Inhibition of Low Molecular Weight Protein Tyrosine Phosphatase by an Induced-Fit Mechanism

Article abstract of DOI:10.1021/acs.jmedchem.6b00993

The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a regulator of a number of signaling pathways and has been implicated as a potential target for oncology and diabetes/obesity. There is significant therapeutic interest in developing potent and selective inhibitors to control LMW-PTP activity. We report the discovery of a novel class of LMW-PTP inhibitors derived from sulfophenyl acetic amide (SPAA), some of which exhibit greater than 50-fold preference for LMW-PTP over a large panel of PTPs. X-ray crystallography reveals that binding of SPAA-based inhibitors induces a striking conformational change in the LMW-PTP active site, leading to the formation of a previously undisclosed hydrophobic pocket to accommodate the α-phenyl ring in the ligand. This induced-fit mechanism is likely a major contributor responsible for the exquisite inhibitor selectivity.

Full text of DOI:10.1021/acs.jmedchem.6b00993

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Article
Inhibition of low molecular weight protein tyrosine
phosphatase by an induced-fit mechanism
Rongjun He, Jifeng Wang, Zhi-Hong Yu, Ruo-Yu Zhang, Sijiu Liu, Li Wu, and Zhong-Yin Zhang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Sep 2016
Downloaded from http://pubs.acs.org on September 28, 2016
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Inhibition of low molecular weight protein tyrosine phosphatase by an induced-fit
mechanism
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Rongjun He , Jifeng Wang , ZhiꢀHong Yu, RuoꢀYu Zhang, Sijiu Liu, Li Wu, ZhongꢀYin Zhang
Department of Medicinal Chemistry and Molecular Pharmacology, Department of Chemistry, Center for
Cancer Research, and Institute for Drug Discovery, Purdue University, 720 Clinic Drive, West
Lafayette, IN 47907
Running Title: Highly Selective Inhibitors of LMWꢀPTP
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Abstract
The low molecular weight protein tyrosine phosphatase (LMWꢀPTP) is a regulator of a number
of signaling pathways and has been implicated as a potential target for oncology and diabetes/obesity.
There is significant therapeutic interest in developing potent and selective inhibitors to control LMWꢀ
PTP activity. We report the discovery of a novel class of LMWꢀPTP inhibitors derived from
SulfoPhenyl Acetic Amide (SPAA), some of which exhibit greater than 50ꢀfold preference for LMWꢀ
PTP over a large panel of PTPs. Xꢀray crystallography reveals that binding of SPAAꢀbased inhibitors
induces a striking conformational change in the LMWꢀPTP active site, leading to the formation of a
previously undisclosed hydrophobic pocket to accommodate the αꢀphenyl ring in the ligand. This
inducedꢀfit mechanism is likely a major contributor responsible for the exquisite inhibitor selectivity.
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Introduction
As a major posttranslational modification mechanism, protein tyrosine phosphorylation regulates
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numerous cellular processes including growth, differentiation, migration, and survival. Proper level of
protein tyrosine phosphorylation, which is dynamically maintained by protein tyrosine kinases (PTKs)
and protein tyrosine phosphatases (PTPs), is essential for normal cellular homeostasis. Perturbation to
the balance between PTK and PTP activity induces either excessive or diminished substrate
phosphorylation, which is associated with a wide range of pathological conditions such as cancer,
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diabetes and autoimmune disorders. Consequently, the ability to selectively modulate signaling
pathways mediated by protein tyrosine phosphorylation holds enormous potential for therapeutic
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intervention, as evidenced by the development of PTK inhibitors for various clinical indications. Given
the substantial success of PTK inhibitors, there is heightened interest in targeting the opposing PTPs for
therapeutic applications.
