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Introduction
Protein tyrosine phosphatase 1B (PTP1B) is a member of
the family of protein tyrosine phosphatase (PTPase) and is
further classified as a non-receptor PTPase for its location to
the cytoplasmic face of the endoplasmic reticulum through
the C-terminal 35 residues. Considerable progress has been
made recently in the understanding of the relationship
between PTP1B and type 2 diabetes. In
vitro, PTP1B associates with tyrosine residues 1162 and 1163 of the insulin
receptor (IR)[1,2]. Many other studies have shown that PTP1B
can directly interact with the activated IR or IR substrate-1
(IRS-1) to dephosphorylate phosphotyrosine residues,
resulting in the downregulation of insulin
action[1_4]. Compelling data also came from PTP1B knockout mice, which
displayed increased insulin sensitivity in a tissue-specific
manner[5,6]. Enhanced tyrosine phosphorylation of the IR was
observed in the muscle and liver, suggesting that the
receptor might be a direct substrate of
PTP1B[5].
The increased expression of PTP1B in the adipose tissue
and muscle of obese humans and rodents is thought to be
related to insulin resistance[7], whereas the increased insulin
sensitivity from weight loss is accompanied by reduced
PTP1B activity[8].
Further evidence for the involvement of PTP1B in
insulin resistance was provided by cell line studies. PTP1B
overexpression in rat primary adipose tissues and 3T3/L1
adipocytes has been shown to decrease
insulin-sensitive Glut4
translocation[9] and IR and IRS-1
phosphorylation[10], respectively.
PTP1B has been widely recognized as an attractive
target for therapy of type 2 diabetes. The development of
PTP1B inhibitors has become a promising way to treat type
2 diabetes, even though it has been demonstrated that it is
not easy to find a selective, safe, and effective PTP1B
inhibitor[11].
We developed a molecular level, high-throughput
screening assay for PTP1B inhibitors. After the screening of our
compound library, we identified LGH00081 as a novel
inhibitor of PTP1B.
Materials and methods
Materials and instruments The plasmid pGEX-KG was
a kind gift from Dr Kun-liang GUAN of the University of
Michigan (Ann Arbor, MI, USA). The restriction enzymes
and Ex Taq polymerase were from TaKaRa (Dalian, China).
Escherichia coli strain BL21-CodonPlus (DE3) was
purchased from Stratagene (La Jolla, CA, USA). GSTrap FF was
obtained from Amersham Pharmacia Biotech (Uppsala,
Sweden). Substrate p-nitrophenyl phosphate
(pNPP) was from Calbiochem (San Diego, CA, USA) and
3-O-methylfluorescein phosphate (OMFP) was from Sigma
Aldrich (St Louis, MO, USA). Other reagents and solvents
used in experiments were of analytical grade. Ham's F12
medium (F12) was from Invitrogen (Carlsbad, CA, USA).
Fetal bovine serum (FBS) was purchased from Hyclone
(Logan, UT, USA). pY20 and anti-IR (IRβ) were from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin,
horseradish peroxidase(HRP)-linked antimouse
immunoglobulin (IgG) and antirabbit IgG antibodies were from Cell
Signal Technology (Danvers, MA, USA). The polyvinylidene
fluoride (PVDF) membranes were from Millipore (Billerica,
MA, USA) and the enhanced chemiluminescence (ECL)
reagents were from Calbiochem (USA).
Construction, expression, and purification of the PTP1B
catalytic domain The nucleotide fragment encoding the
human PTP1B catalytic domain was amplified by RT-PCR
using RNA from human placenta. The cDNA of the PTP1B
catalytic domain (91_1053 according to gi190741) was cloned
into the pGEX-KG expression vector with EcoR
I/Sac I. The nucleotide sequence cloned into the recombinant
plasmid was confirmed by DNA sequencing. The recombinant
plasmid was then transformed into
Escherichia coli BL21-CodonPlus (DE3) for expression.
