Discovery and evaluation of the hybrid of bromophenol and saccharide as potent and selective protein tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.04.004

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signaling pathway. Inhibition of PTP1B is expected to improve insulin action. Appropriate selectivity and permeability are the gold standard for excellent PTP1B inhibitors. In this work, molecular hybridization-based screening identified a selective competitive PTP1B inhibitor. Compound 10a has IC₅₀ values of 199?nM against PTP1B, and shows 32-fold selectivity for PTP1B over the closely related phosphatase TCPTP. Molecule docking and molecular dynamics studies reveal the reason of selectivity for PTP1B over TCPTP. Moreover, the cell permeability and cellular activity of compound 10a are demonstrated respectively.

Full text of DOI:10.1016/j.ejmech.2017.04.004

Accepted Manuscript
Discovery and evaluation of the hybrid of bromophenol and saccharide as potent and
selective protein tyrosine phosphatase 1B inhibitors
Renshuai Zhang, Rilei Yu, Qi Xu, Xiangqian Li, Jiao luo, Bo Jiang, Lijun Wang, Shuju
Guo, Ning Wu, Dayong Shi
PII:
S0223-5234(17)30258-1
10.1016/j.ejmech.2017.04.004
EJMECH 9351
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2017
Revised Date: 21 March 2017
Accepted Date: 2 April 2017
Please cite this article as: R. Zhang, R. Yu, Q. Xu, X. Li, J. luo, B. Jiang, L. Wang, S. Guo, N. Wu, D.
Shi, Discovery and evaluation of the hybrid of bromophenol and saccharide as potent and selective
protein tyrosine phosphatase 1B inhibitors, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.04.004.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery and evaluation of the hybrid of bromophenol and
saccharide as potent and selective Protein tyrosine
phosphatase 1B inhibitors
Renshuai Zhang,ab Rilei Yu,d Qi Xu,ab Xiangqian Li,ab Jiao luo,ab Bo Jiang,ab Lijun
Wang,ab Shuju Guo,ab Ning Wu,ab Dayong Shi*abc
aKey Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese
Academy of Sciences, Qingdao, China.
bLaboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for
Marine Science and Technology, Qingdao, China.
cUniversity of Chinese Academy of Sciences, Beijing, China.
dKey Laboratory of Marine Drugs, The Ministry of Education of China, School of
Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.
*Corresponding author: Tel. +86-0532-8289-8719; Fax: +86-0532-8289-8741: E-Mail:
shidayong@qdio.ac.cn.
Abstract
Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin
signaling pathway. Inhibition of PTP1B is expected to improve insulin action.
Appropriate selectivity and permeability are the gold standard for excellent PTP1B
inhibitors. In this work, molecular hybridization-based screening identified a selective
competitive PTP1B inhibitor. Compound 10a has IC50 values of 199 nM against
PTP1B, and shows 32-fold selectivity for PTP1B over the closely related phosphatase
TCPTP. Molecule docking and molecular dynamics studies reveal the reason of
selectivity for PTP1B over TCPTP. Moreover, the cell permeability and cellular
activity of compound 10a are demonstrated respectively.
Key words: PTP1B; bromophenol; saccharide; selectivity; permeability
Introduction
Diabetes mellitus is a comprehensive metabolic syndrome, with the progress of the
disease, which may lead to blurring vision, cardiovascular diseases, kidney or other
organ damage, dysfunction and many more.[1] Type-II diabetes accounts for more
than 90% of the total in all diabetics.[2] PTP1B down regulates insulin signaling by
catalyzing the dephosphorylation of phosphotyrosine residues, and precise control of

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the phosphorylation levels of proteins involved in insulin signal pathways.[3, 4]
Elchebly, M. and Klaman, L. D. et al have disclosed that PTP1B knockout mice
revealed phenotypes of enhanced sensitivity to insulin, lower plasma glucose and
insulin levels in 1999 and 2000.[5, 6] Thus, PTP1B was recognized as an attractive
therapeutic target for type-II diabetes.[7] PTP1B was the first validated PTP target,
which has been cloned and fully characterized in 1988. However, as of 2016, there
were not any of small molecule PTP1B inhibitors, as therapeutic drug for type-II
diabetes, approved over the world. This is in sharp contrast to that several small
molecule kinase inhibitors have been approved in recent years.[8] Why is this so?
Three significant challenges to the development of excellent small molecule PTP1B
inhibitors are: (1) the relatively shallow nature of the catalytic pocket of PTP1B,
which is disadvantageous for binding of small molecule inhibitors.[9] (2) the catalytic
pocket consists of several polar amino acids (Ser216, Cys215 and Arg221 et al),
which result in that small molecules only containing polar groups could effectively
bind to the catalytic site.[10] The initial PTP1B inhibitors were pTyr mimetics based
on the electrostatic properties of active site. The phosphonates, carboxylic acids,
sulphonamides and sulphanamic acids were initially used as pTyr mimetics for
designing of PTP1B inhibitors.[11-13] These pTyr mimetics with negative charge
were hydrophilic, which resulted in poor cell permeability and oral bioavailability. (3)
TCPTP is the most homologous to PTP1B, with 74% sequence identity in the
catalytic region. However, the knockout of TCPTP would result in abnormalities in B
cell and T cells functions.[14] Therefore, it is difficult but important to achieve high
selectivity for PTP1B over TCPTP based on above observation.
Figure 1. Structures of PTP1B inhibitors
A number of PTP1B inhibitors including synthetic and natural small molecules
have been discovered and structure-based design (Fig 1).[15-18] Compound 1, 2 and
3 were classic PTP1B inhibitors, which were designed on the basis of catalytic pocket
characteristics. Compound 4, 5 and 6 were derived from natural products and have
also been proved to be active against PTP1B. The compound 1 and
2-oxalylarylaminobenzoic acid derivative 2 showed potent activity (1 : IC50=120 nM,

