculosis, diabetes, cancer, and immune dysfunctions.1,2 Many stu-
dies validate the potential of PTPs in drug discovery for the treatment of
these diseases.3–5 Despite the efforts to target PTPs, the highly con-
served active site among the family becomes one of the main obstacles
to successful drug discovery. However, to overcome these difficulties,
new drug design strategies have emerged, including the development of
bidentate inhibitors, which can specifically target PTP active sites and
also unique peripheral regions, and allosteric inhibitors, which bind out
of the PTP active-site.6–8
ability to promote HER2-dependent tumorigenesis in vivo. MSI-1436 is
being tested in a Phase I clinical trial in metastatic breast cancer pa-
tients.12,13 Recently, Li et al. reported the identification of an allosteric
inhibitor of lymphoid-specific tyrosine phosphatase (LYP) via structural
modifications of a previous competitive inhibitor.14 LYP inhibition has
aroused great interest in the autoimmunity therapeutics community,
since this PTP has been implicated in many autoimmune diseases.8,14,15
The PTP Yersinia outer protein H (YopH) is a well-known virulence
factor of Yersinia, bacterial genus that includes three species that causes
human illness, ranging from gastrointestinal disease to bubonic
plague.16,17 Phan et al. modeled a phosphotyrosyl mimetic hexapeptide
in YopH structure, identifying a second non-catalytic substrate binding
site, which presents a possible alternative to the search for selective
enzyme inhibitors.18 Some studies have confirmed this finding by
modeling inhibitors at the allosteric site of YopH.19,20
The search for protein tyrosine phosphatase 1B (PTP1B) inhibitors,
a PTP associated with obesity, diabetes, cancers, and cardiovascular
diseases,9–11 has made great strides in the discovery of allosteric PTP
inhibitors.12 A well-elucidated case is trodusquemine (MSI-1436), a
reversible non-competitive inhibitor of PTP1B, that attenuates its
Martins et al. previously identified four synthetic chalcones as
competitive inhibitors of Yersinia enterocolitica YopH.21 Therapeutic
applications of chalcones may have emerged thousands of years ago
through the use of plants and herbs to treat different diseases such as
⁎ Corresponding authors at: Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Campus Trindade,
Florianópolis, SC 88040-900, Brazil (H. Terenzi). Universidade Federal do Piauí, CPCE, Bom Jesus, PI 64900-000, Brazil (A.C.O. Menegatti).
E-mail addresses: hernan.terenzi@ufsc.br (H. Terenzi), angelamenegatti@yahoo.com.br (A.C.O. Menegatti).
1 In memoriam, Professor Maurizio Botta died August 2, 2019.
https://doi.org/10.1016/j.bmcl.2020.127350
Received 15 April 2020; Received in revised form 8 June 2020; Accepted 9 June 2020
Availableonline12June2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

A.C.A. de Souza, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127350
Table 1
Synthesis of chalcones 3a-r.
Compound
R
% LYP Inhibition
Compound
R
% LYP Inhibition
3a
3b
3c
3d
3e
3f
Ph
4.2
9.5
13
2.5
1.8
5.0
0.5
1.0
3.9
3.0
3j
3-NO2-C6H4
2-OH-C6H4
3-OH-C6H4
3-OCH3,4-OH-C6H3
4-Br-C6H4
57.9
11.7
7.9
3.6*
4-CH3-C6H4
2-OCH3-C6H4
3-OCH3-C6H4
4-OCH3-C6H4
3,4-OCH3-C6H3
3,5-OCH3-C6H3
2,5-OCH3-C6H3
2,4-OCH3-C6H3
3k
3l
3.1
1.6
6.8
8.4
4.2
4.6
15.8
3.9
3m
3n
3o
3p
3q
3r
9.1
3.7
15.7
10.3
11.4
25.7
33.2
4.2
1-naphthyl
4-F-C6H4
3.6
2.4
2.2
3.3
3g
3h
3i
4.0
0.8
(i) Ethanol, KOH 50%, r.t., 24 h *Compound in bold showed phosphatase inhibition greater than 50%, at 25 µM using pNPP as substrate (Supporting Data). Data are
expressed as means SD of three independent experiments.
cancer, inflammation and diabetes.22,23
Table 2
Here, we studied a new series of chalcone derivatives as PTPs in-
hibitors. We identified a new chalcone derivative as a potential allos-
teric inhibitor of YopH, opening possibilities for the search for new
molecules targeting this allosteric site not usually conserved among the
PTPs.
