Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

Article abstract of DOI:10.1002/cbic.201100414

Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic substrate enhancement is described targeting the secondary binding sites of PTPs. By screening four different PTPs from bacterial (MptpA) and human origin (PTP1B, HePtp, Shp2) with this assay, specific fragments were identified. One highly specific fragment that binds to the secondary site of Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) was characterized in order to validate the assay concept. Finally by covalently linking the secondary fragment to a phosphotyrosine mimetic, a moderately active but highly specific inhibitor of MptpA was obtained.

Full text of DOI:10.1002/cbic.201100414

DOI: 10.1002/cbic.201100414
Dynamic Substrate Enhancement for the Identification of
Specific, Second-Site-Binding Fragments Targeting a Set of
Protein Tyrosine Phosphatases
Marco F. Schmidt,[b, c, e] Matthew R. Groves,[d] and Jçrg Rademann*[a, b]
Protein tyrosine phosphatases (PTPs) are key regulators in
living systems and thus are attractive drug targets. The devel-
opment of potent, selective PTP inhibitors has been a difficult
challenge mainly due to the high homology of the phospho-
tyrosine substrate pockets. Here, a strategy of dynamic sub-
strate enhancement is described targeting the secondary bind-
ing sites of PTPs. By screening four different PTPs from bacteri-
al (MptpA) and human origin (PTP1B, HePtp, Shp2) with this
assay, specific fragments were identified. One highly specific
fragment that binds to the secondary site of Mycobacterium
tuberculosis protein tyrosine phosphatase A (MptpA) was char-
acterized in order to validate the assay concept. Finally by co-
valently linking the secondary fragment to a phosphotyrosine
mimetic, a moderately active but highly specific inhibitor of
MptpA was obtained.
Introduction
Protein tyrosine phosphorylation plays a central role in living
systems as the dominant on/off switch of protein function. Ty-
rosine phosphorylation is both dynamic and tightly regulated,
controlled by the opposing actions of protein tyrosine kinases
(PTKs) and protein tyrosine phosphatases (PTPs).[1] Several PTKs
and PTPs have been identified as attractive therapeutic targets
in human diseases including autoimmunity, obesity, diabetes,
and cancer.[2,3] Potent PTK inhibitors have been developed and
are used successfully in the clinic.[4] On the other hand, the de-
velopment of potent, selective PTP inhibitors remains a difficult
task mainly due to the high homology of the catalytic domains
of PTPs.[5–7] In addition, the active sites of PTPs are smaller than
those of PTKs and highly charged, thus posing additional chal-
lenges for the development of potent inhibitors with good
pharmacokinetic properties. Recently, Knapp’s group compared
the crystal structures of 22 human PTPs and concluded that
protein pockets next to the active sites are less homologous
among PTPs.[8] Exploiting these “secondary binding sites”
might offer new opportunities to identify specific PTP inhibi-
tors.
This method employs a generic PTP substrate carrying an alde-
hyde functionality that enables dynamic, template-assisted li-
gation with nucleophiles. With this setup fragments recognized
specifically by the secondary binding site of the enzyme
should be identified by a significantly enhanced turnover of
the generic substrate.
This novel methodology builds on the recently established
concept of dynamic ligation screening (DLS). DLS has been
used to identify fragments in a high-throughput assay either
through enhancing the inhibition of a fluorogenic substrate,[11]
or enhancing binding as detected in an anisotropy (fluores-
cence-polarization) assay.[12] In addition, dynamic substrate en-
hancement, as demonstrated here, supports the recent finding
of Ellman’s group that screening substrate libraries can be
useful for the development of improved enzyme inhibitors.[13]
[a] Prof. Dr. J. Rademann
Classically, small molecular entities that bind to secondary
binding sites were identified by biophysical fragment screen-
ing.[9] With either NMR spectroscopy or X-ray crystallography
for detection, this approach is capable of identifying low-affini-
ty fragments that bind to a defined site. It does not, however,
provide sufficient information on how to convert low-affinity
hit fragments into specific inhibitors. Moreover, the biophysical
methods require expensive equipment and are operated with
low throughput.[10]
Medicinal Chemistry, Institute of Pharmacy, Leipzig University
Brꢀderstrasse 34, 04103 Leipzig (Germany)
E-mail: rademann@uni-leipzig.de
[b] Dr. M. F. Schmidt, Prof. Dr. J. Rademann
Leibniz-Institut fꢀr Molekulare Pharmakologie (FMP)
Robert-Rçssle-Strasse 10, 13125 Berlin (Germany)
[c] Dr. M. F. Schmidt
Institute of Chemistry and Biochemistry, Freie Universitꢁt Berlin
Takustrasse 3, 14195 Berlin (Germany)
[d] Dr. M. R. Groves
European Molecular Biology Laboratory (EMBL)
Notkestrasse 85, 22603 Hamburg (Germany)
In this contribution, a concept is established that exploits
the screening of enzyme substrates generated by dynamic
fragment ligation for the development of second-site-selective
inhibitors. We propose the term “dynamic substrate enhance-
ment” for this method in order to describe its characteristics.