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2: IC50 = 18 nM). Unfortunately, they showed little selectivity (1: 1.0-fold; 2: 3.6-fold)
for PTP1B over TCPTP.[19, 20] The compound 3 was a heterocyclic pTyr Mimetic
without electronegative groups, showed nanomolar activity (IC50=32 nM) and
demonstrated modest caco-2 permeability (0.4×10-6 cm/s), but without selectivity.[15,
21, 22] Additionally, a number of PTP1B inhibitors from natural product have also
been discovered. The natural products 4, 5 and 6 exhibited a micromolar level of
activity (4: IC50 = 0.18 µM; 5: IC50 = 2.42 µM; 6: IC50 = 4.8 µM) against PTP1B,
however, the selectivity or membrane permeability of these compounds were not
further reported.[23-25] All of above observations indicated that (1) some PTP1B
inhibitors derived from plants or microorganism without charged groups only showed
modest activity, (2) some artificially designed inhibitors exhibited excellent inhibitory
activity, but lacked adequate membrane permeability or selectivity. It is relatively easy
to obtain highly active PTP1B inhibitors. The real greater challenge is to enhance the
selectivity and permeability of PTP1B inhibitors.
Our laboratory has reported a number of bromophenols (BPN, BDB et al) derived
from marine alga Rhodomela confervoides, which displayed sub-micromolar PTP1B
activity.[26-31] They shared similar structural features, that was, containing one or
more bromophenol functional groups. Similarly, a large number of polyphenols have
also been found in terrestrial plants, which showed a variety of biological activity,
including anti-parasite, antitumor, antioxidant, antibacterial and anti-diabetic.[25,
32-35] Unlike natural products derived from the sea, terrestrial plant metabolites are
usually free of halogens because of the lack of halogen sources. Pentagalloyl glucose
was discovered in roots and fruits of a variety of terrestrial plants, which showed
micromolar activity against PTP1B, but without further study of selectivity and
permeability.[25, 36] Would it work if polyphenols derived from terrestrial plants
were replaced by bromophenols from marine algae? The initial design strategy of
these hybrid molecules was shown in Fig S1.
Figure 2. Compounds 8-11a were first designed.
Compound 10a and 11a were originally designed, which incorporated four
molecules of bromophenol functional groups that were coupled to ribose or xylose

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respectively. In order to investigate the effect of number of functional groups on
activity, compounds 8a and 9a were also designed (Fig 2). The synthetic route of title
molecules 8-11a was shown in Scheme S1. Interestingly, only β-anomer of 11a was
obtained. The effect of synthesized compounds (3a-11a) on recombinant PTP1B
inhibition was evaluated using colorimetric assay. The inhibitory rate was measured at
concentration of 10 µM, and the compounds with good inhibition rate (>50% at 10
µM) were selected for IC50 assay. The results of the enzymatic assay were shown in
Table 1. The intermediates (3a-7a), which contain one or more bromophenol groups
protected by TBDMS, do not exhibit PTP1B inhibitory activity with the inhibition
rate < 50% at 10 µM. The targeted compounds (9a-11a) with 3 or 4 bromophenol
groups are ideal to potent inhibitors of PTP1B with IC50 values 0.19-0.24 µM. The
compound 8a, with two bromophenol groups coupled by ethylene glycol, is
significantly less active toward PTP1B. Interestingly, the increase of bromophenol
group leads to enhanced compound activity, whereas the xylose or ribose linker has
little effect on activity. Does an analog containing 5 bromophenol groups have the
higher activity? Unfortunately, we failed to synthesize glucose bromophenol
derivatives, which contain 5 bromophenol groups and coupled by glucose. That was
probably due to the presence of bromine atoms which greatly increased the steric
hindrance. Additionally, the compound 6 (Fig 1) with five polyphenol groups coupled
by glucose, showed IC50 value of 4.8 µM against PTP1B. The activity of 9-11a was
20-fold greater than that of 6.
Table 1. Inhibition of PTP1B by Inhibitors 3-11a
% PTP1B inhibition
(at 10 µM )
% PTP1B inhibition
(at 10 µM )
IC50
compd
compd
IC50 (µM)
(µM)
-
-
-
-
-
25.04±6.33
95.45±11.20
96.37±9.76
99.00±15.38
-
3a
4a
5a
6a
7a
24.23±3.00
32.83±3.24
12.02±5.43
43.56±5.86
41.94±10.32
8a
9a
0.24±0.02
0.19±0.04
0.20±0.03
4.8
10a
11a
6a
a6: data from literature.[25]

