A focused library of protein tyrosine phosphatase inhibitors

Article abstract of DOI:10.1021/jm100528p

Protein tyrosine phosphatases such as PTP1B and YopH are potential targets for the development of therapeutic agents against a variety of pathological conditions including diabetes, obesity, and infection by the bacterium Yersinia pestis. A focused library of bidentate α-ketoacid-based inhibitors has been screened against several tyrosine phosphatases. Compound 2a has IC ₅₀ values of 43 and 220 nM against YopH and PTP1B, respectively, and shows a 30-fold selectivity for PTP1B over the closely related phosphatase TCPTP.

Full text of DOI:10.1021/jm100528p

6768 J. Med. Chem. 2010, 53, 6768–6772
DOI: 10.1021/jm100528p
A Focused Library of Protein Tyrosine Phosphatase Inhibitors
Anthony B. Comeau,† David A. Critton,‡ Rebecca Page,‡ and Christopher T. Seto*,†
†Department of Chemistry, Brown University, 324 Brook Street, Box H, Providence, Rhode Island 02912, and
‡Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Box G-E4, Providence, Rhode Island 02912
Received April 30, 2010
Protein tyrosine phosphatases such as PTP1B and YopH are potential targets for the development of
therapeutic agents against a variety of pathological conditions including diabetes, obesity, and infection
by the bacterium Yersinia pestis. A focused library of bidentate R-ketoacid-based inhibitors has been
screened against several tyrosine phosphatases. Compound 2a has IC50 values of 43 and 220 nM against
YopH and PTP1B, respectively, and shows a 30-fold selectivity for PTP1B over the closely related
phosphatase TCPTP.
Introduction
selectivity for PTP1B over TCPTP despite the similarity in the
structures of their active sites.
Intracellular signal transduction pathways are controlled
by the reciprocal activities of protein kinases and phospha-
tases. Significant success has been achieved in the develop-
ment of protein tyrosine kinase inhibitors. For example, the
drugs imatinib and gefitinib are kinase inhibitors that are
currently used to treat several cancers. In contrast, the devel-
opment of drugs that target protein tyrosine phosphatases
(PTPsa) is less advanced.1 The FDA has approved sodium
stibogluconate, which is a potent inhibitor of the Src homo-
logy PTP 1 (SHP-1), SHP-2, and PTP1B,2 as an orphan drug
for cutaneous leishmaniasis. Isis Pharmaceuticals is develop-
ing an antisense drug that targets PTP1B for the treatment of
type 2 diabetes,3 and a number of medicinal chemistry programs
are underway to discover small molecule PTP inhibitors.1
Two significant challenges to the development of orally bio-
available, small molecule inhibitors are: (1) many PTPs share
similar structures and sequences in the region of their active
sites, making it difficult to design inhibitors that are specific
for their designated target, and (2) many small molecules that
bindwith high affinity in theseactivesites are hydrophilic, and
as a result have poor cell permeability.1
PTP1B was the first validated PTP target. It down regulates
insulin signaling by dephosphorylating the insulin receptor
and several of its downstream signaling proteins.4 PTP1B
knockout mice show enhanced sensitivity to insulin and are
resistant to diet induced weight gain, making this PTP an
important target for both diabetes and obesity.5 T-Cell PTP
(TCPTP) ishighly homologous toPTP1B, with74% sequence
identity in the catalytic region. Unlike the PTP1B knockout
mice that develop normally, the TCPTP knockout results in
embryonic lethality caused by abnormalities in B- and T-cells.6
This observation suggests that it is desirable to achieve high
A number of bacteria, including Yersinia and Salmonella,
rely on PTPs for their pathogenicity.7,8 The Yersinia pestis
bacterium, which causes bubonic plague, employs several
virulence factors that it injects into the cytosol of eukaryotic
cells using a contact-dependent type III secretion apparatus.9
One of these factors is the Yersinia PTP or YopH (Yersinia
outer protein H). This PTP is essential for virulence of the
bacterium; plasmids that lack a functional gene for this protein
are not virulent.10 YopH compromises the host immune
system by dephosphorylating host proteins including FAK
(focal adhesion kinase), FYB (Fyn binding protein), the focal
adhesion protein Cas, and SKAP-HOM (SKAP55 homo-
logue).11 In addition, YopH impairs T- and B-cell activation
by inhibiting early phosphorylation reactions in the antigen
receptor signaling complex.12 Thus, YopH is a target for deve-
lopment of antibacterial agents against the bubonic plague.
