A. Ju et al.
Bull. Chem. Soc. Jpn. Vol. 87, No. 3 (2014)
421
for 3 to 16 h. Cells were harvested and then lysed by sonication
in 50 mM Tris-HCl (pH 8), 300 mM NaCl, 1% Tergitol-
type NP-40, 1 mM phenylmethanesulfonyl fluoride (PMSF).
The lysates were clarified at 11000 rpm for 30 min at 4 °C. The
supernatants were applied by gravity flow to a column of Ni-
NTA resin (PEPTRON, Daejon, Korea). The resin was washed
with 20 mM Tris-HCl (pH 8), 500 mM NaCl, 50 mM imidazole
and eluted with 20 mM Tris-HCl (pH 8), 500 mM NaCl, 200-
300 mM imidazole. The eluted proteins were dialyzed overnight
against 20 mM Tris-HCl (pH 8), 100 mM NaCl, 30% glycerol,
0.5 mM PMSF before storage at ¹80 °C.
In Vitro Phosphatase Assays and Kinetic Analysis.
Activities of PTPs were measured using the substrate 3-O-
methylfluorescein phosphate (OMFP; Sigma-Aldrich) in a 96-
well microtiter plate assay. MLS-001 and OMFP were solu-
bilized in DMSO. All reactions were performed at a final
concentration of 1% DMSO. The final incubation mixtures
(100 ¯L) were optimized for enzyme activity and composed
of 30 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM ethylenedi-
aminetetraacetic acid (EDTA), 0.33% bovine serum albumin
(BSA), and 100 nM of PTP. Reactions were initiated by
addition of 100 ¯M OMFP and incubated for 30 min at 37 °C.
Fluorescence emission from product was measured with a
multiwell plate reader (Synergy H1; excitation at 485 nm;
emission at 535 nm). The reaction was linear over the time
period of the experiment and was directly proportional to both
enzyme and substrate concentrations. Half-maximal inhibition
constant (IC50) was defined as the concentration of an inhibitor
that caused a 50% decrease in the PTP activity. Half-maximal
inhibition constants and best curve fit for Lineweaver-Burk
plots were determined by using the curve fitting program Prism
3.0 (GraphPad Software). All experiments were performed in
triplicate and were repeated at least three times.
Effects of MLS-001 on ERK Phosphorylation Mediated
by SHP-2 in Cells. Transfected HEK 293 cells were treated
with MLS-001 (0 and 5 ¯M) for 3 h. Cells were lysed in lysis
buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
1% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF,
1 mM Na3VO4, and 1 mM NaF. The samples were separated by
SDS-PAGE, followed by immunoblotting analysis.
Immunoblotting Analysis. Samples were run in SDS-12%
polyacrylamide gels and transferred to a nitrocellulose mem-
brane. The membrane was blocked in 5% nonfat skim milk and
incubated with an appropriate antibody, followed by incubation
with secondary antibody conjugated to horseradish peroxidase.
The immunoreactive bands were visualized using an ECL sys-
tem (Pierce, Rockford, IL).
Results and Discussion
Owing to its role in human diseases such as neurodegen-
eration, cancer, and diabetes,17 SHP-2 is a target for drug
development.18 Among the several potent SHP-2 inhibitors that
have been developed so far,19 NSC-87877 is one of the most
well-known, and inhibits SHP-2 phosphatase activity with
strong potency.20 However, NSC-87877 lacks inhibition speci-
ficity as it also inhibits other phosphatases.21-26 Therefore, it
is necessary to discover novel SHP-2-specific inhibitors. In an
effort to identify a novel SHP-2-specific inhibitor, we tested
many organic compounds for SHP-2 inhibition by in vitro
phosphatase assays using OMFP as a substrate. Of them, MLS-
001 (Figure 1) inhibited SHP-2 phosphatase activity in vitro.
An inhibition curve was plotted for SHP-2 and SHP-2 was
inhibited with an IC50 value of 3.54 « 0.61 ¯M (Figure 2A).
The PTP inhibitory activity of MLS-001 was then assessed
against several other PTPs (ACP1, CDC25A, DUSP3,
DUSP14, DUSP18, DUSP22, DUSP23, DUSP26, TCPTP,
SHP-1, and SSH3) that belong to different subclasses of PTPs
using in vitro phosphatase assays. Recombinant human PTPs
were overexpressed in bacteria as 6 © His-tagged proteins and
purified by nickel-chelate column chromatography as previ-
ously described.27 As shown in Table 1 and Figure S2, MLS-
001 did not show any inhibitory effects on other PTPs. Among
those, SHP-1 and TCPTP that are closely related to SHP-2 were
not also inhibited by MLS-001. Therefore, the results suggest
that MLS-001 selectively inhibits SHP-2.
Inhibition Study. The inhibition constant (Ki) to SHP-2
for the inhibitor was determined through Michaelis-Menten
enzyme analysis by measuring the initial rates at several OMFP
concentrations for each fixed concentration of the inhibitor. The
data were fitted to the following equation to obtain the inhibi-
tion constant of competitive inhibitors. The slopes obtained
were replotted against the inhibitor concentrations. The Ki
value was obtained from the slopes of these plots.
1=V ¼ Kmð1 þ ½Iꢀ=KiÞ=Vmax½Sꢀ þ 1=Vmax
ð1Þ
To study the binding mechanism of SHP-2 and MLS-001,
kinetic studies based on the Michaelis-Menten equation were
performed with SHP-2 and MLS-001. Kinetic studies using
SHP-2 and MLS-001 revealed a competitive inhibition, thereby
suggesting that MLS-001 binds to the catalytic cleft of SHP-2
(Figure 2B). The Km value of SHP-2 phosphatase for OMFP
was 179 « 43 ¯M and the Lineweaver-Burk plot shows that
the Ki was 5.4 « 1.6 ¯M.
Synthesis of MLS-001. MLS-001 was synthesized by
means of the synthesis pathways shown in S1 of Supporting
Information.
Effects of MLS-001 on SHP-2 Phosphatase Activity in
Cells. HEK 293 cells were transfected with FLAG-SHP-2
phosphatase expression plasmid. After 48 h of transfection,
cells were pretreated with MLS-001 (20 ¯M) for 3 h. Cells were
washed twice with phosphate buffered saline (PBS) buffer and
lysed in PTP lysis buffer (0.5% NP-40, 0.5% Triton X-100,
150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1%
N
N
S
N
¹1
glycerol, 1 mM PMSF, and 1 ¯g mL aprotinin) for 30 min at
N
4 °C. Cleared cell lysates from centrifugation were mixed with
FLAG M2-agarose (Sigma-Aldrich, St.Louis, MO) and incu-
bated for 1 h at 4 °C using a rotation device. After incubation,
FLAG M2-agarose was washed three times with PTP lysis
buffer and their phosphatase activities were measured.
N
N
Figure 1. Chemical structure of MLS-001.
Ju, Anna