IC50, Selectivity and Ki values for compound 3j.
PTP
IC50 (μM)
SI*
Ki (µM)
LYP
11.5
10.9
3.2
2.4
3.2
Ref value = 1
0.94
1.9
1.0
1.4
0.8
0.4
1.0
0.5
PTP-PEST
PTP1B
PtpA
0.4
0.3
0.30
0.2
0.2
Firstly, 18 chalcones 3a-r were synthesized by aldol condensation,
using a methodology already described by our research group, and were
characterized by melting point (mp), 1H and 13C nuclear magnetic re-
sonance (NMR) spectroscopy, and had their structures confirmed by
high-resolution mass spectrometry (HRMS) (Supporting Data). To the
best of our knowledge, chalcones 3c, 3g, 3j, 3l, 3q-r have not been
reported in the literature previously. As these chalcones have 4-carboxy
groups and we were interested in broad spectrum PTP inhibitors, in-
itially, we screened the in vitro inhibitory activity of the chemical li-
brary against the recombinant LYP, since carboxylic groups are
common pharmacophores described in previously LYP inhibitors
(Table 1).24–26
0.2
0.28
3.0
1.2
0.6
0.8
0.26
PtpB
2.3
0.19
YopH
2.6
0.22
The results are shown as the average of the individual mean
SD for three
independent experiments. *The selective index (SI) is given by SI (IC50PTP/
IC50LYP).
and non-competitive mode of inhibition for PtpA, YopH and PTP1B
(Fig. 1 and Table 2, Fig. S71 presents the Lineweaver–Burk plots). While
PtpA is significantly different in structure from YopH and PTP1B, these
latter share a certain degree of structural similarity particularly within
the catalytic site as well as the so-named second phosphate binding site,
although protein sequences differ to some extent.
Inhibition results in Table 1 suggest that the presence of electron-
donating groups in the chalcone moiety negatively affected the in-
hibitory activity. In addition, the best inhibitory activities were found
in compounds that present electron withdrawing groups, such as 3j
which has the nitro group known as a strongly electron withdrawing
group by inductive and resonance effect. This conclusion is more evi-
dent comparing the results of chalcones 3d (R = 3-OCH3-C6H4), 3j
(R = 3-NO2-C6H4) and 3l (R = 3-OH-C6H4).
PTP1B second phosphate binding site was identified by co-crystal-
lization with a synthetic non-peptide substrate (BPPM).27 From this
finding, other works have demonstrated inhibitors binding to this al-
losteric site, which are associated to a significant improvement in
druggability, suggesting that allosteric modulation may become an
important pillar for the future of PTPs drug discovery.12,28–32 Most of
the reported PtpA inhibitors act as competitive inhibitors, however, our
group recently described the first non-competitive inhibitor of PtpA.33
Despite the identification of YopH second substrate site and the report
of bidentate inhibitors,18,34 the allosteric inhibition of bacterial PTPs is
largely unexplored.19
Based on the initial screening, one compound, 3j, showed sig-
nificant LYP inhibition (greater than50%) (Table 1). In order to better
understand and assess the molecular aspects involved in PTP inhibition,
3j was selected for further assays; the IC50 values were determined
against a panel of PTPs including human and bacterial PTPs (from Y.
enterocolitica and Mycobacterium tuberculosis) (Table 2), and the speci-
ficity of inhibition was determined. Although the chalcone derivative 3j
did not show selectivity in relation to the PTPs studied, it appears to be
more potent for bacterial enzymes (YopH, PtpA and PtpB) and for
human PTP1B.