HePTP is a phosphatase that is expressed in white blood
cells in bone marrow, thymus, spleen, and lymph nodes. It
represses T-cell activation and proliferation by binding to and
dephosphorylating the activation loop of the MAP kinases
Erk1, Erk2, and p38.13 HePTP is overexpressed in patients
with the preleukemic disorder myelodysplastic syndrome and
acute myelogenous leukemia, suggesting a link between ab-
normal HePTP activity and leukemia.14 Taken together, these
observations reinforce the notion that PTPs are attractive
targets for the development of small molecule inhibitors.
Table 1 shows a comparison of the sequences among four
PTPs, PTP1B, TCPTP, HePTP, and YopH, in sections of the
enzymes that are important for binding and catalysis. The P-
and WPD-loops comprise the active site region of PTPs, while
residues of the secondary site often interact with anionic func-
tional groups on substrates and inhibitors. PTP1B and TCPTP
are identical in these regions, while HePTP shows some varia-
tion from PTP1B in the secondary site and WPD-loop but not
in the P-loop. YopH has differences from PTP1B in the WPD-
and P-loops but has matching Arg residues in the secondary site.
We have been investigating aryl R-ketocarboxylic acids
as nonhydrolyzable analogues of aryl phosphate esters.15
*To whom correspondence should be addressed. Phone: 401-863-
3587. Fax: 401-863-9368. E-mail: christopher_seto@brown.edu.
aAbbreviations: PTP1B, protein tyrosine phosphatase 1B; YopH,
Yersinia outer protein H; TCPTP, T-cell protein tyrosine phosphatase;
PTP, protein tyrosine phosphatase; SHP-1, Src homology protein
tyrosine phosphatase 1; SHP-2, Src homology protein tyrosine phos-
phatase 2; HePTP, hematopoietic protein tyrosine phosphatase.
r
pubs.acs.org/jmc
Published on Web 08/23/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6769
Scheme 1. Synthesis of Anilinesa
Table 1. Sequence Similarities among Four PTPsa
a Sequence differences from PTP1B are highlighted in red.
a Reagents: (a) K2CO3, DMSO, microwave irradiation, 195 °C, 10 min;
(b) 1 atm H2, 10% Pd(OH)2/C, EtOH. See Figure1 for structures of the
R groups.
Scheme 2. Synthesis of Inhibitorsa
Figure 1. Structures of inhibitors.
The R-ketocarboxylate is designed to have reduced negative
charge at physiological pH when compared to phosphate
esters and phosphonates and may give improved pharmaco-
kinetic characteristics. However, this functional group retains
enough anionic character to make strong interactions with the
active and secondary sites of PTPs. In a recent study, we
examined the activity of compound 1 (Figure 1),16 which
incorporates two aryl R-ketoacid groups that are designed
to contact both the active site and the secondary pTyr binding
site.17 This inhibitor has IC50 values of 240 nM and 1.5 μM
against YopH and PTP1B, respectively. However, it displays
less than 2-fold selectivity for PTP1B over TCPTP. In an effort
to improve the potency and specificity of 1, we have construc-
ted a focused library of inhibitors that incorporates substitu-
ents on the terminal diphenyl ether ring (2a-o). We have also
examined compound 3, which incorporates a 3-benzyloxy
substituent. These compounds are optimized to interact with
regions of PTPs outside of the catalytic site, including the
secondary binding site.18 The inhibitors were screened in
crude form using YopH, and four compounds were selected
for purification and further analysis against YopH, PTP1B,
TCPTP, and HePTP.
a Reagents: (a) SOCl2, benzene; (b) CH2Cl2 or THF, anilines 7a-o;
(c) NaOH, H2O; (d) 3-(benzyloxy)aniline. See Figure 1 and Scheme 1 for
structures of the R groups. 3-(Benzyloxy)aniline and anilines 7f, g, i, k,
and n were obtained from commercial sources.
irradiation at 195 °C for 10 min. The resulting nitroben-
zene derivatives 6 were reduced by catalytic hydrogenation
using palladium hydroxide on carbon to give the desired
anilines.
The inhibitors were synthesized as shown in Scheme 2.