BL21-CodonPlus (DE3) cells containing the recombinant plasmid were grown in 1 L
Luria-Bertani (LB) medium in the presence of ampicillin (100
mg/L) by shaking at 37 °C, and the protein expression was
induced by adding isopropyl β-D-thiogalac-topyranoside
(IPTG) to 500 nmol/L when the cell density reached optical
density at 600 nm (OD600) of 0.4_0.6. The cells were
harvested after 4 h by centrifugation for 2 min at
7000×g and washed twice with
phosphate-buffered saline (PBS) buffer (140 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L
Na2HPO4, and 1.8 mmol/L
KH2PO4), and resuspended in 30 mL PBS
including 0.1% Triton X-100, 1 mmol/L EDTA, and 2 mmol/L
dithiothreitol (DTT). The cells were lysated by sonication
for 3 min on ice. After centrifugation at 12
000×g for 15 min, the supernatant was loaded onto a GSTrap FF column
equilibrated previously with PBS buffer with 1 mmol/L EDTA and
2 mmol/L DTT. The loaded column was washed with PBS
buffer, and then the bound active protein was eluted with
elution buffer (50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L
glutathione, 1 mmol/L EDTA, and 2 mmol/L DTT). The
protein samples from the purification procedure were analyzed
by 10% reducing SDS-PAGE gel and the protein
concentration was determined by the Bradford method with bovine
serum albumin (BSA).
PTP1B enzymatic assay The enzymatic activities of the
PTP1B catalytic domain were determined at 30 °C by
monitoring the hydrolysis of pNPP. Dephosphorylation of
pNPP generates product pNP, which was monitored at an
absorbance of 405 nm by the EnVision multilabel plate reader
(PerkinElmer Life Sciences, Boston, MA, USA). In a typical
100 µL assay mixture containing 50 mmol/L
3-[N-morpholino] propane-sulfonic acid (MOPs), pH 6.5, 2 mmol/L
pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously
monitored and the initial rate of the hydrolysis was
determined using the early linear region of the enzymatic reaction
kinetic curve.
PTP1B inhibitor screening High-throughput
screening (HTS) optimization and validation were undertaken
according to our standard operation process before the
primary screening. A total of 48 000 pure chemicals collected
from different sources with wide structural diversity were
screened. Two microliters of stock solution of compounds
in DMSO was transferred into the wells of a 96-well
flat-bottom plate (Greiner, Frickenhausen, Germany);
this yielded a final compound concentration of 2 µg/mL
and 2% DMSO. After initializing the enzymatic reaction, the plate was then
read every 20 s for 2 min in the EnVision multilabel plate
reader at 405 nm. All screening operations were
performed by SAGIAN core integrated robotic system (Beckman
Coulter, Fullerton, CA, USA). We used
Na3VO4 as the positive control and DMSO as the negative control to evaluate
our HTS system. The slope of the linear portion of the
kinetic curve generated from each well was used to determine
the activity of PTP1B. Both the negative and positive
controls were set in each plate for calculating. The inhibition
effect of the compound was represented by the percentage
of the slope of the linear portion of its kinetic curve relative
to that of the negative control. Quality control parameters
involving coefficient of variation (CV) and Z´ factor were
calculated in real time[12].
For the 50% percentage inhibition concentration
(IC50) caculation, inhibition assays were performed with 30 nmol/L
recombinant enzyme, 2 mmol/L pNPP in 50 mmol/L MOPS at
pH 6.5, and the inhibitors diluted around the estimated
IC50 values. The IC50 was calculated with Prism 4 software
(Graphpad, San Diego, CA, USA) from the non-linear curve
fitting of the percentage of inhibition (% inhibition) versus
the inhibitor concentration [I] by using the following
equation: % Inhibition =
100/(1+[IC50/[I]]k), where
k is the Hill coefficient.
Characterization of the PTP1B inhibitor identified from
the screening To characterize the hit identified from the
HTS, the assay was carried out in a 100 µL system
containing 50 mmol/L MOPS, pH 6.5, 30 nmol/L PTP1B,
pNPP in 2-fold dilution from 80 mmol/L, and different concentrations
of the inhibitor. In the presence of the competitive inhibitor,
the Michaelis-Menten equation is described as
1/v=(Km/[Vmax
[S]])(1+[I]/Ki)+1/Vmax
, where v is the initial rate,
Vmax is the maximum rate, and [S] is the substrate concentration.
The Ki value was obtained by the linear replot of apparent
Km/Vmax (slope) from the primary reciprocal plot versus the
inhibitor concentration [I] according to the equation
Km/Vmax=1+[I]/
Ki.
Molecular docking The docking was completed by
the Discovery Studio program (Accelrys, San Diego, CA,
USA). The crystal structure of PTP1B (Protein Data Bank
code 2hb1) was defined as the receptor, and the active site
of PTP1B was regarded as the binding site of our
competitive inhibitor LGH00081. LGH00081 was docked in a
flexible manner, and conformations are determined by the
stochastic Monte Carlo (MC) conformation search method.