The PTPs constitute a large family of signaling enzymes (>100) that can be broadly classified
into three groups: tyrosineꢀspecific, dualꢀspecific, and the low molecular weight protein tyrosine
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phosphatase (LMWꢀPTP). The common feature of all PTPs is the active site sequence C(X)
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R(S/T),
also known as the PTP signature motif or Pꢀloop, which specifically binds the phosphoryl moiety of the
substrate. As an enigmatic member of the PTP family, the LMWꢀPTP, encoded by Acp1, is an 18 kDa
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cytosolic enzyme found ubiquitously in both prokaryotic and eukaryotic organisms. LMWꢀPTP has
been shown to either positively or negatively regulate the function of plateletꢀderived growth factor
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receptor (PDGFR),
fibroblast growth factor receptor (FGFR), epidermal growth factor receptor
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(EGFR), insulin receptor (IR),
and is involved in numerous signaling cascades including focal
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18,19
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adhesion kinase (FAK),
STAT5,
and Epherin.
However, the mechanism(s) by which these
pathways are controlled by LMWꢀPTP is not yet fully understood.
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A number of studies suggest LMWꢀPTP as a potential oncoprotein. Increased expression of
LMWꢀPTP mRNA and protein levels were observed in malignant samples of breast, colon, bladder and
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kidney.
More recently, LMWꢀPTP was reported to enhance the metastatic potential of prostate
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cancer cells, resulting in worse patient survival. LMWPTP is also overexpressed and active in primary
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colorectal cancer. Interestingly, LMWꢀPTP has also been implicated in the development of type II
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diabetes and obesity.
For example, LMWꢀPTP was implicated as a negative regulator of insulin
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signaling through dephosphorylation of components in the IR pathway.
Dietꢀinduced obese mice
treated with LMWꢀPTP antisense oligonucleotide exhibited decreased plasma insulin, glucose,
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triglyceride and cholesterol levels, and showed increased glucose and insulin tolerance.
In view of the emerging recognition of LMWꢀPTP as a potential target for oncology and
diabetes/obesity, there is considerable interest in obtaining small molecule inhibitors of LMWꢀPTP as
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,29ꢀ37
potential therapeutics.
Moreover, potent and selective inhibitors of LMWꢀPTP would be invaluable
tools for interrogating LMWꢀPTP biology and therapeutic hypothesis testing. Unfortunately, reported
inhibitors lack robust selectivity for LMWꢀPTP and therefor are not suitable as chemical probes for
further mechanistic studies and ultimately, for the development of therapeutic agents targeting LMWꢀ
PTP. We describe the identification and characterization of a novel series of LMWꢀPTP inhibitors,
which exhibit greater than 50ꢀfold preference for LMWꢀPTP over a large panel of PTPs. Crystal
structures of LMWꢀPTPꢀinhibitor complexes reveal a ligand induced conformational change in LMWꢀ
PTP that is responsible for the exquisite inhibitor selectivity.
Results and Discussion
Acquisition of potent and selective LMWꢀPTP inhibitors. The PTPs, as a target class, have
presented significant difficulties for drug discovery. The high sequence and structure homology within
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the PTP active site creates a challenge in achieving inhibitor selectivity among closely related family
members. In addition, the positiveꢀcharged environment of the PTP active site poses unique drug
discovery challenges, as most competitive inhibitors by necessity are negatively charged, which
complicates drug development due to low cell permeability and bioavailability. Since existing drugs are
already endowed with favorable pharmacokinetic properties, we sought to harness the privileged drug
space for novel PTP inhibitory scaffolds that can be further optimized into potent and selective PTP
inhibitors with improved drugꢀlike properties. To this end, we have recently discovered SulfoPhenyl
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Acetic Amide (SPAA), a fragment originated from cefsulodin, as a novel pTyr mimetic. We found that
SPAA inhibited LMWꢀPTP with an IC of 2 mM, as opposed to an IC of >50 mM for PTP1B and
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SHP2. LMWꢀPTP exists in two isoforms (namely LMWꢀPTPA and LMWꢀPTPB, respectively) which
differ only by a loop that flanks the catalytic site. This study concerns mostly the LMWꢀPTPA isoform.