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Figure 3. Compounds were designed for SAR study.
To investigate the effect of phenolic hydroxyl and bromine atoms on the activity,
analogues (8-11b, 4-7c, 8-11d) bearing varying substituted benzene ring were
prepared (Fig 3). The structures and synthetic route of these compounds were shown
in Scheme S2. The compounds 11b and 11d were β-anomer, which was same to that
of 11a. However, α and β-anomer of 7c were obtained under the same condition.
These new analogues were then evaluated for their ability to inhibit PTP1B in
colorimetric assay (Table 2). The compound 8b, 4c and 8d, containing different
substituted benzene rings which were coupled by ethylene glycol, showed no activity
(IC50 > 20 µM). The observation was consistent with compound 8a, which revealed
that compounds containing two bromophenol groups did not have PTP1B inhibitory
activity. Compound 9b, one of the hydroxyl groups on every substituted benzene ring
was replaced by a methoxy group, showed modest inhibition against PTP1B (IC50 =
8.45 µM). However, the activity of compound 9b is significant decreased compared
with the corresponding compound 9a, which contains two hydroxyl groups on every
substituted benzene ring. Further, the compound 5c, all hydroxyl groups were
replaced by methoxy groups, was complete inactive against PTP1B. The same
conclusion was observed for compounds 10a, 10b, 6c and 11a, 11b, 7c. It revealed
that the compounds with free hydroxyl groups were more potent PTP1B inhibitors
than the corresponding methoxyl compounds. The compounds 10b and 6c displayed
obviously improvement in potency compared with the corresponding compounds 9b
and 5c, and it is consistent to that the PTP1B inhibitory activity of 10a is greater than
9a. It indicated that the increase of bromophenol group improved PTP1B inhibitory
activity. The compound 9d containing 3, 4-dihydroxybenzene ring exhibited weaker
activity than that of compound 9a and 9b bearing bromine-substituted benzene ring. It

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was similar that the inhibitor 10d and 11d showed higher IC50 values than 10a and
11a, respectively. Reduction of bromine atom led to drop in PTP1B inhibitory potency,
suggested that the existence of bromine substitution on the phenyl ring had a certain
influence on the inhibitory activity against PTP1B. It was unclear why inhibitor 7c
and 11b were more active than the closely related inhibitors 6c and 10b, on the
contrast, the compound 11d and 11a were less active than 10d and 10a. It was
ambiguous that the effect of saccharide on activity against PTP1B. In general,
compound 10a bearing dihydroxy bromobenzene group exhibited the best inhibitory
activity and was selected for further study.
Table 2. Inhibition of PTP1B by Inhibitors 8-11b, 4-7c, 8-11d
compd
8b
IC50 (µM)
> 20
compd
6c
IC50 (µM)
3.41±0.13
1.11±0.08
> 20
9b
8.45±0.11
1.50±0.13
0.75±0.06
> 20
7c
10b
11b
4c
8d
9d
12.3±0.26
2.20±0.22
3.00±0.32
10d
11d
> 20
5c
Figure 4. (A) Compound 10a is a classical competitive inhibitor. (B) Interaction of
inhibitors (10a, 11b, 5c and 7c) with PTP1B by SPR. Data is presented as maximum
binding.
A detailed kinetic analysis of 10a was then conducted using p-NPP as the
small-molecule substrate in a continuously monitored colorimetric assay.
Lineweaver-Burk analyses of compound 10a against PTP1B, confirmed that it was a
competitive inhibitor with classical kinetic behavior (Fig 4A). The surface plasmon
resonance (SPR) could characterize the interaction between drug targets and
small-molecules and has been exploited as a powerful tool for drug discovery. Here,

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the SPR technology was used to analyse small molecule (10a, 11b, 5c and 7c) binding
to PTP1B. SPR traces were presented in Fig S2. The response values of representative
inhibitors were displayed in Fig 4B. The compound 10a contained two phenolic
hydroxyl in one bromophenol functional group showed most potent binding to PTP1B.
The inhibitor 11b bearing a phenolic hydroxyl in one bromophenol group showed a
significant reduction in binding to PTP1B. Conversely, no binding was observed in
compounds 5c and 7c which did not contain free phenolic hydroxyl groups.
Additionally, the inhibitors (10a, 11b, 5c and 7c) displayed no binding to the blank
(Fig S3), in which there was no immobilized protein. The SPR binding data
demonstrated that the compound 10a could be bond to PTP1B and supported an
important role for phenolic hydroxyl in the interaction with PTP1B.
Figure 5. Model of inhibitor 10a bound to PTP1B (PDB: 1QXK). (A) The surface of
catalytic pocket of PTP1B. (B) H-bonds between 10a and key amino acids. (C) The
distance between PHE182 and 10a.
Molecular docking was performed to predict the binding mode of 10a with PTP1B
and explain the interaction of 10a with PTP1B. The X-ray structure of PTP1B with a
difluorophosphonate inhibitor as the ligand was used as the starting point (PDB code
1QXK).[37] After the difluorophosphonate inhibitor was removed from the protein
structure, 10a was minimized using Tripos force field of SYBYL-X 2.0 software. Fig
5A showed the binding orientation of 10a minimized in the catalytic binding pocket
of PTP1B. The binding site of the PTP1B was shown in the opaque molecular surface
colored by atom type representation. One of the substituted benzene ring was
embedded into the catalytic pocket, and the remaining substituted benzene rings were
located at the inlet of the catalytic pocket. The dihydroxybenzene ring moiety plays an
important role in the binding, as a plurality of hydroxyl groups involved in hydrogen
bonding interactions with amino acid residues Lys120, Asp181 and Arg47, 48, 221 et
al (Fig 5B). The key distance (3.30 - 3.44 Å) between a substituted benzene ring
which was not involved in the formation of H-bonds and Phe182 was shown in Fig
5C. A π-π stacking interaction was observed between the phenyl ring and Phe182. All
of the substituted benzene rings of inhibitor 10a formed an interaction with