[e] Dr. M. F. Schmidt
Current address:
University Chemical Laboratory, University of Cambridge
Lensfield Road, Cambridge, CB2 1EW (UK)
2640
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Identification of PTP-Targeting Fragments
acts as a negative regulator of insulin and is therefore an at-
tractive target for the treatment of type II diabetes and obesi-
ty.[16] HePTP (PTPN7) has been shown to be over-expressed in
some patients with acute myeloblastic leukemia, thus suggest-
ing a link between increased HePTP activity and this disease.[17]
Finally, activating mutations in Shp2 (PTPN11) cause Noonan
syndrome and have been identified in human cancer. In addi-
tion, Shp2 is under consideration as a drug target against
cancer-cell metastasis.[18]
Results and Discussion
A generic PTP substrate, 4-(formyl)phenylphosphate (1), which
contains an aldehyde group, was investigated as a dynamic
ligation probe. The O-aryl phosphate could be hydrolyzed by
the catalytic active sites of all tested PTPs. For dynamic sub-
strate enhancement, substrate 1 was incubated with an excess
of one amine fragment per well and the PTP of interest. After
a fixed time, the enzymatic reaction was terminated, and the
released phosphate (Pi) was quantified as the green ammoni-
um phosphomolybdate complex of Malachite Green at 630 nm
(Figure 1).[14] In agreement with our recent work on caspase-
3,[12] three outcomes of this experiment are conceivable. A) The
Initially, substrate 1 was assayed against every PTP in 50 mm
Tris·HCl (pH 7.0) in order to determine the KM values and to
optimize enzyme concentrations and incubation times (see the
Experimental Section). The conventional addition of dithio-
threitol (DTT) to the buffer was avoided in order to exclude
any interaction of the dithiol with the aldehyde electrophile.
For the dynamic substrate-enhancement assay, a concentration
of 250 mm of 1 was selected. A diverse set of 110 fragments
(primary amines) was assembled[19] and screened at a concen-
tration of 500 mm against each PTP. In the case of MptpA, two
enhancing fragments and one potent competitive inhibitor
were identified; for PTPN7, six enhancing molecules and one
potent competitive inhibitor were observed; seven enhancing
binders and one potent competitive inhibitor were found to
be active in the case of PTPN11; while two amines enhanced
the activity of PTP1B and one amine showed total inhibition of
PTP1B. The identified enhancing binders were structurally di-
verse (Scheme 1); all but one only displayed enhancing effects
against a single PTP. As a control, all hit fragments were tested
against their target PTPs without substrate 1 in the established
pNPP and DiFMUP assays.[20] None of the enhancing fragments
increased substrate turnover by PTP alone, thereby excluding
the possibility of allosteric activation and supporting the
postulated substrate enhancement by template-assisted liga-
tion. On the other hand, the identified competitive inhibitor 2
was active towards all tested PTPs, and similar 2,4-thiazolidi-
nones have been recently described as PTP inhibitors.[21] Ac-
cording to our results, compound 2 is an unselective and com-
petitive PTP inhibitor—KI values have been determined for the
four PTPs (Scheme 1). Moreover, the nonselectivity of 2 is in
agreement with the high homology of the active sites of PTPs.
Amine fragment 3, which accelerated the turnover of sub-
strate 1 by MptpA phosphatase cooperatively as well as selec-
tively, was selected for further validation. To quantify the ob-
served enhancing effect, different concentrations of substrate
1 (0, 100, 200, 300, 400, 500, 750, and 1000 mm) were incubat-
ed with different concentrations of 3 (0–1000 mm), and the
reactions were started by adding 0.3 mm MptpA in 50 mm
Tris·HCl. The reactions were terminated by adding Malachite
Green solution, and the amount of phosphate generated was
determined. For each concentration of compound 3, the initial
rate of the enzymatic reaction, v0, was plotted against the log-
arithm of the substrate concentration to provide an apparent
KM value (KMapp) for each of the fragment concentrations
(Figure 2). Interestingly, the observed Vmax values were not
modified significantly at different concentrations of 3, not even
at the highest excess of 3 (Figure 2). From this observation it
could be concluded that the kcat value was not changed by
Figure 1. Differential detection of cooperatively binding or inhibitory nucleo-
philic fragments RꢀNH2 in one dynamic substrate-ligation assay of PTPs. The
turnover of substrate 1 can be monitored by measuring the Malachite
Green phosphomolybdate complex formed from released phosphate anion
(Pi). Three alternative scenarios of fragment interactions can be distinguished
in the assay. A) (Inhibition) binding of fragment RꢀNH2 leads to decreased
phosphate formation. B) In contrast, nonbinding RꢀNH2 fragments have no
effect on substrate conversion or the formation of the Malachite Green com-
plex. C) Cooperative binding of the nucleophilic fragment and substrate 1 to
the protein results in enhanced substrate turnover by the PTP and thus in-
creased the phosphate signal.