Regarding the competitive inhibition of LYP and PTP-PEST by 3j,
this result is consistent with the structural similarities between
them,35,36 further PTPs present a highly polar active site, which is easily
targeted by small molecules endowed with polar groups,24 such as the
carboxylic and the nitro groups in 3j.35 To the best of our knowledge,
most inhibitors reported for PTPB are competitive inhibitors, including
chalcone derivatives.37
To further understand this broad inhibition by 3j we sought to de-
termine the mechanism of inhibition for each enzyme evaluating the
kinetic parameters. Interestingly, detailed kinetic analyses revealed that
3j has a competitive mode of inhibition for LYP, PTP-PEST and PtpB,
Furthermore, to determine whether the inhibition was caused by the
formation of a reversible or irreversible enzyme-inhibitor complex, we
2

A.C.A. de Souza, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127350
Fig. 1. Michaelis–Menten plots representing inhibitory profile of compound 3j against the panel of PTPs. Results are presented as means
SD for three independent
experiments.
investigated the recovery of the phosphatase activity by a preincubation
and dilution assay (Supporting Data). Compound 3j seems to be a re-
versible inhibitor of LYP, PTP-PEST, PtpB and PtpA with recovery of
76%, 71%, 81%, and 76% of enzymatic activity, respectively. 45% of
PTP1B activity was recovered, which might suggest that 3j acts as
slowly dissociating inhibitor. Different from other PTPs, 3j irreversibly
inhibited YopH, after the dilution only 10% of the enzymatic activity
was recovered (Fig. S72).
observed a substantial difference in LYP cleavage profile, the intensity
of the peptides 2155 m/z and 3414 m/z were strongly reduced in the
presence of the inhibitor, indicating that 3j might reduce the formation
of these fragments due to protein-inhibitor interaction (Figs. S77-S79).
Together these peptides contain the P-loop residues 226–233, therefore
we may assume that LYP active site is being protected in the presence of
the competitive inhibitor 3j. The non-competitive inhibitor 3j also led
to changes in the cleavage profile of YopH (Figs. S80). Visibly, the most
prominent difference found was the intensity of the peptide 2022 m/z
that decreases considerably on 3j presence. This peptide corresponds to
YopH amino acids residues 255 to 272 (Fig. S81), which are located in
part of sheet β2 and helix α2.
Interestingly, two non-competitive inhibitors with irreversible in-
hibition mechanisms have been reported recently for PTP1B and
YopH.19,30 Kuban-Jankowska et al. identified that chicoric acid (CA) is
an irreversible inhibitor of YopH, and the inhibition is not induced by
the oxidation of the catalytic cysteine. We corroborated this finding for
CA, we observed only 0.6% recovery of enzymatic activity (Fig. S73),
thus CA and 3j are irreversibly inhibitors of YopH. Regarding the in-
hibitory potency, 3j was about 145-fold more potent than CA, with IC50
value of 2.6 µM and 379 µM, respectively.19,30 Although CA is a chal-
cone-like compound and has a carboxyl group, it differs from 3j by the
presence of electron-donor groups in the aromatic ring. As the mole-
cular docking results indicated that CA might induce allosteric inhibi-
tion of YopH,19 we sought to investigate its mechanism of inhibition by
kinetics assays. As shown in Fig. S74, CA showed a non-competitive
inhibition of the mixed type (α greater than 1), with a Ki value of
Examining the peptides corresponding to the catalytic pocket of
YopH, peptide 993 m/z, we did not observe major changes when 3j was
present (Fig. S82). Further studies of limited proteolysis were per-
formed with the Glu-C protease. In general, we observed changes in
peptides that represent amino acid residues 268–290, similar to that
found for trypsin (Figs. S83-S87).