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corresponding amino acid residues, that might explain why compound 10a exhibited
excellent activity against PTP1B.
Table 3. Inhibition of Phosphatases by Inhibitors 9a and 10a
IC50 (µM)
compd
9a
PTP1B
0.24±0.02
0.19±0.04
4.8
TCPTP
1.98
SHP2
4.53
PTPLAR
NAa
Selectiveb
8.0
5.94
15.86
> 50
NA
32.0
10a
6c
0.07
> 50
0.01
aNA: no activity (the inhibitor % in 20 µg/mL < 25.0%). Selective: IC50
b
(TCPTP)/IC50 (PTP1B). c6: data from literature.[25]
Compound 10a is the best inhibitor in all title compounds against PTP1B. Does it
have selectivity for PTP1B over other phosphatases? Further, the selectivity of the 9a
and 10a was determined by measuring their inhibitory activity against a panel of
several phosphatases including TCPTP, SHP-2 and PTPLAR. The IC50 values of
inhibitors 9a and 10a against the phosphatases, and the selectivity ratios were shown
in Table 3. The compound 9a and 10a showed no activity against PTPLAR, and only
exhibited moderate activity for SHP2. We were gratified to find that compound 9a
and 10a were selective for PTP1B over PTPLAR and SHP2. On the basis of amino
acid sequence homology, TCPTP was the phosphatase most closely related to PTP1B.
The inhibitor 9a had 8-fold selectivity for PTP1B over TCPTP. Furthermore, the
compound 10a showed 32-fold selectivity for PTP1B over TCPTP. It was a
significant level of selectivity given the 74% sequence homology in their catalytic
active site. The compound 10a was more excellent than the vast majority of PTP1B
inhibitors reported in literatures.[15, 16, 38] Additionally, the compound 6 did not
exhibit selectivity for PTP1B over TCPTP, by contrast, displayed the lower IC50 value
against TCPTP than that of PTP1B.

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Figure 6. (A), (B) and (C): The binding mode and interactions of compound 10a with
TCPTP (PDB: 1L8K). The surface of catalytic pocket of TCPTP, the key amino acids
and the distance between Phe183 and 10a are shown separately. (D) The structure of
TCPTP (pink) has been aligned with PTP1B (green).
A positive effort was made to explain the selectivity of inhibitor 10a by performing
molecular docking and molecular dynamics (MD) simulations. The binding mode of
compound 10a with TCPTP [39] was displayed in of Fig 6A. Some main interactions
between inhibitor 10a and the receptor can be observed in of Fig 6B and 6C. The
superimposed images of secondary structures of TCPTP and PTP1B (PDB: 1QXK)
was shown in of Fig 6D, as the Phe182 of PTP1B and Phe183 of TCPTP were
displayed separately. Only one crystal structure of TCPTP was available in the Protein
Data Bank, and this structure has the WPD loop in the open position, which formed a
larger catalytic pocket than that of PTP1B. The substituted benzene rings were loosely
distributed in the binding pocket. Only the oxygen atoms of the ribose and the ester
bonds formed hydrogen bonding with amino acid residues Arg222 and Phe183, that
was different from the binding pattern of 10a with PTP1B (Fig 5A and 5B). In
addition, in contrast to the closed WPD loop of PTP1B, the WPD loop of TCPTP is
open, which led to the opposite orientation of Phe183 (Fig 6D). The closest distance
between the substituted benzene ring and Phe183 is 5.16 Å, which resulted in the loss
of π-π stacking or T-shape interaction.
Unlike the only TCPTP structure with an open WPD loop did not bind with any
ligands, the PTP1B (PDB: 1QXK) with a closed WPD loop had a
difluorophosphonate inhibitor as the ligand. A ligand-free PTP1B crystal structure
(PDB: 3A5J) was chosen for further investigation of the mode of binding to the

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inhibitor 10a, considering that the presence of the ligand might affect the position of
loop. The binding mode and H-bond interaction were shown in Fig S4. The
orientation of 10a in binding pocket of PTP1B (PDB: 3A5J) was very similar to that
in TCPTP (Fig 6A). However, there are three substituted benzene rings of compound
10a that formed H-bonds with PTP1B (Fig S4B), which was completely different
from the bonding between 10a and TCPTP. Although the WPD loop of PTP1B (PDB:
3A5J) without ligands was also in the open position which was the same to TCPTP,
the orientation of Phe182 in PTP1B (PDB: 3A5J) showed a significant difference with
that of Phe183 in TCPTP (Fig. S5A). Most notably, the distance between Phe182 and
one of substituted benzene ring was appropriate for the formation of π-π stacking
interaction (Fig. S5B).
Figure 7. Evolution of the distance between the pairwising interacting atoms that can
form H-bonds from 10a and residues of TCPTP (1L8K) or PTP1B (3A5J).
The interaction of the protein with ligands is a dynamic process that results in a
lower binding energy of the complex and a more stable complex. Molecular docking
is a static process, it can only show the interaction of ligand and protein at a certain
moment. While the dynamic process of ligand-protein binding could be displayed by
MD simulation, which take into account the influence of water molecules and ions et
al. The binding modes of 10a and PTP1B (3A5J) or TCPTP (1L8K) were shown in