quantity of Pi is decreased; this would suggest that the tested
fragment acts as an inhibitor of the PTP. B) No change in the Pi
concentration is observed; this would indicate no binding of
the amine. C) The observed Pi concentration increases; this
would indicate enhanced activity of substrate 1, presumably
by ligation of the fragment. Accordingly, it should be possible
to identify competitive inhibitors and cooperatively binding,
substrate-enhancing fragments in one primary screen.
To demonstrate the approach, we selected a set of thera-
peutically relevant PTPs: Mycobacterium tuberculosis protein ty-
rosine phosphatase A (MptpA), protein tyrosine phosphatase
1B (PTP1B), protein tyrosine phosphatase N7 (PTPN7 also
known as HePTP for hematopoietic PTP), and protein tyrosine
phosphatase 11 (PTPN11 also known as Shp2). MptpA is be-
lieved to mediate M. tuberculosis survival in host cells.[15] PTP1B
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J. Rademann et al.
free aldehyde substrate 1, [A], and fragment ligation product
(imine) 4, [I], contributed equally to the overall phosphate gen-
eration at a total fragment concentration, [Ft], of 181.4 mm and
a total aldehyde concentration, [At], of 181.4 mm. Considering
the different KM values of 1 and 4 and taking into account the
unchanged kcat value, this condition was reached at a ratio of
concentrations [I]/[A]=104/259. Inserting this ratio into the
sum equation for the total aldehyde concentration [At]=[A]+
[I] resulted in the free aldehyde concentration [A]=129.4 mm;
insertion of this concentration into the sum equation for the
total fragment concentration yielded the free fragment con-
centration [F]=129.4 mm and [I]=52 mm. The obtained concen-
trations finally indicated an equilibrium constant for the forma-
tion of the imine as
½Iꢂ
½4ꢂ
Keq
¼
¼ 3:31 mmꢀ1
½Aꢂ½Fꢂ ½free 1ꢂ½free 3ꢂ
in the presence of the protein.
For further confirmation, the postulated ligation product of
1 and 3 was converted into a potential inhibitor by replacing
the phosphate ester with a phosphate mimic. Cooperative
binder 3 was linked covalently with the phosphotyrosine mim-
etic
N-(4-carboxyphenyl)trifluoromethylsulfonamide[23]
(5)
through an amide bond in 6 or through a reduced amide in 7.
Compounds 3, 5, 6, and 7 were tested separately in functional
enzyme assays based on pNPP and DiFMUP.[20] Phosphotyro-
sine mimetic 5 was equally active against all four PTPs, with a
KI value of approximately 300 mm (Scheme 2). In contrast, com-
pounds 6 and 7 only showed activity towards MptpA with sim-
ilar KI values of 13ꢁ6 mm (amide 6) and 11ꢁ7 mm (amine 7). In
agreement with the initial screening results for fragment 3,
compounds 6 and 7 showed no activity at a concentration of
300 mm against the other tested PTPs.
Scheme 1. Unspecific, competitive inhibitor 2 and specific, cooperative en-
hancer fragments were identified in ligation assays of substrate 1 with four
different PTPs. KI values were determined for inhibitor 2. The effect of specif-
ic enhancing fragments is quantified by calculating the relative rate en-
hancement: (Abs630 with fragment+substrateꢀAbs630 blank)/(Abs630 without
fragmentꢀAbs630 blank).
varying the concentration of enhancing fragment 3 (kcat =Vmax
/
[E]t). Therefore, the observed decrease in the apparent KM
Molecular modeling of the protein–ligand complexes of 5
and 6 was performed in order to propose potential binding
modes of the MptpA inhibitors. Two MptpA structures have
been published, one contains a bound ligand (PDB ID: 1U2Q),
the second is without a ligand (1U2P).[24] Compounds 5 and 6
were docked into the active site of the ligand-bound MptpA
structure (1U2Q) by using AutoDock4. Phosphate mimetic 5
(magenta) in this model was able to bind direct with the cata-
lytic residues of the protein (Figure 3A); compound 6 (blue)
also fitted into the active site, as the overlay in Figure 3B
shows, in addition, it was able to access a secondary binding
site adjacent to the active pocket.