Using a phosphotyrosyl hexapeptide as substrate, Phan et al. iden-
tified YopH second substrate binding site in a long and shallow groove
alongside helix α4 and the β6/β7, this site is composed by residues
R278, R295, R337, T343, K386, S389, K396.18 Taken together, our data
indicate that 3j is an irreversible non-competitive inhibitor of YopH
that binds to a site different than the catalytic site. The change in helix
α2 cleavage pattern by trypsin and Glu-C could indicate that 3j is
binding nearby. Since the C-terminal of helix α2 is oriented towards the
helix α4 and β6/β7 and contains the residues R278, we speculate that
3j interacts close to the second binding site of YopH and inhibits the
enzyme allosterically.
68.3
20 and α of 12.5
6.9 µM. Consequently, CA and 3j show
irreversible allosteric inhibition of YopH.
In addition, YopH stability in the presence of 3j was followed by
fluorescence measurements and thermal denaturation. YopH fluores-
cence spectrum presents an emission maximum (λmax) at 328 nm, the
binding of 3j resulted in fluorescence quenching without shift the λmax
,
indicating that 3j did not cause structural changes in YopH. YopH
Similarly, to those data reported by Phan et al., Kuban-Jankowska
et al. showed, through molecular docking studies, that CA could bind to
YopH second phosphate binding site. Accordingly, R255 interacts with
the aromatic ring of CA by a cation-π interaction, while R380 from helix
α4 is in the close proximity to the small molecule.19 Further, the
cleavage profile of YopH in the presence of CA supports the proposed
binding model (Figs S88). Although it seems difficult to determine a
specific point of interaction between YopH and CA, we noted that
tryptic peptides from the active site (i.e. the fragment 2550 m/z con-
taining the WPD-loop) and the second binding site (i.e. R278, T343 and
R337) are affected by the presence of the inhibitor. Additional, Kuban-
Jankowska et al. proposed that CA binds to both catalytic and secondary
thermal denaturation profile showed a melting temperatures (Tm) of
36.8
0.2 °C, while 3j presence (at 50 µM) indicated a decrease of
1.5 °C in the Tm, the difference observed suggests that the addition high
concentration of 3j did not affect YopH stability (Figs. S75-S76). Thus,
the observed enzyme inhibition is not caused by changes in protein
structure stability.
Further we sought to investigate the possible binding site of com-
pound 3j in YopH structure. To this aim, we carried out limited pro-
teolysis assays. First, we analyzed LYP and YopH peptide mass finger-
print (PMF) in the presence or absence of 3j, to compare the effect of a
competitive (LYP) or non-competitive (YopH) binding mechanism. We
3

A.C.A. de Souza, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127350
further corroborate this binding hypothesis, an intermolecular re-
cognition experiment was carried out by means of extended molecular
dynamics (MD) simulations. Briefly, YopH and 3j were included in a
simulation box of TIP3P-type water molecules at a distance higher than
40 Å each other. Following energy minimization, heating, and density
equilibration, two replicas of unrestrained MD simulations were run
and trajectories were produced for 500 ns in each replica for a total
simulation time of 1 µs. Notably, 3j quickly approaches YopH, most
likely driven by electrostatic interactions that led the molecule to in-
teract within the same positively charged cleft al-ready identified by
molecular docking. Cluster analysis of MD frames showed that 3j spend
more than 75% of its time in that site. In the most representative pose
extrapolated from MD trajectories, 3j establishes direct H-bond inter-
actions with R278, and S345, whereas a water-bridged H-bond inter-
action is established with K379. The chalcone core is inserted in a hy-
drophobic cleft bounded by I344, M382, Y383, and A390, whose
conformation is adapted to the presence of the ligand (Fig. 2B).
Overall, computational modeling results are in line with experimental
evidence and further substantiate the non-competitive allosteric mode
of action of 3j at a molecular level.