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Fig S6 after 50 nanoseconds of MD simulation. Compound 10a bound with the
TCPTP and PTP1B in a similar mode with three aromatic rings embedded into the
binding site and the forth one exposed outside. Three benzene rings of 10a were
loosely distributed in the binding pocket of TCPTP, whereas the three benzene rings
embedded in PTP1B form π-π stacking interaction which made the ligand more stable.
Further, the hydroxyl groups at the aromatic ring formed hydrogen bonds with
Glu115 and Asp46 of TCPTP, whereas four amino acids were involved in the
interaction between the PTP1B and inhibitor 10a. The H-bonds between Glu115,
Asp46 and inhibitor 10a were newly generated during MD simulation, and the
original interactions between Phe183, Arg22 and 10a were lost in complex
TCPTP/10a (Fig 6B and Fig S6A). The H-bonds between Glu115, Arg222 and the
inhibitor were always present throughout the MD simulation in complex PTP1B/10a
(Fig S4B and Fig S6B). The stability of these hydrogen bonds was evaluated by
calculating the evolution of distances between H-bond interacting atoms in 50 ns MD
simulations (Fig 7). The distances of the pairwise interacting residues substantially
fluctuated at the beginning of the MD except that between Glu115, Arg222 and 10a in
complex PTP1B/10a. Afterwards, the distances reached equalization and maintained
constant in the last 20 ns of the MD, which suggested that these hydrogen bonds were
stable. Additional, the distance between benzene rings of 10a and phenylalanine of
PTP1B or TCPTP was more than 5 Å, which resulted in the loss of π-π stacking or
T-shape interaction (Fig S7). The observation was different from the result found in
molecule docking (Fig 5C and Fig S5B), and indicated that the interaction of the
ligand with phenylalanine was unstable and not durable.
Figure 8. (A)PTP1B (3A5J, in yellow) was aligned with itself after dynamic
simulation (in blue). (B) TCPTP (1L8K, in yellow) was aligned with itself after
dynamic simulation (in blue). (C) Molecular dynamic simulation of 10a bound with
TCPTP (1L8K, in black) and PTP1B (3A5J, in red).
The change of protein conformation reflects its stability in MD simulation. The
WPD loop is highly correlated with the catalytically active site of PTP1B and TCPTP.
The WPD loop of PTP1B (3A5J) has always been in open position before and after

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dynamic simulation (Fig 8A), in contrast, the position of loop of TCPTP (1L8K)
changes from open to half-close (Fig 8B). The root-mean-squared deviation (RMSD)
is widely used to assess the distance between two aligned objects. The backbone
RMSD value of PTP1B was lower than that of TCPTP, which suggested that the
PTP1B/10a was more stable than TCPTP/10a in 50 ns MD simulation (Fig 8C). All
the above observations suggested that the interaction between 10a and PTP1B was
more potent and stable than that between 10a and TCPTP. That may be the reason, at
least in part, why the compound 10a displayed significant selectivity for PTP1B over
TCPTP.
Figure 9. Cell uptake of compound 10a in C2C12 myotubes (10 µM exposure for 8
h). (A) DMSO treat group (control). (B) The internal standard (10 mM × 10 µL)
was added after cell was disrupted.
The permeability of inhibitor 10a was assayed in C2C12 myotubes by HPLC (Fig.
9). Cell was treated with DMSO (control group) or 10a (0.1 µmol) and subsequently
incubated for 8 h. The medium was then removed and cell was disrupted. An equal
amount of the internal standard[40] was added, and then the cell debris was removed
by filtration. The amount of compound 10a ingested into the cell was analyzed by
HPLC to obtain cell uptake. The result suggested that the proportion of compound
10a ingested into the cells was about 36.9%. Approximately 74.0 pmmol of
compound 10a could be absorbed per million cells, which was considered to be an
appropriate degree of cell uptake.[41]

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Figure 10. (A) Activation of insulin signaling during exposure of C2C12 myotubes to
10a. C2C12 myotubes were treated with 10a or 100 nM insulin. (B), (C) and (D)
Phosphorylation levels of IRS1, IRβ and Akt. Band density was quantified and
normalized with β-actin. The results shown are means ± SD (n = 3). (*P < 0.05, **P <
0.01 versus DMSO-treated group)
To determine the molecular mechanism underlying the effects of inhibitor 10a on
insulin signaling, the total and phosphorylated protein expression levels were
performed by western blotting. Protein band intensities were quantified by
densitometric analysis. The result showed that compound 10a (0.25, 0.5 and 1.0µM)
increased p-IRS1 expression in an insulin-independent manner (Fig. 10). Inhibitor
10a also significantly increased the relative abundances of p-IRβ and p-Akt compared
with that in the control group (DMSO-treated) without affecting the expression levels
of total IRβ and Akt. The results demonstrated that compound 10a activates insulin
signaling through up-regulating the phosphorylation level of IRS-1, IRβ and Akt.
Conclusion
In summary, the incorporation of bromophenol functional groups derived from
marine algae and saccharide by simple molecular hybridization led to the discovery of
some PTP1B inhibitors. Inhibitor 10a, which incorporated bromophenol and ribose,
emerged as the most potent PTP1B inhibitor in all targeted compounds with IC50
values of 199 nM and 32-fold selectivity over TCPTP. Molecular modeling and SPR
studies indicated that the phenolic hydroxyls in benzene rings played an important
role in the interaction of inhibitors and PTP1B. The MD studies revealed that the