values (KM =(kcat +k )/k1) had to be a consequence of the in-
ꢀ1
creased binding affinity KD (KD =kꢀ1/k1) of the ligation product
to MptpA. This finding supported our hypothesis that 3 acted
solely as a cooperative binder, increasing substrate affinity
without accelerating the catalytic reaction itself. Similar correla-
tions between inhibitor binding, KI, and substrate conversion,
KM/kcat, have been demonstrated previously for the phospho-
rus-containing peptide inhibitors of thermolysin, carboxypepti-
dase A, and pepsin.[22]
Finally, the apparent KM values were analyzed as a function
of the concentration of fragment 3. While the KM of 1 alone
(KM1) was 258.8ꢁ29 mm, KMapp decreased with increasing con-
centrations of 3. Fitting of the data with the reciprocal func-
tion
In summary, we have demonstrated the development of se-
lective protein tyrosine phosphatase inhibitors based on the
detection of cooperative, second-site binding fragments by dy-
namic substrate enhancement. Compounds 6 and 7 can com-
pete well with the potency and selectivity of earlier MptpA in-
hibitors.[25–27]
KM1ꢀKM2
KM appðFÞ ¼ KM1
ꢀ
1 þ KM50=F
indicated that KMapp approached a minimal, KM2, at 104ꢁ
10 mm that was interpreted as being the KM2 of the ligation
product 4. The “KM50 value”, defined by a KMapp of (KM1 +KM2)/2,
was 181.4ꢁ19.5 mm; this indicated that the concentrations of
Conclusion
The development of specific inhibitors of protein tyrosine
phosphatases (PTP) has been hampered by the high similarity
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Identification of PTP-Targeting Fragments
tored in an enzyme assay. Al-
though it had already been
demonstrated that the screening
of substrates can be successful
in the definition of inhibitor
structures, our approach allows
a strongly facilitated variation of
fragments by using the incuba-
tion of an electrophilic substrate
with nucleophilic fragments.
Experimental Section
Materials and general methods:
Commercially available reagents
and solvents were obtained from
Sigma Aldrich and used as re-
ceived, unless otherwise noted. Re-
actions were monitored by analyti-
cal thin-layer chromatography on
Merck silica gel 60 F524 plates.
Compounds were visualized with a
UV lamp. For HPLC analysis, an
Agilent 1100 system was used with
a reversed-phase column (Nucleo-
sil 100 C-18, 5 mm, 2ꢁ250 mm
(Grom, Herrenberg, Germany) op-
erated with acetonitrile/water mix-
tures containing 0.1% formic acid),
Figure 2. To determine the observed enhancing effect, various concentrations of substrate 1 were incubated with
~
!
&
&
*
^
various concentrations of compound 3 ( : 0, : 100, : 250, : 500, : 750, and : 1000 mm). After the reactions
had been started by adding MptpA, A) initial velocities were recorded and B) apparent KM values were obtained.
The latter were plotted as a function of [3] and were used to determine the equilibrium of 3 and 1. While the KM
value of 1 alone was 259ꢁ29 mm, the apparent KM as [3] increased approached the KM of the ligation product 4
at 104ꢁ10 mm. Nonlinear fitting of this saturation curve revealed a 50% value of the apparent KM at a total frag-
ment concentration of 181.4 mm, thus indicating equal contributions of substrates 1 and 4 to the phosphate re-
lease. Calculation yielded an equilibrium constant for the imine formation of Keq =3.31 mmꢀ1 in the presence of
the protein.
between the phosphotyrosine binding pocket of different
a diode array detector, and an ESI mass spectrometer employing
single quadrupole detection. Microwave-assisted synthesis was per-
formed on an Initiator Microwave Synthesizer from Biotage (Uppsa-
la, Sweden). Bioassaying of PTPs was performed on a microtiter
plate reader (UV absorption and fluorescence intensity: Safire2,
Tecan, Crailsheim, Germany). The protein tyrosine phosphatase
(PTP) from Mycobacterium tuberculosis (MptpA) was isolated from a
bacterial expression system in E. coli. Human PTPs (PTP1B, PTPN7,
PTPN11) were provided by Prof. Dr. Stefan Knapp (University of
Oxford, Structural Genomics Consortium).