Further, 3j efficacy in inhibiting the growth of Y. enterocolitica was
evaluated in vitro. Interestingly a direct effect of 3j in bacterial growth
was not observed, the maximum inhibition was nearly 20% after 48 h at
the evaluated concentrations (8 to 250 µM) (Fig. S90). YopH is essential
for virulence but not for bacterial growth, thus we assumed that 3j
inhibition would impair the intracellular survival of Y. enterocolitica
rather than its viability.
In summary, we report herein the synthesis and PTP inhibitory ac-
tivity of chalcones derivatives, while the inhibitory mechanism of the
derivative 3j toward a small collection of PTPs was characterized. We
found that compound 3j is a low-micromolar (Ki < 2 µM) PTPs in-
hibitor, although having different inhibition mechanisms on some PTPs.
Compound 3j is indeed a competitive inhibitor of LYP, PTP-PEST and
PtpB, and a non-competitive inhibitor of PTP1B, PtpA and YopH.
Remarkably, the mechanism of YopH inhibition by 3j was proven to be
irreversible and non-competitive. Further, 3j inhibition mechanism was
corroborated by the result of limited proteolysis. In addition, we
showed that CA is an irreversible mixed type inhibitor of YopH. Our
limited proteolysis analysis indicated that 3j binding alters the cleavage
pattern of YopH helix α2, suggesting that the binding site of 3j might be
in proximity of the well-known second substrate binding site of YopH.
Finally, molecular docking studies revealed that 3j bind within the
second binding site of YopH, and in the catalytic site for LYP, in line
with experimental evidence. Thus, we propose that 3j binds closely to
the second binding site of YopH, and irreversibly inhibits its activity.
Accordingly, the second binding site of YopH and compound 3j present
potential for the development of new allosteric inhibitors with a higher
potency and specificity for this enzyme.
Fig. 2. Binding mode of 3j to YopH predicted by molecular docking (A) or MD
simulations (B). A) the crystallo-graphic structure of YopH coded by PDB:
4YAA, after reverting the W345Y mutation, is shown as green cartoon. The
catalytic site is colored magenta, the catalytic cysteine is shown as sticks.
Residues of the second substrate binding site are colored orange; 3j binding
posed by GOLD is shown as yellow stick, that from AutoDock is shown as cyan
sticks. B) Magnification of the binding mode of 3j within the allosteric site of
YopH such as predicted by MD simulations. The molecule is shown as yellow
sticks, the protein as green cartoon. Residues contacted by 3j and described in
the text are showed as green sticks and are labeled. Polar contacts are high-
lighted by black dashed lines.
binding sites of YopH, a behavior that could be justified by the large
change in cleavage profiles presented herein and the mixed type in-
hibition displayed by CA. The MS results found for 3j and CA (Figs. S80
and S88) show a reduction in intensity of the fragment 2022 m/z,
suggesting that helix α2 may be strongly affected by a non-competitive
inhibitor.
Given the irreversible non-competitive inhibition and change in
helix α2 cleavage pattern, we next sought to determine the binding
mode of 3j in the three-dimensional structure of YopH using compu-
tational modeling tools. To this aim, we performed a blind docking
experiment with GOLD and AutoDock to scan the whole surface of the
crystallographic structure of YopH17 with 3j. Notably, both programs
converged to dock the molecule in a highly-basic groove (colored or-
ange in Fig. 2A) that is far from the catalytic cavity (colored magenta in
Fig. 2A). It is worth mentioning that the docking site of 3j is occupied
by anions in multiple X-ray crystallography structures of YopH, as well
as it has been identified in other works as putative second binding site
for substrates or inhibitors.18,38 Although the docking poses of GOLD
and AutoDock were slightly different, none of the programs placed 3j
within the catalytic site, in agreement with experimental evidences
described above. The same docking protocol was used to monitor the
interaction of 3j to LYP (Fig. S89), clearly suggesting that the molecule
binds exclusively within the catalytic site of this protein, in agreement
with the competitive mechanism of action observed experimentally. To
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We dedicate this work to the memory of Professor Maurizio Botta, a
friend and mentor who loved to share knowledge. The authors would
like to thank the following facilities for their contributions to this work,
the Chemistry Department, Central Laboratory of Structural Biology
and Clinical Analysis Laboratory Unit (Sector Microbiology, University
Hospital) of the Federal University of Santa Catarina. We gratefully
acknowledge financial support from CNPq and CAPES (Brazil).