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PTP1B/10a complex was more stable than TCPTP/10a, which was the potent proof
for observed 32-fold selectivity of 10a for PTP1B over TCPTP, although TCPTP and
PTP1B shared high degree of homology in catalytic site. Additionally, the moderate
permeability of inhibitor 10a was directly demonstrated by HPLC. Moreover, 10a
displayed cellular activity in an IR phosphorylation assay. These results provided
further evidence that bromophenol compounds incorporating saccharides can be
potent inhibitors of PTP1B. Further studies on these compounds in cell permeability,
selectivity and cellular activity would be necessary to provide PTP1B inhibitors
suitable for in vivo proof of animal studies in diabetes.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
41506169), and Key research and development project of Shandong province
(2015GSF115029, 2016ZDJS07A13), and the Project of Discovery, Evaluation and
Transformation of Active Natural Compounds, Strategic Biological Resources Service
Network Programme of Chinese Academy of Sciences (ZSTH-026), and the Scientific
and Technological Innovation Project Financially Supported by Qingdao National
Laboratory for Marine Science and Technology (No. 2015ASKJ02), and Aoshan
Talents Program Supported by Qingdao National Laboratory for Marine Science and
Technology (NO. 2015ASTP).
Notes
The authors declare no competing financial interest.
References:
[1] D.W.C.A. Plotnick. Type 1 Diabetes Mellitus in Pediatrics. Pediatr. Rev. 29 (2008), 374-385.
[2] Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 36 (2012), S67-S74.
[3] S. Yip, S. Saha, J. Chernoff. PTP1B: a double agent in metabolism and oncogenesis. Trends
Biochem Sci. 35 (2010), 442-449.
[4] K.A. Kenner, E. Anyanwu, J.M. Olefsky, J. Kusari. Protein-tyrosine Phosphatase 1B Is a Negative
Regulator of Insulin- and Insulin-like Growth Factor-I-stimulated Signaling. 271 (1996),
19810-19816.
[5] L.D. Klaman, O. Boss, O.D. Peroni, J.K. Kim, J.L. Martino, J.M. Zabolotny, N. Moghal, M.
Lubkin, Y.B. Kim, A.H. Sharpe, A. Stricker-Krongrad, G.I. Shulman, B.G. Neel, B.B. Kahn.
Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in
protein-tyrosine phosphatase 1B-deficient mice. Mol Cell Biol. 20 (2000), 5479-5489.
[6] M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A.L. Loy, D. Normandin, A.
Cheng, J. Himms-Hagen, C.C. Chan, C. Ramachandran, M.J. Gresser, M.L. Tremblay, B.P.
Kennedy. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine

ACCEPTED MANUSCRIPT
phosphatase-1B gene. Science. 283 (1999), 1544-1548.
[7] V.S. Murthy, V.M. Kulkarni. 3D-QSAR CoMFA and CoMSIA on protein tyrosine phosphatase
1B inhibitors. Bioorg Med Chem. 10 (2002), 2267-2282.
[8] R. Zhang, S. Chen, X. Zhang, R. Yu, S. Wan, M. Geng, T. Jiang. Synthesis and evaluation of
novel non-covalent binding quinazoline glycoside derivatives targeting the L858R and T790M
variants of EGFR. RSC Adv. 6 (2016), 36857-36862.
[9] S.L. McGovern, E. Caselli, N. Grigorieff, B.K. Shoichet. A Common Mechanism Underlying
Promiscuous Inhibitors from Virtual and High-Throughput Screening. J Med Chem. 45 (2002),
1712-1722.
[10] D. Barford, A.J. Flint, N.K. Tonks. Crystal structure of human protein tyrosine phosphatase 1B.
Science. 263 (1994), 1397-1404.
[11] K. Shen, Y.F. Keng, L. Wu, X.L. Guo, D.S. Lawrence, Z.Y. Zhang. Acquisition of a specific and
potent PTP1B inhibitor from a novel combinatorial library and screening procedure. J Biol Chem.
276 (2001), 47311-47319.
[12] S. Shrestha, B.R. Bhattarai, K.H. Lee, H. Cho. Mono- and disalicylic acid derivatives: PTP1B
inhibitors as potential anti-obesity drugs. Bioorg Med Chem. 15 (2007), 6535-6548.
[13] K.K. Amarasinghe, A.G. Evdokimov, K. Xu, C.M. Clark, M.B. Maier, A. Srivastava, A.O. Colson,
G.S. Gerwe, G.E. Stake, B.W. Howard, M.E. Pokross, J.L. Gray, K.G. Peters. Design and
synthesis of potent, non-peptidic inhibitors of HPTPbeta. Bioorg Med Chem Lett. 16 (2006),
4252-4256.
[14] K.E. You-Ten, E.S. Muise, A. Itie, E. Michaliszyn, J. Wagner, S. Jothy, W.S. Lapp, M.L.
Tremblay. Impaired bone marrow microenvironment and immune function in T cell protein
tyrosine phosphatase-deficient mice. J Exp Med. 186 (1997), 683-693.
[15] A.P. Combs, W. Zhu, M.L. Crawley, B. Glass, P. Polam, R.B. Sparks, D. Modi, A. Takvorian, E.
McLaughlin, E.W. Yue, Z. Wasserman, M. Bower, M. Wei, M. Rupar, P.J. Ala, B.M. Reid, D.
Ellis, L. Gonneville, T. Emm, N. Taylor, S. Yeleswaram, Y. Li, R. Wynn, T.C. Burn, G. Hollis,
P.C.C. Liu, B. Metcalf. Potent Benzimidazole Sulfonamide Protein Tyrosine Phosphatase 1B
Inhibitors Containing the Heterocyclic (S
Mimetic. J Med Chem. 49 (2006), 3774-3789.
)-Isothiazolidinone Phosphotyrosine
[16] B.G. Szczepankiewicz, G. Liu, P.J. Hajduk, C. Abad-Zapatero, Z. Pei, Z. Xin, T.H. Lubben, J.M.
Trevillyan, M.A. Stashko, S.J. Ballaron, H. Liang, F. Huang, C.W. Hutchins, S.W. Fesik, M.R.
Jirousek. Discovery of a Potent, Selective Protein Tyrosine Phosphatase 1B Inhibitor Using a
Linked-Fragment Strategy. J Am Chem Soc. 125 (2003), 4087-4096.
[17] V.M. Balaramnavar, R. Srivastava, N. Rahuja, S. Gupta, A.K. Rawat, S. Varshney, H. Chandasana,
Y.S. Chhonker, P.K. Doharey, S. Kumar, S. Gautam, S.P. Srivastava, R.S. Bhatta, J.K. Saxena,
A.N. Gaikwad, A.K. Srivastava, A.K. Saxena. Identification of novel PTP1B inhibitors by
pharmacophore based virtual screening, scaffold hopping and docking. Eur J Med Chem. 87
(2014), 578-594.
[18] T.N. Doman, S.L. McGovern, B.J. Witherbee, T.P. Kasten, R. Kurumbail, W.C. Stallings, D.T.
Connolly, B.K. Shoichet. Molecular Docking and High-Throughput Screening for Novel Inhibitors
of Protein Tyrosine Phosphatase-1B. J Med Chem. 45 (2002), 2213-2221.
[19] C. Dufresne, P. Roy, Z. Wang, E. Asante-Appiah, W. Cromlish, Y. Boie, F. Forghani, S.
Desmarais, Q. Wang, K. Skorey, D. Waddleton, C. Ramachandran, B.P. Kennedy, L. Xu, R.
Gordon, C.C. Chan, Y. Leblanc. The development of potent non-peptidic PTP-1B inhibitors.

ACCEPTED MANUSCRIPT
Bioorg Med Chem Lett. 14 (2004), 1039-1042.
[20] G. Liu, Z. Xin, Z. Pei, P.J. Hajduk, C. Abad-Zapatero, C.W. Hutchins, H. Zhao, T.H. Lubben, S.J.
Ballaron, D.L. Haasch, W. Kaszubska, C.M. Rondinone, J.M. Trevillyan, M.R. Jirousek. Fragment
screening and assembly: a highly efficient approach to a selective and cell active protein tyrosine
phosphatase 1B inhibitor. J Med Chem. 46 (2003), 4232-4235.
[21] B. Douty, B. Wayland, P.J. Ala, M.J. Bower, J. Pruitt, L. Bostrom, M. Wei, R. Klabe, L.
Gonneville, R. Wynn, T.C. Burn, P.C. Liu, A.P. Combs, E.W. Yue. Isothiazolidinone inhibitors of
PTP1B containing imidazoles and imidazolines. Bioorg Med Chem Lett. 18 (2008), 66-71.
[22] R.B. Sparks, P. Polam, W. Zhu, M.L. Crawley, A. Takvorian, E. McLaughlin, M. Wei, P.J. Ala, L.
Gonneville, N. Taylor, Y. Li, R. Wynn, T.C. Burn, P.C. Liu, A.P. Combs. Benzothiazole
benzimidazole (S)-isothiazolidinone derivatives as protein tyrosine phosphatase-1B inhibitors.
Bioorg Med Chem Lett. 17 (2007), 736-740.
[23] C. Seo, J.H. Sohn, H. Oh, B.Y. Kim, J.S. Ahn. Isolation of the protein tyrosine phosphatase 1B
inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060. Bioorg Med
Chem Lett. 19 (2009), 6095-6097.
[24] X. Fan, N.J. Xu, J.G. Shi. Bromophenols from the Red AlgaRhodomelaconfervoides. J Nat Prod.
66 (2003), 455-458.
[25] R.R. Baumgartner, D. Steinmann, E.H. Heiss, A.G. Atanasov, M. Ganzera, H. Stuppner, V.M.
Dirsch. Bioactivity-Guided Isolation of 1,2,3,4,6-Penta-O
-galloyl-d
-glucopyranose fromPaeonia lactiflora
73 (2010), 1578-1581.
Roots As a PTP1B Inhibitor. J Nat Prod.
[26] D. Shi, J. Li, B. Jiang, S. Guo, H. Su, T. Wang. Bromophenols as inhibitors of protein tyrosine
phosphatase 1B with antidiabetic properties. Bioorg Med Chem Lett. 22 (2012), 2827-2832.
[27] B. Jiang, D. Shi, Y. Cui, S. Guo. Design, synthesis, and biological evaluation of bromophenol
derivatives as protein tyrosine phosphatase 1B inhibitors. Arch Pharm (Weinheim). 345 (2012),
444-453.
[28] B. Jiang, S. Guo, D. Shi, C. Guo, T. Wang. Discovery of novel bromophenol
3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol as protein
tyrosine phosphatase 1B inhibitor and its anti-diabetic properties in C57BL/KsJ-db/db mice. Eur J
Med Chem. 64 (2013), 129-136.
[29] D. Shi, S. Guo, B. Jiang, C. Guo, T. Wang, L. Zhang, J. Li. HPN, a Synthetic Analogue of
Bromophenol from Red Alga Rhodomela confervoides: Synthesis and Anti-Diabetic Effects in
C57BL/KsJ-db/db Mice. Marine Drugs. 11 (2013), 350-362.
[30] N. Wu, J. Luo, B. Jiang, L. Wang, S. Wang, C. Wang, C. Fu, J. Li, D. Shi. Marine Bromophenol
Bis (2,3-Dibromo-4,5-dihydroxy-phenyl)-methane Inhibits the Proliferation, Migration, and
Invasion of Hepatocellular Carcinoma Cells via Modulating β1-Integrin/FAK Signaling. Marine
Drugs. 13 (2015), 1010-1025.
[31] Y. Cui, D. Shi, Z. Hu. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two
new synthetic bromophenols and their methoxy derivatives. Chinese Journal of Oceanology and
Limnology. 29 (2011), 1237-1242.
[32] M.T. Engström, M. Karonen, J.R. Ahern, N. Baert, B. Payré, H. Hoste, J.P. Salminen. Chemical
Structures of Plant Hydrolyzable Tannins Reveal Their in Vitro Activity against Egg Hatching and
Motility ofHaemonchus contortus
840-851.
Nematodes. J Agr Food Chem. 64 (2016),