PTPs. Addressing secondary binding sites might offer a solu-
tion to the problem. Here, specific PTP inhibitors were devel-
oped by a novel, fragment-based-ligation approach. Fragments
that bind specifically to the secondary binding sites of four dif-
ferent PTPs were identified in a dynamic substrate-enhance-
ment assay. A generic substrate for PTPs containing an alde-
hyde functionality was incubated together with one nucleo-
philic fragment per well and one phosphatase. The released in-
organic phosphate was quantified by detecting its phospho-
molybdate complex. The assay format can detect substrate-
enhancing fragments that accelerate substrate cleavage and
distinguish them from competitive inhibitors of the enzymes
in the same set of experiments.
Phosphoric acid mono-(4-formyl-phenyl) ester (1): Diethyl chloro-
phosphate (2.36 mL, 16.3 mmol, 1 equiv) was added dropwise to a
cooled (08C) solution of 4-hydroxylbenzaldehyde (2 g, 16.3 mmol,
1 equiv) and triethylamine (2.3 mL, 19.6 mmol, 1.2 equiv) in dry
CH2Cl2 under nitrogen. The reaction mixture was warmed to RT,
and stirring was continued until completion of the reaction (3–6 h).
The reaction mixture was extracted with HCl (1m) and a saturated
aqueous solution of Na2CO3. The organic phase was dried (MgSO4)
and concentrated in vacuo. A septum-sealed microwave tube
charged with the protected phosphate (300 mg, 1.14 mmol,
1 equiv) and trimethylsilylbromide (300 mL, 2.3 mmol, 2 equiv) in
CH3CN was heated in the microwave synthesizer (608C, 10 min).
After completion of the reaction, as indicated by TLC, the reaction
mixture was quenched with MeOH/H2O (95:5, v/v) and concentrat-
ed to give phosphate ester 1 in a yield of 3.0 g (91%). 1H NMR
(300 MHz, [D6]DMSO): d=2.02 (brs, 2H; PO(OH)2), 6.92 (d, J=2 Hz,
2H; CH), 7.64 (d, J=2 Hz, 2H; CH), 9.87 ppm (brs, 1H; CHO);
13C NMR (75 MHz, [D6]DMSO): d=116.4, 129.5, 131.3, 156.1,
191.0 ppm (CHO); 31P NMR (121 MHz, [D6]DMSO): d=22.3 ppm;
ESI-MS: calcd for C7H7O5P: 202.1 Da; found, m/z 201.1 Da [MꢀH]+.
The mechanism of dynamic substrate enhancement was an-
alyzed in greater detail for one selected fragment combination.
The concentrations of the fragment and substrate were varied
systematically, and the substrate turnover was monitored for
each concentration. The enhancing fragment was found to in-
crease the binding affinity of the generic substrate strongly,
whereas it had no effect on the rate of the enzymatic reaction
itself. The most strongly enhancing fragment was then con-
verted into a PTP inhibitor by linking it irreversibly to a phos-
photyrosine mimetic. The obtained inhibitor was fully selective
for one of the four tested PTPs. In summary, the developed
methodology should have significant potential for the frag-
ment-based optimization of enzyme substrates and the frag-
ment-based development of enzyme inhibitors. In principal,
the method could be adapted to other enzymes, such as pro-
teases, for which the conversion of substrates can be moni-
5-Amino-2-(2,4-dimethyl-phenyl)isoindole-1,3-dione
(3):
A
septum-sealed microwave tube charged with 4-aminophthalic acid
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2643

J. Rademann et al.
Figure 3. A) Model of 4-(trifluoromethanesulfonylamino)benzoic acid 5 (ma-
genta) binding into the active site of MptpA. B) Overlay of 5 (magenta) and
7 (blue) using AutoDock4 with AutoDockTools indicating that 7 addresses
the active site and the potential second site docked in MptpA (1U2Q). The
structural data suggest that a secondary binding site next to the phospho-
tyrosine binding site exists and can be used for selective PTP inhibitor
design. The figures were generated with PyMOL v.0.99.[29]
Scheme 2. Inhibitor development starting from the enhancing fragment 3
and the phosphotyrosine isoster fragment 5. Irreversible fragment condensa-
tion products 5 and 6 are specific inhibitors of MptpA in preference to the
other three tested PTPs.
was stirred until neutral pH was reached, then lyophilized from
acetonitrile/water to yield 1.71 g of compound 5 (95%). 1H NMR
(300 MHz, [D6]DMSO): d=7.61 (m, 2H; CH), 7.95 (m, 2H; CH),
10.61 ppm (brs, 1H; COOH); 13C NMR (75 MHz, [D6]DMSO): d=
106.21, 110.39, 114.56, 123.62 (CF3), 121.19, 121.92, 133.74, 144.10
(Ar), 169.80 ppm (COOH); ESI-MS: calcd for C8H6F3NO4S: 269.2 Da;
found: m/z 268.2 Da [MꢀH]+.