4

A.C.A. de Souza, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127350
Appendix A. Supplementary data
20. Vazquez J, Tautz L, Ryan JJ, Vuori K, Mustelin T, Pellecchia M. Development of
molecular probes for second-site screening and design of protein tyrosine phospha-
tase inhibitors. J Med Chem. 2007;50(9):2137–2143. https://doi.org/10.1021/
jm061481l.
Materials and Methods, characterizations, 1H and 13C NMR spectra,
Supplementary Figures and Tables can be found in the Supplementary
Data. (PDF). Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bmcl.2020.127350.
21. Martins PGA, Menegatti ACO, Chiaradia-Delatorre LD, et al. Synthetic chalcones and
sulfonamides as new classes of Yersinia enterocolitica YopH tyrosine phosphatase
inhibitors. Eur J Med Chem. 2013;64:35–41. https://doi.org/10.1016/j.ejmech.2013.
04.018.
22. Zhou B, Xing C. Diverse Molecular Targets for Chalcones with Varied Bioactivities.
Med Chem (Los Angeles). 2015;5(8):388–404. https://doi.org/10.4172/2161-0444.
1000291.
References
23. Zhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z. Chalcone: A Privileged
Structure in Medicinal Chemistry. Chem Rev. 2017;117(12):7762–7810. https://doi.
org/10.1021/acs.chemrev.7b00020.
1. He R, Yu Z, Zhang R, Zhang Z. Protein tyrosine phosphatases as potential therapeutic
targets. Acta Pharmacol Sin. 2014;35(10):1227–1246. https://doi.org/10.1038/aps.
2014.80.
24. Yu X, Sun J-P, He Y, et al. Structure, inhibitor, and regulatory mechanism of Lyp, a
lymphoid-specific tyrosine phosphatase implicated in autoimmune diseases. Proc
Natl Acad Sci U S A. 2007;104(50):19767–19772. https://doi.org/10.1073/pnas.
0706233104.
2. He R, Zeng L-F, He Y, Zhang S, Zhang Z-Y. Small molecule tools for functional in-
terrogation of protein tyrosine phosphatases. FEBS J. 2013;280(2):731–750. https://
doi.org/10.1111/j.1742-4658.2012.08718.x.
3. Lazo JS, McQueeney KE, Burnett JC, Wipf P, Sharlow ER. Small molecule targeting of
PTPs in cancer. Int J Biochem Cell Biol. 2018;96:171–181. https://doi.org/10.1016/j.
biocel.2017.09.011.
25. Vang T, Xie Y, Liu WH, et al. Inhibition of lymphoid tyrosine phosphatase by ben-
zofuran salicylic acids. J Med Chem. 2011;54(2):562–571. https://doi.org/10.1021/
jm101004d.
4. Zhao BT, Nguyen DH, Le DD, Choi JS, Min BS, Woo MH. Protein tyrosine phosphatase
1B inhibitors from natural sources. Arch Pharm Res. 2018;41(2):130–161. https://doi.
org/10.1007/s12272-017-0997-8.
26. He Y, Liu S, Menon A, et al. A potent and selective small-molecule inhibitor for the
lymphoid-specific tyrosine phosphatase (LYP), a target associated with autoimmune
diseases. J Med Chem. 2013;56(12):4990–5008. https://doi.org/10.1021/
jm400248c.
5. Dutta NK, He R, Pinn ML, et al. Mycobacterial Protein Tyrosine Phosphatases A and B
Inhibitors Augment the Bactericidal Activity of the Standard Anti-tuberculosis
Regimen. ACS Infect Dis. 2016;2(3):231–239. https://doi.org/10.1021/acsinfecdis.