Compound 8, which was prepared as reported previously,16
contains two trifluoromethyloxazolone groups. These tri-
fluoromethyloxazolones, which are conveniently prepared
by reaction of the corresponding 4-substituted phenylglycine
derivative with trifluoroacetic anhydride,19 represent masked
R-ketocarboxylic acids. The R-ketoacids can be unmasked at
the end of thesynthesis withaqueous base. The carboxylicacid
group in 8 was converted to the acid chloride with thionyl
chloride and subsequently reacted with anilines 7a-o to give
amides 9a-o. The trifluoromethyloxazolones were then hy-
drolyzed with aqueous sodium hydroxide to give the crude
inhibitors 2a-o. In a similar manner, the acid chloride of 8 was
reacted with 3-(benzyloxy)aniline to give 10, which was hydro-
lyzed to generate inhibitor 3. The purity of the crude inhibitors
was determined to be g90% by 1H NMR spectroscopy.20
Chemistry
Before constructing the library, we needed to prepare the
4-phenoxyaniline analogues 7a-e, h, j, l, m, and o (Scheme 1).
These anilines were synthesized by SNAr reaction of 4-chlor-
onitrobenzene 4 with various phenols in the presence of
potassium carbonate. The reactions were heated by microwave

6770 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Table 2. Inhibition of YopH by Crude Inhibitors 1, 2a-o, and 3a
Comeau et al.
IC50
(nM)
IC50
(nM)
compd
R
compd
R
2a
2b
2c
2d
3
4-Ph
30
50
2i
3,4-Cl2
3-F
210
220
4-SCF3
4-Et
4-n-hexyl
2j
70
90
2k
1
2-Me
H
230
240
100
110
110
190
200
2l
4-iPr
2,4-F2
4-OMe
4-CN
800
910
2e
2f
2g
2h
4-SCH3
4-F
4-Me
2m
2n
2o
1100
1300
4-n-butyl
a Average of two measurements. The purity of the crude inhibitors
was determined to be g90% by 1H NMR spectrometry. See Figure 1 for
the structure of compound 3.
Table 3. Inhibition of Phosphatases by Purified Inhibitors 2a-da
Figure 2. Model of inhibitor 2a bound to PTP1B (pink). The struc-
ture of TCPTP (green) has been aligned with PTP1B. Top: Overlay
of the ribbon diagrams of the two phosphatases. Bottom: The sur-
face of PTP1B is superimposed on the stick diagram of TCPTP.
YopH and PTP1B. Importantly, it has a 30-fold selectivity for
PTP1B over TCPTP. This is a significant level of selectivity
given the high sequence homology shared by these two PTPs
in their active site regions. By contrast, the parent inhibitor 1
has less than 2-fold preference for PTP1B over TCPTP.16 The
added 4-phenyl substituent in 2a when compared to 1 leads to
improved selectivity by both increasing the compound’s
potency against PTP1B and decreasing potency against
TCPTP. Compounds 2a-d are modest inhibitors of TCPTP
IC50 (μM)
PTP1B
0.043 ( 0.006 0.22 ( 0.04 6.5 ( 0.9 21 ( 5
0.063 ( 0.011 1.6 ( 0.2 4.0 ( 0.8 53 ( 4
0.053 ( 0.010 0.49 ( 0.07 3.5 ( 0.6 52 ( 8
1.2 ( 0.2 2.3 ( 0.4 28 ( 3
compd
R
YopH
TCPTP HePTP
2a
2b
2c
2d
4-Ph
4-SCF3
4-Et
4-n-hexyl 0.089 ( 0.021
a Average of two measurements.
(IC50 = 2.3-6.5 μM) and poor inhibitors of HePTP (IC50
=
21-53 μM). Lineweaver-Burk analyses of compound 2b
against YopH, and compound 2a against PTP1B, confirm
that they are reversible competitive inhibitors.20
Enzyme Inhibition and Modeling Studies
The crude R-ketoacids were screened against YopH to
gauge their potential as PTP inhibitors (Table 2). Many of
the compoundsshowimprovedactivity whencomparedtothe
parent inhibitor 1. Only compounds that incorporated highly
polar groups such as 2,4-F2 (2m), 4-OMe (2n), and 4-CN (2o),
or a bulky 4-iPr substituent (2l), are weaker inhibitors than 1.