In order to increase the potency and selectivity of SPAA for LMWꢀPTP, we sought to capture additional
interactions outside of the active site pocket. To this end, we prepared a SPAAꢀbased focused library by
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reacting αꢀsulfophenyl acetic acid with a set of 192 amines of different size, charge, and lipophilicity.
The library was assembled directly on 96ꢀwell plates under standard HBTU amide coupling conditions.
Reactions from representative wells were monitored by LCꢀMS, which indicated a 70ꢀ80% product
conversion.
Library compounds were screened for inhibitory activity against LMWꢀPTP at a compound
concentration of ~10 ꢁM, and counterꢀscreened against PTP1B and SHP2 to provide preliminary
specificity information. Several compounds, 1, 2, 3, 4, 5, 6, 7, 8 (Figure 1), exhibited >50% inhibition
against LMWꢀPTP, but <10% inhibition against PTP1B and SHP2, indicating significant specificity for
LMWꢀPTP. These hits were resynthesized and purified with reversedꢀphase HPLC, and their IC values
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against LMWꢀPTP were measured, which are summarized in Figure 1. To our delight, all of the hits
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display IC values less than 10 ꢁM, consistent with our initial screening results. This represents a more
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than 200ꢀfold gain in binding affinity compared to the parent SPAA. Kinetics studies with compound 7
were carried out to probe its mode of inhibition. Figure 2 showed that 7 is a competitive inhibitor against
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LMWꢀPTPA with a K of 3.2 ± 0.1 ꢁM. We also determined that compound 7 inhibits the LMWꢀPTPB
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isoform with a K of 1.7 ± 0.1 ꢁM, indicating that the SPAAꢀbased inhibitors likely inhibit both LMWꢀ
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PTP isoforms with similar potency. Structure and activity survey revealed that the aniline containing
compound 9 and the 2ꢀaminothiazole containing compound 10 have significantly reduced activities,
indicating that substituents on aniline (compound 9 vs compounds 1, 2, 3, 4, 5, 8) and the second
benzene ring (compound 10 vs compound 7) are required for high affinity binding. The propyl amine
containing compound 11 is inactive as expected given that it does not have an aromatic group at the
amine terminal, and it was inactive in our initial screening. To gain a more comprehensive picture of
how selective these compounds are to the LMWꢀPTP, we broadened the scope of selectivity profiling by
inclusion of an additional 22 phosphatases in addition to PTP1B and SHP2. As shown in Table 1,
compounds 3, 5, and 6 exhibit no inhibitory activity against any of the phosphatases in the panel at 200
ꢁ
M concentration. In particular, compound 3 possesses a greater than 35ꢀfold preference for LMWꢀPTP
over all other PTPs.
Structural basis of LMWꢀPTP inhibition by the SPAAꢀbased inhibitors. To elucidate the
structural basis of LMWꢀPTP inhibition and to aid the design of more potent and selective LMWꢀPTP
inhibitors, we sought to determine the crystal structures of LMWꢀPTP in complex with the SPAAꢀbased
inhibitors. We initially obtained LMWꢀPTP crystals under previously published conditions including
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00 mM MES buffer, 30% PEGME 5,000 and 0.2 M ammonium sulfate in the presence of 1 mM
compound 7. The structure was determined by molecular replacement, using the published LMWꢀPTP
structure as the search model (PDBID:5PNT). However, compound 7 was nowhere to be found. Instead,
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a MES molecule occupied the active site (Figure 3A). MES is a week inhibitor of LMWꢀPTP, with an
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IC50 greater than 10 mM. The sulfonic acid moiety mimics the interaction of the PO group of a
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substrate within the phosphatase active site. The rest of the molecule interacts with the surrounding