5b00133.
27. Puius YA, Zhao Y, Sullivan M, Lawrence DS, Almo SC, Zhang Z-Y. Identification of a
second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: A paradigm
for inhibitor design. Proc Natl Acad Sci 1997; 94(25): 13420 LP - 13425. doi:10.
1073/pnas.94.25.13420.
6. Zhang Z-Y. Drugging the Undruggable: Therapeutic Potential of Targeting Protein
Tyrosine Phosphatases. Acc Chem Res. 2017;50(1):122–129. https://doi.org/10.
1021/acs.accounts.6b00537.
28. Baskaran SK, Goswami N, Selvaraj S, Muthusamy VS, Lakshmi BS. Molecular
Dynamics Approach to Probe the Allosteric Inhibition of PTP1B by Chlorogenic and
Cichoric Acid. J Chem Inf Model. 2012;52(8):2004–2012. https://doi.org/10.1021/
ci200581g.
7. Irving E, Stoker AW. Vanadium Compounds as PTP Inhibitors. Molecules.
2017;22(12):2269. https://doi.org/10.3390/molecules22122269.
8. Stanford SM, Bottini N. Targeting Tyrosine Phosphatases: Time to End the Stigma.
Trends Pharmacol Sci. 2017;38(6):524–540. https://doi.org/10.1016/j.tips.2017.03.
004.
29. Meng G, Zheng M, Wang M, et al. Design and synthesis of new potent PTP1B in-
hibitors with the skeleton of 2-substituted imino-3-substituted-5-heteroarylidene-
1,3-thiazolidine-4-one: Part I. Eur J Med Chem. 2016;122:756–769. https://doi.org/
10.1016/j.ejmech.2016.05.060.
9. Hale AJ, ter Steege E, den Hertog J. Recent advances in understanding the role of
protein-tyrosine phosphatases in development and disease. Dev Biol.
2017;428(2):283–292. https://doi.org/10.1016/j.ydbio.2017.03.023.
10. Tonks NK. Protein tyrosine phosphatases - from housekeeping enzymes to master
regulators of signal transduction. FEBS J. 2013;280(2):346–378. https://doi.org/10.
1111/febs.12077.
30. Krishnan N, Konidaris KF, Gasser G, Tonks NK. A potent, selective, and orally
bioavailable inhibitor of the protein-tyrosine phosphatase PTP1B improves insulin
and leptin signaling in animal models. J Biol Chem. 2018;293(5):1517–1525. https://
doi.org/10.1074/jbc.C117.819110.
31. Zhang J, Sasaki T, Li W, et al. Identification of caffeoylquinic acid derivatives as
natural protein tyrosine phosphatase 1B inhibitors from Artemisia princeps. Bioorg
Med Chem Lett. 2018;28(7):1194–1197. https://doi.org/10.1016/j.bmcl.2018.02.
052.
11. Thiebaut P-A, Besnier M, Gomez E, Richard V. Role of protein tyrosine phosphatase
1B in cardiovascular diseases. J Mol Cell Cardiol. 2016;101:50–57. https://doi.org/
10.1016/j.yjmcc.2016.09.002.
12. Kumar AP, Nguyen MN, Verma C, Lukman S. Structural analysis of protein tyrosine
phosphatase 1B reveals potentially druggable allosteric binding sites. Proteins Struct
Funct Bioinforma. 2018;86(3):301–321. https://doi.org/10.1002/prot.25440.
13. Krishnan N, Koveal D, Miller DH, et al. Targeting the disordered C terminus of PTP1B
with an allosteric inhibitor. Nat Chem Biol. 2014;10(7):558–566. https://doi.org/10.
1038/nchembio.1528.
32. Ghattas MA, Raslan N, Sadeq A, Al Sorkhy M, Atatreh N. Druggability analysis and
classification of protein tyrosine phosphatase active sites. Drug Des Devel Ther.