The most potent compounds with IC50 values less than 100 nM
incorporate hydrophobic substituents at the 4-position
(compounds 2a-d). It is unclear why inhibitor 2h, which
incorporates a 4-n-Bu substituent, is less active than the
closely related inhibitors 2c and d, which incorporate 4-Et
and 4-n-hexyl substituents, respectively.
We purified the four most active compounds (2a-d) by
HPLC and assayed them against four PTPs; YopH, PTP1B,
TCPTP, and HePTP (Table 3). There is a good correlation
between the IC50 values obtained using crude and purified
inhibitors, suggesting that the data obtained with the crude
compoundsprovides a reliable indication oftheirtrue activity.
In general, 2a-d are potent YopH inhibitors with IC50 values
in the range of 43-89 nM. They also have reasonable activity
against PTP1B, with values in the 0.22-1.6 μM range.
Compound 2a is the best inhibitor in the series against both
We examined the potential binding mode of 2a with PTP1B
using molecular modeling. The X-ray structure of PTP1B
bound to a difluorophosphonate inhibitor was used as the
starting point (PDB code 1QXK).21 After the difluoropho-
sphonate inhibitor was removed from the structure, 2a was
minimized in the active site using Autodoc Vina.22 The
resulting model was aligned with the structure of TCPTP
(PDB code 1L8K)23 in an effort to identify features that may
explain the selectivity of 2a for PTP1B over TCPTP.
Figure 2 shows the model of 2a minimized in the active site
of PTP1B and superimposed on the structure of TCPTP. The
top panel in Figure 2 highlights the high degree of similarity
of these two phosphatases and emphasizes the difficulty in
designing inhibitors that are selective for one enzyme over
the other. One significant difference between the two struc-
tures is the position of the WPD loop. Only one crystal
structure of TCPTP is available in the Protein Data Bank,23
and this structure has the WPD loop in the open position. In
contrast, the structure of PTP1B that we used for the mini-
mization had the WPD loop closed onto a difluorophospho-
nate inhibitor.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6771
corresponding TCPTP residues are insideparentheses. Thesix
residues are Ala17 (Gln19), Gln21 (Leu23), Cys32 (His34),
Lys39 (Glu41), Phe52 (Tyr54), and Ala264 (Pro262). As
shown in detail in the top panel of Figure 3, our modeling
results suggest that Cys32 (His34) may play an important role
in determining the 30-fold selectivity of compound 2a for
PTP1B over TCPTP. The side chain of Cys32 of PTP1B
resides below and parallel to the plane of the terminal phenyl
ring of 2a. In contrast, the side chain of His34 of TCPTP
occupies the same region of space as the terminal phenyl ring
in the 2a/PTP1B model. Thus, a plausible explanation for the
selectivity of 2a for PTP1B over TCPTP is that His34 of
TCPTP pushes the biphenyl subunit of 2a into a different and
presumably less favorable conformation in the binding site,
which results in a lower overall binding energy between 2a and
TCPTP when compared with PTP1B. We note that the
position of Cys32 in crystal structures of PTP1B with the
WPD loop in the open (PDB code 2HNP)24 and closed
states21 are identical.
The middle panel in Figure 3 shows a more detailed view of
the contacts between residues in the catalytic site of PTP1B
and one of the R-ketocarboxylate groups of inhibitor 2a. The
carboxylate and ketone form a number of hydrogen bonds
with backbone N-H atoms in both the P- and WPD-loops,
including those of residues Phe182, Arg221, and Ser216. The
inhibitor also forms hydrogen bonds with the side chains of
Arg221 and Asp181. The benzene ring of the inhibitor forms a
hydrophobic contact with the side chain of Phe182. The
bottom panel of Figure 3 shows that the second R-ketoacid
can hydrogen bond with the side chains of Arg24 and Gln21
near the secondary site. The model also suggests a cation-π
interaction between the neighboring phenyl ring of the inhi-
bitor and the side chain of Arg24.
Conclusion
In summary, we have constructed a focused library of PTP
inhibitors that are based upon the structure of compound 1.
Inhibitor 2a, which incorporates a biphenyl substituent,
emerged as the most potent compound in the library with
IC50 values of 43 and 220 nM against YopH and PTP1B,
respectively. This inhibitor displays a significant level of
selectivity for PTP1B over TCPTP. Molecular modeling
studies indicate that the two R-ketocarboxylic acid groups in
2a contact the catalytic and secondary sites and that the side
chain of His34 of TCPTP may play an important role in the
observed 30-fold selectivity of 2a for PTP1B over TCPTP.