residues by van der Waals interactions. We then systematically screened alternate crystallization
conditions without MES and finalized 25ꢀ30% PEGME 5,000 in 100 mM BisꢀTris, pH 6.0ꢀ6.5 for
optimal crystal growth of the LMWꢀPTP•7 complexes. Again, the structure of LMWꢀPTP•7 was
determined by molecular replacement, and compound 7 was unambiguously identified around the
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C(X) R(S/T) PTP signature motif (Figure 3B). For comparison, we also crystallized and solved the
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structure of LMWꢀPTP in complex with compound 9, which has an IC > 100 ꢀM, under the same
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conditions (Figure 3C). Finally, by soaking the LMWꢀPTP•MES crystals in 100 mM BisꢀTris buffer, we
obtained the crystal structure of apo LMWꢀPTP (Figure 3D). In the apo LMWꢀPTP structure, the MES
molecule is replaced by three water molecules, which are well organized to form hydrogen bonds with
the amides of the Pꢀloop residues. The data collection and refinement statistics for all four crystal
structures are summarized in Table 2. The overall LMWꢀPTP structures are very similar and the root
mean square derivation (rmsd) for all Cα atoms is 0.341 Å between LMWꢀPTP•7 and LMWꢀPTP•MES,
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.529 Å between LMWꢀPTP•7 and LMWꢀPTP•9, and 0.630 between LMWꢀPTP•7 and the apo
enzyme.
Although the overall threeꢀdimensional structures are quite similar between LMWꢀPTP•7 and
LMWꢀPTP•MES, superimposition of both structures revealed a localized conformational change in the
active site. In the LMWꢀPTP•MES complex, the side chain dihedral angle χ1 (∠CꢀCAꢀCBꢀSG) of
Cys17, which is located in the Pꢀloop, is 167.2 degree, while in the presence of compound 7, the side
chain of Cys17 is pushed 114.3 degree away by the αꢀphenyl ring in 7, adapting a new χ1 of 52.9 degree
(Figure 4A). In addition, the movement of the Cys17 side chain also shifts the Pꢀloop downward by 1.4
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Å from its original position (Figure 4A). Interestingly, the conformation of the side chain of Cys17 in
the apo form of LMWꢀPTP is identical to that observed in LMWꢀPTP•MES structure (Figure 4B), while
the side chain of Cys17 exists in the same conformation in both LMWꢀPTP•9 and LMWꢀPTP•7
structures (Figure 4C). The net result of this SPAA ligand induced conformational change is the creation
of a hydrophobic cavity constituted by residues Ile16, Cys17, Pro130, Tyr131, Phe138 and the sideꢀ
chain aliphatic atoms of Asp56, which is well suited for the αꢀphenyl ring in compound 7 and 9 (Figure
4D). To the best of our knowledge, no other PTPs undergo this structural change in the active site upon
ligand binding.
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To better understand the unique preference of SPAA for LMWꢀPTP, we compared the active site
features of LMWꢀPTP with those of PTP1B, a representative of the classical PTPs. As shown in Figure
5A, sequence alignment and structure superimposition of the signature motif C(X) R(S/T) of LMWꢀ
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PTP•7 with that of the PTP1B structure (PDBID: 1PXH) revealed that: 1) although the backbones of the
two signature motifs align very well but only 3 of 7 residues are conserved between LMWꢀPTP and
PTP1B, making the active site pockets rather different in shape and electrostatic potential between the
two enzymes; 2) Cys17 plays a key role in the induceꢀfit process to create a hydrophobic cavity
accommodating the αꢀphenyl ring of compound 7¸ but the corresponding residue in PTP1B (and other
classical PTPs) is Gly220, which is incapable of doing the same; 3) a severe steric clash exists between
the αꢀphenyl ring and Gln262, and another between the benzothiazole and Tyr46. Since the conserved
Gln262 and Tyr46 come from the adjacent active site Qꢀloop and pYꢀloop of PTP1B, steric clashes may
exist whether the WPDꢀloop is in the closed (Figure 5B) or open conformation (Figure 5C).