The number of MC trials was fixed to 50 000.
Selectivity of LGH00081 on other PTPase family
members We evaluated the inhibitory effects of LGH00081 on
other PTPase family members, T-cell PTPase (TCPTP), Src
homology domain 2 (SH2) _containing tyrosine
phosphatase-1 (SHP1), SHP2, leukocyte antigen-related
phosphatase D1 (LAR D1), protein tyrosine phosphatase a (PTPa)
and vaccinia virus VH-1-related dual-specific protein
phosphatase (VHR). TCPTP (41_1075 according to BC008244),
SHP1 (244_570 according to BC002523), SHP2 (1116_2162
according to NM002834.3), LAR D1 (1275_1613 according
to gi18860871), PTPα D1 (697_1707 according to gi20073056),
and VHR (56_613 according to BC002682) cDNA were cloned
into pGEX-KG. GST-PTPase were overexpressed as
GST-fusion proteins in Escherichia coli
BL21-CondenPlus (DE3) and purified by affinity chromatography. Assays were
performed for the majority of PTPase using 2 mmol/L
pNPP as the substrate around their
Km value (TCPTP: 1.12 mmol/L, SHP1: 11.76 mmol/L, SHP2: 7.82 mmol/L, LAR
D1: 0.87 mmol/L, and PTPα D1: 1.25 mmol/L) and for
VHR, which is insensitive to pNPP, using 2.5 µmol/L OMFP
as the substrate with a Km value of 20 µmol/L at their optimal
pH, respectively.
Cell culture The Chinese hamster ovary (CHO) cell line
transfected with an expression plasmid-encoding human IR
(CHO/hIR) was a kind gift from Dr Michel TREMBLAY of
McGill University (Montreal, Canada). The cells were grown
in F12 medium supplemented with 10% (v/v) FBS, 2
mmol/L L-glutamine, 50 units/mL penicillin and 50 µg/mL
streptomycin.
Western blotting The cells were rinsed twice with PBS,
terminated immediately by liquid nitrogen, then lysed with
1× SDS loading buffer. The samples were electrophoresed
on 10% SDS-polyacrylamide gels and transferred to PVDF
membranes. The membranes were blocked for 1 h with 5%
(w/v) BSA and incubated with the primary antibodies
overnight at 4 ºC and the secondary antibodies for 1 h at room
temperature. Antigen-antibody complexes were detected
by the ECL kit.
Results
HTS and quality control To find novel, small molecular
inhibitors, we developed a HTS with PTP1B. The screening
assay included 30 nmol/L GST-fusion PTP1B protein and 2
mmol/L pNPP substrate in order to obtain a good
signal-to-noise ratio. After optimization, the intraplate and interplate
CV were both less than 10%, suggesting that the liquid
handling and compound transfer procedure was precise, and
the Z´ factor, a standard statistical measure of assay quality,
reached 0.69. A total of 48 000 compounds were screened in
automated format in 1 d. The assay performed well with an
averaged Z´ factor of 0.63; fifty compounds with an
inhibition rate higher than 50% at a final concentration of 2 µg/mL
were identified.
Discovery of a novel inhibitor of PTP1B from HTS
After the hit validation and dose-response curve determination,
compound LGH00081 with a novel structure (Figure 1A,
chemical name:
5-(3-((Z)-((Z)-2-(4-chlorophenylimio)-4-oxothiazolidin-5-ylidene)
methyl)-2,5-dimethyl-1H-pyrrol-1-yl) isophthalic acid) was discovered to potently inhibit
PTP1B with an IC50 of 1.6 μmol/L (Figure 1B).
Characterization of compound LGH00081 LGH00081
demonstrated a time-independent inhibition of PTP1B (Figure
2A). We further determined the inhibition modality of
LGH00081 for PTP1B; it inhibited PTP1B with the
characteristics typical of a competitive inhibitor, as indicated by
increased Km values and constant
kcat values when the inhibitor concentration was increased (Figure 2B). Meanwhile,
the result of the Lineweaver-Burk plot confirmed LGH00081
as a competitive inhibitor of PTP1B for intersecting at
the y-axis of a nest of lines with increased inhibitor
concentration (Figure 2C). The
Ki value was calculated as 309
nmol/L from Figure 2D.
Interaction mode of LGH00081 and PTP1B by docking
analysis As the docking result show (Figure 3), the
compound could enter the catalytic pocket of PTP1B flexibly
(Figure 3A) with the highest dock score of 64.724, and the
predicted hydrogen bonds of LGH00081 were formed with
Tyr46, Lys 120, and Gly220, respectively (Figure 3B).