ACCEPTED MANUSCRIPT
[33] C. Xu, Y. Yagiz, W. Hsu, A. Simonne, J. Lu, M.R. Marshall. Antioxidant, Antibacterial, and
Antibiofilm Properties of Polyphenols from Muscadine Grape (Vitis rotundifolia
Michx.) Pomace against Selected Foodborne Pathogens. J Agr Food Chem. 62 (2014), 6640-6649.
[34] C. Huang, S. Lee, C. Lin, T. Tu, L.H. Chen, Y.J. Chen, H. Huang. Co-treatment with Quercetin
and 1,2,3,4,6-Penta-O
-galloyl-β-d
-glucose Causes Cell Cycle
Arrest and Apoptosis in Human Breast Cancer MDA-MB-231 and AU565 Cells. J Agr Food
Chem. 61 (2013), 6430-6445.
[35] V.C. Lin, P. Kuo, Y. Lin, Y. Chen, Y. Hseu, H. Yang, J. Kao, C. Ho, T. Way. Penta-O
-galloyl-β-d
-glucose Suppresses EGF-Induced eIF3i Expression through
Inhibition of the PI3K/AKT/mTOR Pathway in Prostate Cancer Cells. J Agr Food Chem. 62
(2014), 8990-8996.
[36] L.T.M. Ngan, J. Moon, T. Shibamoto, Y. Ahn. Growth-Inhibiting, Bactericidal, and Urease
Inhibitory Effects of Paeonia lactiflora Root Constituents and Related Compounds on
Antibiotic-Susceptible and -Resistant Strains of Helicobacter pylori. J Agr Food Chem. 60 (2012),
9062-9073.
[37] Z. Xin, G. Liu, C. Abad-Zapatero, Z. Pei, B.G. Szczepankiewicz, X. Li, T. Zhang, C.W. Hutchins,
P.J. Hajduk, S.J. Ballaron, M.A. Stashko, T.H. Lubben, J.M. Trevillyan, M.R. Jirousek.
Identification of a monoacid-based, cell permeable, selective inhibitor of protein tyrosine
phosphatase 1B. Bioorg Med Chem Lett. 13 (2003), 3947-3950.
[38] Y. Wei, Y. Chen, L. Shi, L. Gao, S. Liu, Y. Cui, W. Zhang, Q. Shen, J. Li, F. Nan. Discovery and
structural modification of novel inhibitors of PTP1B inspired by the ACT fragment of
scleritodermin A. MedChemComm. 2 (2011), 1104.
[39] L.F. Iversen. Structure Determination of T Cell Protein-tyrosine Phosphatase. J Biol Chem. 277
(2002), 19982-19990.
[40] The structure of internal standard was showed in supporting information.
[41] M. Patra, T.C. Johnstone, K. Suntharalingam, S.J. Lippard. A Potent Glucose-Platinum Conjugate
Exploits Glucose Transporters and Preferentially Accumulates in Cancer Cells. Angew Chem Int
Ed Engl. 55 (2016), 2550-2554.

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ò the hybrid of bromophenol and saccharide was prepared
ò compound 10a exhibited remarkable selectivity for PTP1B over TCPTP
ò molecule docking and molecular dynamics studies reveal the reason of selectivity
ò the cell permeability and cellular activity of compound 10a were demonstrated