Collectively, the active site specificity manifested by the inducedꢀfit mechanism is unique for LMWꢀ
PTP, and likely responsible, in large part, for SPAA’s preference to the LMWꢀPTP over PTP1B, SHP2
and other PTPs as well.
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In addition to revealing a novel inducedꢀfit mechanism for LMWꢀPTP inhibition by the SPAAꢀ
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containing inhibitors, the structure of LMWꢀPTP•7 also uncovers the basis for its potency: the sulfonic
acid moiety is in close proximity to the Pꢀloop and is stabilized by six hydrogen bonds with main chain
amides of Leu13, Gly14, Ile16, Cys17, Arg18 and the side chain amide of Arg18 (Figure 6) with a
distance ranging from 2.8 to 3.2 Å. In addition, the amide NH of compound 7 forms a hydrogen bond
with carboxylate O of the general acid Asp129 with a distance of 3.0 Å. This is a new interaction that
was never observed in previously reported LMWꢀPTP inhibitors. Moreover, a water molecule bridges
another two hydrogen bonds between the N atom within thiazole and the carboxylate O of Asp129.
Besides the hydrogen bond interactions mentioned above, compound 7 is also involved in a network of
πꢀπ interactions with LMWꢀPTP (Figure 6). πꢀπ interaction has long been acknowledged as an integral
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force in maintaining protein stability
as well as in differential recognition of medicinal agents
4
2
containing aromatic substituents by their protein targets. In the unliganded state, the phenyl ring of
Tyr131 is parallel to that of Tyr49 on the opposite side of the empty active site, with a distance of ~9.9
Å between the two aromatic rings. When compound 7 is bound in the active site, the benzothiazole ring
of 7 is sandwiched between the phenyl rings of Tyr49 and Tyr131. The distance of the closest atoms
between the phenyl ring of Tyr49 and the benzothiazole ring is ~3.5 Å. Similarly, the distance of the
closest atoms between the phenyl ring of Tyr131 and the benzothiazole ring is ~3.8 Å. Hence, the
43
benzothiazole ring falls within the London dispersion distance with both of these tyrosine residues.
The position and orientation of the Tyr131 side chain is constrained by Tyr132 through an edge to face
πꢀπ interaction, with the distance of ~4.0 Å (mass center to mass center) (Figure 6). Tyr131 not only
forms a πꢀπ interaction with the phenyl ring of benzothiazole moiety, it also forms an edge to face πꢀπ
interaction with the αꢀphenyl ring of 7, with a distance of ~4.2 Å (Figure 6). Taken together, these πꢀπ
interactions form a network involving three tyrosine residues from LMWꢀPTP (Tyr49, Tyr131 and
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Tyr132) and the benzothiazole and αꢀphenyl ring from compound 7. These interactions, in addition to
those mentioned above, likely play a major role for tight binding of compound 7.
In order to understand why installation of a double aromatic ring to the SPAA moiety generated
much better inhibitors for LMWꢀPTP than did the single ring counterparts (Figure 1), we also
determined the structure of LMWꢀPTP•9. Superimposition of the LMWꢀPTP•9 structure to that of
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LMWꢀPTP•7 revealed that the sulfonic acid moiety and the αꢀphenyl ring of compound 9 are located at
exactly the same positions in the active site as the corresponding groups in compound 7 (Figure 4C).
However, the aniline ring of compound 9 overlaps with only half of the double aromatic benzothiazole
ring of compound 7. Although it still forms a πꢀπ interaction with Tyr131, the distance between the
aniline ring and Tyr49 increases to about 4.5 Å, significantly longer than what is required for optimal
London dispersion interaction. Moreover, the nonpolar benzene ring also eliminates the waterꢀbridged
hydrogen bonds interaction with Asp129. Thus, the reduction in πꢀπ interactions and waterꢀbridged polar
interactions between LMWꢀPTP and the single benzene ring at the acetamide position may account for
the >15ꢀfold decrease in inhibitory activity for compound 9 versus compound 7. Compared to compound
7
, the thiazole group in 10 may still retain the waterꢀbridged hydrogen bond interactions but likely loses
the πꢀπ interactions due to the absence of the terminal benzene ring. This may explains why compound
10 is superior to compound 9 even though it has a smaller single aromatic ring, suggesting that
appropriately positioned polar atoms in the first aromatic ring will benefit binding affinity.