Inhibitory effect of LGH00081 on other PTPase family
members To test the selectivity of the compound on other
PTPase family members, we prepared human TCPTP, SHP1,
SHP2, CD45D1D2, VHR, LAR D1, and PTPαD1 (Figure 4)
and determined the IC50 values for these enzymes. The
results are shown in Table 1. LGH00081 showed better
inhibition on PTP1B, TCPTP, SHP1, and SHP2 than VHR, with no
visible inhibitory effects to both receptor-type PTPase LAR
and PTPα.
LGH00081 increased the IR tyrosine phosphorylation
level in CHO/hIR cells To determine whether LGH00081
could exert its effect in the cell level to activate the insulin
signaling pathway, we tested the enhanced effect of
LGH00081 on IR phosphorylation in CHO/hIR cells by
Western blotting. As shown in Figure 5, LGH00081 could
synergistically increase the insulin_induced tyrosine
phosphorylation level of IRβ in a dose-dependent manner and reached
maximal effect at 10 µmol/L.
Discussion
Data from previous studies support the concept that PTP1B
is a key negative regulator of insulin signal
transduction. The termination of insulin signaling requires the
dephosphorylation of IRβ, and its downstream effector is involved in insulin
signal termination, thus the overactivation or
overexpression of PTP1B can attenuate the insulin signal, resulting in
insulin resistance[13,14]. Therefore reducing the activity of PTP1B,
which dephosphorylates IRβ, would be expected to increase
insulin sensitivity[15_18]. Consequently, compounds that
inhibit PTP1B may have potential therapeutic use for the
treatment of type 2 diabetes and obesity.
Since being developed approximately 20 years ago, HTS
has become key technique used in drug discovery. HTS
provides a fast, effective, and convenient tool to test
hundreds of thousands of structural diverse compounds for their
ability to modulate disease-relevant targets, and identifies
novel compounds with interesting biological activities, which
yields molecules that could be optimized into drugs. HTS
assay for PTP1B was established to discover small-molecule
inhibitors. The average Z´ factor is 0.63, which fulfills the
demand for HTS quality control.
Through large-scale screening, several hits with good
activity and novel structures were discovered. Here,
compound LGH00081 was reported for the first time as a PTP1B
inhibitor. LGH00081 shows an inhibition manner
independent of incubation time, which may indicate that it is a
fast-binding inhibitor to PTP1B. We demonstrated that it was a
typical competitive inhibitor to PTP1B and the result was
confirmed by the docking analysis in which LGH00081 was
shown to enter into the catalytic pocket of PTP1B and
behave as a competitor to the substrate.
Selectivity may be the biggest problem in the
development of PTPase inhibitors. We measured the inhibitory
effect of LGH00081 on different types of PTPase. We found
that it inhibited non-receptor-type PTPase (such as TCPTP,
SHP1, and SHP2 with an IC50 value around 2 µmol/L). For the
dual-specific PTP VHR, LGH00081 showed similar inhibition,
with an IC50 value of 5.77 µmol/L.
No visible inhibitory effects on both receptor-type
PTPase LAR and PTPα were found even at the highest concentration of 40 µmol/L in the
test. Its effects on PTPase should be studied further to
allow the modification on this scaffold to produce more
potent and more selective inhibitors of individual
PTPase.
The critical negative regulatory step in insulin signal
transduction is the dephosphorylation of signaling molecules
by PTPase, such as PTP1B, which helps to terminate insulin
signaling. Changes in the expression levels or activities of
specific PTPase have been reported to influence the insulin
pathway and implicate insulin sensitivity, which is the most
common inherent pathology of type 2 diabetes mellitus and
obesity. IR phosphorylation elevation is a widely used
experiment to validate PTP1B inhibitor bioactivity at the
cellular level. Thus, the activity of LGH00081 was investigated in
CHO/hIR cells. LGH00081 could elevate the tyrosine
phosphorylation level of IRβ after stimulation by 10 nmol/L insulin.
In summary, using well-controlled HTS techniques, we
discovered a PTP1B inhibitor with a novel structure, named
LGH00081. We identified it as a competitive PTP1B inhibitor,
and its interaction mode with PTP1B was predicted by
docking analysis. Furthermore, it enhanced the tyrosine
phosphorylation level of IR in CHO/hIR cells. The ongoing
bioactivity-guided structure modification may lead to the
discovery of more potent and selective PTPase inhibitors.
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