Figure 3. Interactions between inhibitor 2a (white) and PTP1B
(pink). Top: Model of the biphenyl portion of 2a bound to PTP1B
and superimposed on the crystal structure of TCPTP (green).
Residues of TCPTP are listed in parentheses. Middle: Interactions
between one R-ketocarboxylate of 2a bound in the catalytic pocket
of PTP1B. Bottom: Interactions between the second R-ketocarbox-
ylate of 2a and residues near the secondary binding site of PTP1B.
The bottom panel of Figure 2 highlights the differences
between PTP1B and TCPTP that might be used to explain the
selectivity of 2a for PTP1B. One of the R-ketoacid groups of
2a binds deep in the active site pocket, similar to the binding of
a natural pTyr substrate, and the second R-ketoacid binds
near Arg24 (PTP1B numbering) of the secondary site. One
important difference between the two enzymes occurs in the
region occupied by the terminal phenyl ring of the biphenyl
subunit of 2a. In PTP1B, the residue that is in closest
proximity to this phenyl ring is Cys32. However, in TCPTP,
the corresponding residue is His34, which is labeled in the
bottom panel of Figure 2. The imidazole ring on the side chain
of this residue can be seen emerging out of the pink surface of
PTP1B directly underneath the terminal phenyl ring of 2a.
Iversen and co-workers have identified six residues that are
different between PTP1B and TCPTP that might be exploi-
ted for the design of specific inhibitors.23 In the following
list, PTP1B residues are outside of the parentheses, and the
Experimental Section
General Methods. NMR spectra were recorded on Bruker
Avance-300 or Avance-400 instruments. Spectra were calibrated
1
using TMS (δ = 0.00 ppm) for H NMR, CDCl3 (δ = 77.0
ppm), acetone-d6 (δ = 29.5 ppm), or DMSO-d6 for 13C NMR.
Mass spectra were recorded using fast atom bombardment or
electrospray ionization methods. Methylene chloride and
methanol were obtained from a dry solvent dispensing system.
HPLC analyses were performed on a Rainin HPLC system with
C
18 columns and UV detection. All other reagents were used as
received. The inhibitors 2a-d were determined to be g95% pure
as measured by analytical reverse phase HPLC.
PTP Assays. The phosphatase activities of YopH, PTP1B,
and TCPTP were assayed using p-NPP as the substrate at 25 °C,
and the reaction progress was monitored using UV spectrosco-
py. Initial rates were determined by monitoring the hydrolysis of
p-NPP at 420 nm, from 30 to 150 s after mixing. Percent
inhibition assay solutions contained 10% DMSO, substrate

6772 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Comeau et al.
(p-NPP) was at the respective Km for each PTP, 1 mM EDTA, 50
mM NaCl, and 50 mM 3,3-dimethylglutarate buffer at pH 7.0.
The experimentally determined Km values for Yersinia PTP,
PTP1B, and TCPTP were 1.7, 2.3, and 1.7 mM, respectively. The
activity of HePTP was assayed using 4-methylumbelliferyl
phosphate (MUP) at 25 °C and the reaction progress was
monitored using fluorescence spectroscopy. Initial rates were
determined by monitoring the hydrolysis of MUP at excitation
and emission wavelengths of 340 and 460 nm, respectively, from
0 to 150 s after mixing. Percent inhibition assay solutions for
HePTP contained 10% DMSO, 174 μM MUP, 1 mM EDTA,
50 mM NaCl, 50 mM 3,3-dimethylglutarate buffer at pH 7.0.
The experimentally determined Km value of MUP for HePTP
was 174 μM. IC50 values were calculated using the commercial
graphing software Grafit (Erithacus Software Ltd.). Data were
obtained for assays with at least five different concentrations, in
duplicate, of each inhibitor.
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Acknowledgment. This work was supported by an American
Cancer Society Research Scholar Grant (RSG-08-067-01-LIB)
to R.P.
Supporting Information Available: Synthetic procedures,
compound characterization, HPLC traces for 2a-d, Lineweaver-
Burk analyses for compounds 2a and 2b. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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