Initial optimization of compound 7. Based on the structural insights, we carried out an initial
structureꢀactivity relationship study in order to lay the foundation to develop more potent and selective
LMWꢀPTP inhibitors. From the crystal structures, the αꢀphenyl ring is situated in a hydrophobic cavity,
bordered by residues Ile16, Cys17, Pro130, Tyr131, Phe138 and the sideꢀchain aliphatic atoms of
Asp56. We explored whether installation of a small substituent on the αꢀbenzene ring could capture
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additional interactions with the enzyme, leading to increased binding affinity (Figure 7). Substituents to
enhance hydrophobic interactions included orthoꢀchloro (compound 13), orthoꢀmethyl (compound 14),
metaꢀmethyl (compound 16), paraꢀmethyl (compound 20), and paraꢀbromo (compound 21); and
substituents aimed for gaining potential hydrogenꢀbonding interactions included orthoꢀfluoro
(compound 12), metaꢀfluoro (compound 15), metaꢀnitrile (compound 17), metaꢀnitro (compound 18),
and metaꢀtrifluoromethyl (compound 19). We also introduced electron donating substituents (compound
4
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8
9
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4, 16, 20) and electron withdrawing substituents (compounds 17, 18, 19) to ascertain whether
perturbation of electronic properties would influence potential πꢀπ interactions and thereby inhibitor
activity. Unfortunately, we found that modifications to the αꢀphenyl ring led to significant diminished
inhibitory activity toward LMWꢀPTP, indicating that the ligand binding induced hydrophobic pocket
can only accommodate a naked benzene ring.
We next explored substituents on the benzothiazole ring in compound 7. Analysis of the LMWꢀ
PTP•7 crystal structure showed that the amide NH of 7 forms a hydrogen bond with Asp129, and the
benzothiazole moiety forms strong πꢀπ stacking interactions with Tyr49, Tyr131, and Tyr132. We
reasoned that by installing small substituents with appropriate electronic properties and polarities at the
benzene ring of benzothiazole, we could enhance the hydrogen bonding interaction between the amide
NH and Asp129, and/or hydrophobic and polar interactions between the benzothiazole moiety and Tyr
4
9, Tyr131, and Tyr132. Hence we designed and synthesized a series of compounds with electron
donating groups (compounds 23, 24, 25, 29, 31), electron withdrawing groups (compounds 26, 28, 32),
hydrophobic groups (22, 23, 25, 27, 29), and polar groups (compounds 24, 26, 28, 30, 31, 32). As
summarized in Figure 8, most derivatives displayed slightly increased activity over parent compound 7.
Specifically, compound 28 with a nitro group at the 6 position and compound 32 with carboxylate group
at the 6 position are 3.5ꢀ and 2.5ꢀfold more active than 7, with a measured IC at 2.1 ± 0.2 ꢁM and 3.1 ±
50
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.1 ꢁM, respectively. Nitro and carboxylate groups are both electron withdrawing which make the NH
amide bond more acidic, which likely enhances its hydrogen bonding interaction with Asp129. In
addition, the nitro and carboxylate groups are in close proximity to Tyr49, to make a new hydrogenꢀ
bonding interaction with the hydroxyl group of Tyr49. Kinetics studies revealed that compound 28 is
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also a competitive inhibitor for LMWꢀPTP with a K at 1.0 ± 0.1 ꢁM. More importantly, compound 28
i
exhibited at least over 50ꢀfold selectivity for LMWꢀPTP over the panel of 24 phosphatases (Table 1),
making compound 28 the most potent and selective LMWꢀPTP inhibitor reported to date. Deletion of
the sulfonic acid in 28 completely abolishes compound 33’s ability to inhibit LMWꢀPTP, consistent with
the importance of sulfonic acid for inhibition.