2016;10:3197–3209. https://doi.org/10.2147/DDDT.S111443.
33. Sens L, de Souza ACA, Pacheco LA, et al. Synthetic thiosemicarbazones as a new class
of Mycobacterium tuberculosis protein tyrosine phosphatase A inhibitors. Bioorg Med
Chem. 2018;26(21):5742–5750. https://doi.org/10.1016/j.bmc.2018.10.030.
34. Liu F, Hakami RM, Dyas B, et al. A rapid oxime linker-based library approach to
identification of bivalent inhibitors of the Yersinia pestis protein-tyrosine phospha-
tase. YopH Bioorg Med Chem Lett. 2010;20(9):2813–2816. https://doi.org/10.1016/j.
bmcl.2010.03.058.
14. Li K, Hou X, Li R, et al. Identification and structure–function analyses of an allosteric
inhibitor of the tyrosine phosphatase PTPN22. J Biol Chem.
2019;294(21):8653–8663. https://doi.org/10.1074/jbc.RA118.007129.
15. Stanford SM, Bottini N. PTPN22: the archetypal non-HLA autoimmunity gene. Nat
Rev Rheumatol. 2014;10(10):602–611. https://doi.org/10.1038/nrrheum.2014.109.
16. Martins PGA, Mori M, Chiaradia-Delatorre LD, et al. Exploring Oxidovanadium(IV)
Complexes as YopH Inhibitors: Mechanism of Action and Modeling Studies. ACS Med
Chem Lett. 2015;6(10):1035–1040. https://doi.org/10.1021/acsmedchemlett.
5b00267.
35. Hou X, Li K, Yu X, Sun J, Fang H. Protein Flexibility in Docking-Based Virtual
Screening: Discovery of Novel Lymphoid-Specific Tyrosine Phosphatase Inhibitors
Using Multiple Crystal Structures. J Chem Inf Model. 2015;55(9):1973–1983. https://
doi.org/10.1021/acs.jcim.5b00344.
36. Karver MR, Krishnamurthy D, Bottini N, Barrios AM. Gold(I) phosphine mediated
selective inhibition of lymphoid tyrosine phosphatase. J Inorg Biochem.
2010;104(3):268–273. https://doi.org/10.1016/j.jinorgbio.2009.12.012.
37. Chiaradia LD, Martins PGA, Cordeiro MNS, et al. Synthesis, Biological Evaluation,
And Molecular Modeling of Chalcone Derivatives As Potent Inhibitors of
Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB). J Med
Chem. 2012;55(1):390–402. https://doi.org/10.1021/jm2012062.
38. Ivanov MI, Stuckey JA, Schubert HL, Saper MA, Bliska JB. Two substrate-targeting
sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial
virulence. Mol Microbiol. 2005;55(5):1346–1356. https://doi.org/10.1111/j.1365-
2958.2005.04477.x.
17. Moise G, Gallup NM, Alexandrova AN, Hengge AC, Johnson SJ. Conservative tryp-
tophan mutants of the protein tyrosine phosphatase YopH exhibit impaired WPD-
loop function and crystallize with divanadate esters in their active sites. Biochemistry.
2015;54(42):6490–6500. https://doi.org/10.1021/acs.biochem.5b00496.
18. Phan J, Lee K, Cherry S, Tropea JE, Burke Terrence R, Waugh DS. High-Resolution
Structure of the Yersinia pestis Protein Tyrosine Phosphatase YopH in Complex with
a Phosphotyrosyl Mimetic-Containing Hexapeptide. Biochemistry.
2003;42(45):13113–13121. https://doi.org/10.1021/bi030156m.
19. Kuban-Jankowska A, Sahu KK, Gorska M, Tuszynski JA, Wozniak M. Chicoric acid
binds to two sites and decreases the activity of the YopH bacterial virulence factor.
Oncotarget. 2016;7(3):2229–2238. https://doi.org/10.18632/oncotarget.6812.
5