(200 mg, 1.1 mmol, 1 equiv), 2,4-dimethylphenylamine (1.36 mL,
11 mmol, 10 equiv), and triethylamine (1.53 mL, 11 mmol, 10 equiv)
was heated in a microwave (1608C, 30 min). After completion of
the reaction, as indicated by TLC, the reaction mixture was concen-
trated and purified by column chromatography (toluene/acetone
4:1, v/v) to yield 228.5 mg of compound
3
(78%). 1H NMR
N-[2-(2,4-Dimethyl-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-
yl]-4-(trifluoromethylsulfonamido)benzamide (6): 4-(Trifluorome-
thanesulfonylamino)benzoic acid (5) (50 mg, 0.19 mmol, 1 equiv)
was dissolved in dry CH2Cl2 (5 mL). Thionyl chloride (140 mL,
1.6 mmol, 10 equiv) was added dropwise, and the solution was
stirred at 608C for 6 h. Solvents were evaporated, and the residue
was resolved in CH2Cl2. This procedure was repeated three times.
After complete evaporation of thionyl chloride, the product, 4-tri-
fluoromethane sulfonylamino benzoyl chloride, was dissolved in
CH2Cl2 (2 mL) under nitrogen. 5-Amino-2-(2,4-dimethyl-phenyl)iso-
indole-1,3-dione (2, 61 mg, 0.23 mmol, 1.2 equiv) and pyridine
(19 mL, 0.23 mmol, 1.2 equiv) were dissolved in dry CH2Cl2 (1 mL).
The resulting solution was added dropwise to the solution of the
acyl chloride. The reaction mixture was stirred overnight, then the
solvents were evaporated. Product 6 was purified by preparative
HPLC in a yield of 42.3 mg (43%). 1H NMR (300 MHz [D6]DMSO):
d=2.12 (s, 3H; CH3), 2.32 (s, 3H; CH3), 7.01 (m, 1H; CH), 7.27 (m,
1H; CH), 7.81 (m, 4H; CH), 7.94 (m, 1H; CH), 8.14 (m, 1H; CH),
8.37 ppm (m, 1H; CH); 13C NMR (75 MHz [D6]DMSO): d=17.83,
(300 MHz, [D6]DMSO): d=2.10 (s, 3H; CH3), 2.25 (s, 3H; CH3), 4.60
(brs, 2H; NH2), 6.79 (d, J=3 Hz, 1H; CH), 7.04 (d, J=2 Hz, 1H; CH),
7.08 (s, 1H; CH), 7.25 (s, 1H; CH), 7.45 ppm (m 2H; CH); 13C NMR
(75 MHz, [D6]DMSO): d=17.75, 20.75 (CH3), 115.23, 115.81, 119.28,
121.16, 121.83, 123.37, 126.75, 131.32, 131.55, 132.12, 143.67 (CꢀN),
153.26 (CꢀNH2), 166.04, 167.06 ppm (CO); ESI-MS: calcd for
C16H14N2O2: 266.29 Da; found: m/z 267.0 Da [MꢀH]+.
4-(Trifluoromethylsulfonylamido)benzoic acid (5): 4-Aminobenzo-
ic acid methyl ester (1 g, 6.7 mmol, 1 equiv) and pyridine (540 mL,
6.7 mmol, 1 equiv) were dissolved in CH2Cl2 (150 mL) under nitro-
gen, and the mixture was cooled to 08C. Trifluoromethanesulfonic
anhydride (5.96 mL, 35 mmol, 5 equiv) was added dropwise. The
reaction mixture was stirred overnight, warmed up to room tem-
perature, and extracted with 5% HCl and H2O. The organic phase
was dried (MgSO4) and concentrated in vacuo. The obtained mate-
rial was dissolved in LiOH (50 mL, 0.1m), and the mixture was
stirred for 1 h. Ion exchanger was added, and the reaction mixture
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Identification of PTP-Targeting Fragments
20.98, 100.72, 108.66, 115.93, 116.73, 120.08, 121.22, 122.32, 124.82,
125.32, 125.59, 126.82, 128.19, 131.34, 131.78, 132.70, 142.33,
144.68, 165.81 (CONH), 166.10 (CO), 167.03 ppm (CO); ESI-MS:
calcd for C24H18F3N3O5S: 517.5 Da; found: m/z 518.5 Da [M+H]+.
(500 mm) in Tris·HCl (50 mm, pH 7; total volume 20 mL) were incu-
bated for 60 min. Then the Malachit Green solution (70 mL) was
added.
PTPN11: The kinetic parameters of PTP11 were determined by
plotting several substrate concentrations against measured activity.
A KM of 613ꢁ256 mm, a Vmax of 4.406ꢁ1.157 mmminꢀ1, and a kcat of
25.9ꢁ6.8 minꢀ1 were determined at an enzyme concentration of
0.17 mm after end-point measurements (tincubation =60 min). To
screen PTPN11 (0.17 mm), substrate 1 (250 mm) and nucleophilic
fragment (500 mm) in Tris·HCl (50 mm, pH 7; total volume 20 mL)
were incubated for 60 min. Then the Malachit Green solution
(70 mL) was added.
N-(4-((2-(2,4-Dimethyl-phenyl)-1,3-dioxo-1,3-dihydro-1H-isoin-
dol-5-yl)amino-methyl)phenyl)trifluoromethyl-sulfonamide (7): A
septum-sealed microwave tube charged with 4-(4-trifluoromethyl-
sulfonylamino-benzylamino)phthalic acid (10 mg, 24 mmol, 1 equiv),
2,4-dimethylaniline (30 mL, 240 mmol, 10 equiv), and triethylamine
(34 mL, 240 mmol, 10 equiv) was irradiated in the microwave
(1608C, 30 min). After completion of the reaction, as indicated by
TLC, the reaction mixture was concentrated and purified by prepa-
rative HPLC to yield 14.6 mg of 7 (78%). 1H NMR (300 MHz,
[D6]DMSO): d=2.13 (s, 3H; CH3), 2.28 (s, 3H; CH3), 4.24 (s, 2H; CH2),
7.05 (m, 7H; CH), 7.29 (m, 2H; CH), 7.58 ppm (m, 1H; CH); 13C NMR
(75 MHz, [D6]DMSO): d=17.78, 20.82, 50.92, 100.67, 109.72, 115.69,
116.59, 118.98, 120.72, 121.16, 124.13, 124.51, 126.83, 128.89,
131.59, 134.88, 135.47, 137.59, 145.73, 151.45, 166.09 (CO),
167.04 ppm (CO); ESI-MS: calcd for C24H20F3N3O4S: 503.5 Da; found:
m/z 504.5 Da [M+H]+.
PTP1B: The enzyme kinetic parameters of PTP1B were determined
by plotting several substrate concentrations against measured ac-
tivity. A KM of 319ꢁ57 mm, a Vmax of 9.135ꢁ0.800 mmminꢀ1, and a
kcat of 6.5ꢁ0.6 minꢀ1 were determined at an enzyme concentration
of 1.4 mm after end-point measurements (tincubation =15 min). To
screen PTP1B (1.4 mm), substrate 1 (250 mm) and nucleophilic frag-
ment (500 mm) in Tris·HCl (50 mm, pH 7; total volume 20 mL) were
incubated for 15 min. Then the Malachit Green solution (70 mL)
was added.
Malachite Green assay for the determination of phosphate (Pi):
The color reagent for orthophosphate determination was prepared
as previously described.[14] Concentrated sulfuric acid (60 mL, d=
1.84 gLꢀ1) was slowly added to water (300 mL). The solution was
then cooled to room temperature and supplemented with Mala-
chite Green (0.44 g). The resulting orange solution is stable for at
least one year at room temperature. On the day of use, 7.5% am-
monium molybdate (2.5 mL) was added to the dye solution
(10 mL) along with 11% Tween20 (0.2 mL) and water (19.05 mL). Fi-
nally, this solution (70 mL) was added to the phosphate-containing
reaction mixture (20 mL). Assays were performed in untreated,
clear, 384-well microtiter plates (Corning B.V. Life Sciences, Schipol-
Rijk, The Netherlands). All experiments were performed on micro-
plate reader Safire2 (Tecan). Tris·HCl buffer (50 mm, pH 7.0) was
used. A standard curve was measured to determine the correlation
between absorption and phosphate (Pi) concentration with e=
12195mꢀ1 cmꢀ1. Assay conditions were validated specifically for
each of the four protein tyrosine phosphatases. After the amine di-
versity subset had been screened against the PTP of interest, the
identified primary hit compounds (inhibitors and activators) were
validated under the same condition in replicate measurements.
Quantification and characterization of the enhancing effect:
Substrate 1 (0, 100, 200, 300, 400, 500, 750, and 1000 mm) was in-
cubated with different concentrations of amine 3 (0, 100, 250, 500,
750, and 1000 mm) in Tris·HCl (50 mm, pH 7); the reactions were
started by adding MptpA (0.3 mm) resulting in a total volume of
20 mL. The reactions were terminated after 30 min by adding Mala-
chite Green solution (70 mL) in order to determine the final concen-
tration of phosphate. For each concentration of compound 3, the
initial rate of the enzymatic reaction, v0, was plotted against the
logarithm of the substrate concentration to provide an apparent
KM value (KMapp) for each of the fragment concentrations (Figure 2).
While the KM value of 1 alone was 259ꢁ29 mm, KMapp at increasing
concentrations of 3 approached 104ꢁ10 mm, which can be inter-
preted as the KM value of the ligation product. Nonlinear fitting of
this saturation curve by employing GraphPad Prism 4 for Windows
indicated that the 50% value of KMapp was reached at a total frag-
ment concentration of 181.4 mm; at this concentration 50% of the
substrate turnover was contributed by each of the two species.
Enzymatic phosphatase assays: PTP activity was determined in
384-well microtiter plates by using 4-nitrophenyl phosphate (pNPP)
and 6,8-difluoro-4-methyl-umbelliferyl phosphate (DiFMUP)[20] as
substrates and the microtiter plate reader Safire2 (Tecan). Tris·HCl
(50 mm), NaCl (150 mm), and DTT (1 mm) were used as buffer con-
ditions. To determine the KI values of identified compounds, inhibi-
tors (0, 10, 25, 50, 100, 250, 500 mm) were incubated in triplicate
with the PTP of interest (0.3 mm MptpA, 0.3 mm PTPN7, 0.1 mm
PTPN11, 1 mm PTP1B) and pNPP (10 mm) or DiFMUP (10 mm) in a
total volume of 20 mL. The obtained average data were plotted in
an Excel sheet so as to determine KI values by nonlinear regression
fittings, as previously described.[28]
Composition of a fragment library for screening: 110 primary
amines were selected from the FMP ChemBioNet library by diversi-
ty considerations. The members of the ChemBioNet library were
selected by the recently reported maximum common-substructure
concept (MCS).[19]
MptpA: The kinetic parameters of MptpA were determined by
plotting several substrate concentrations against measured activity.
A KM of 259ꢁ29 mm, a Vmax of 1.311ꢁ0.051 mmminꢀ1, and a kcat of
4.37ꢁ0.17 minꢀ1 were determined at an enzyme concentration of
0.3 mm after end-point measurements (tincubation =30 min). To screen
MptpA (0.3 mm), substrate 1 (250 mm) and nucleophilic fragment
(500 mm) in Tris·HCl (50 mm, pH 7; total volume 20 mL) were incu-
bated for 60 min. Then the Malachit Green solution (70 mL) was
added.
Molecular Modeling: Molecular modeling was performed by using
AutoDock 4 with AutoDockTools (http://autodock.scripps.edu). Two
crystal structures of MptpA are available from the Protein Data
Bank (http://www.pdb.org). One with (PDB ID: 1U2Q) and one
without (1U2P) a bound ligand.[24] Compounds 5 and 6 were
docked into the active site of the 1U2Q MptpA structure. The PDB
receptor file 1U2Q was edited by AutoDockTools. All water mole-
cules were removed, and hydrogen atoms were added. The file
was saved as a PDBQT file. In parallel, PDBQT ligand files were pre-
PTPN7: The kinetic parameters of PTPN7 were determined by plot-
ting several substrate concentrations against measured activity. A
KM of 266ꢁ96 mm, a Vmax of 3.437ꢁ0.56 mmminꢀ1, and a kcat of
11.4ꢁ1.9 minꢀ1 were determined at an enzyme concentration of
0.3 mm after end-point measurements (tincubation =60 min). To screen
PTPN7 (0.3 mm), substrate 1 (250 mm) and nucleophilic fragment
ChemBioChem 2011, 12, 2640 – 2646
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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Acknowledgements
We thank Swantje Behnken, Dr. Samuel Beligny, Dr. Jçrn Saupe,
and Carola Seyffarth for technical assistance and fruitful discus-
sions, the DFG (SFB 765, FOR 806, Ra 895/2-5) and the Fonds der
Chemischen Industrie for financial support, and Prof. Stefan
Knapp and Dr. Alastair Barr (Structural Genomics Consortium,
University of Oxford) for providing PTPs.
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Keywords: drug discovery
Mycobacterium tuberculosis · protein tyrosine phosphatases ·
second-site binders
·
dynamic ligation assay
·
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Received: June 27, 2011
Published online on November 3, 2011
2646
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 2640 – 2646