Cellular activity of compound 28. To assess the effects of 28 in insulin signaling pathway, we
pretreated HepG2 cells with 28 for 2 h and then stimulated the cells with 50 nM insulin for 30 min. As
shown in Figure 9A, compound 28 enhanced AKT phosphorylation in a doseꢀdependent manner relative
to the vehicle DMSO. To ensure that the cellular activity of compound 28 is due to inhibition of LMWꢀ
PTP and not nonspecific effects, a structurally related but inactive analog of compound 28, compound
33 (IC50 >> 100 ꢁM for LMWꢀPTP, Figure 8), was also evaluated. As expected, at 16 ꢁM compound 33
exerted no effect on insulin induced AKT phosphorylation while at the same concentration compound
2
8 increased AKT phosphorylation by 260% (Figure 9B). The results indicate that compound 28
promotes insulin induced AKT phosphorylation through specifically inhibiting LMWꢀPTP activity.
In summary, we have developed a novel class of LMWꢀPTP inhibitors based on SPAA by using
a focused library synthesis approach. High throughput screening and kinetic studies of the library
components led to the identification of a number of hits with excellent potency and selectivity for
LMWꢀPTP. Subsequent crystallography studies disclosed that sulfonic acid is in close proximity with
the active site PTP signature motif and stabilized by multiple hydrogen bonds. The structure also
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revealed a striking ligandꢀinduced conformational change in LMWꢀPTP that is essential for inhibitor
specificity. Moreover, the structure also identified a triad of aromatic residues Tyr49, Tyr131, and
Tyr132 that contribute to the potency and specificity of the inhibitors by forming strong πꢀπ stacking
interactions with the aromatic moieties in the ligand. Structureꢀguided optimization of compound 7
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resulted in compound 28, which displayed a K value of 1.0 ± 0.1 ꢁM for LMWꢀPTP and an
i
unprecedented >50 fold selectivity for LMWꢀPTP over 24 other PTPs, marking it the first truly specific
LMWꢀPTP inhibitor. Our study provides a solid foundation upon which more potent and selective
LMWꢀPTP inhibitors can be designed. These compounds are also expected to serve as useful tools to
study LMWꢀPTP’s biological function, and are excellent starting points for developing novel
therapeutics for cancer and type 2 diabetes.
Experimental Section
Materials and General Procedures. pꢀNitrophenyl phosphate (pNPP) was purchased from Fluke.
Crystallization reagents were from Hampton Research. DNA primers were from Genwiz Inc. and other
reagents were obtained from Fisher or Sigma. Reagents were used as purchased from SigmaꢀAldrich and
1
13
Fisher Scientific. H and C NMR spectra were obtained on a BrukerAvance II 500 MHz NMR
spectrometer with TMS or residual solvent as standard. Mass spectra were obtained using an Agilent
Technologies 6130 quadrupole LC/MS. HPLC purification was carried out on a Waters Delta 600
equipped with a Sunfire Prep C18 OBD column (30 mm/150 mm, 5 ꢁm) with methanol water (both
containing 0.1% TFA) as mobile phase (gradient: 50ꢀ100% methanol, flow 10 mL/min). The purity of
all final tested compounds was established to be>95% by Agilent Technologies 6130 quadrupole
LC/MS by using methanol water (both containing 0.1% TFA) as the mobile phase (gradient: 0ꢀ100%
methanol, flow 1.0 mL/min), with UV monitoring at the fixed wavelength of 254 nm.
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Library Synthesis. To all wells of 96ꢀwell plates were added sulfophenylacetic acid (20 µL, 20
mM in DMF), HBTU (20 µL, 20 mM in DMF), HOBt (20 µL, 20 mM in DMF), DIEA (20 µL, 60 mM
in DMF), and corresponding 192 amines (He et al., 2015) from storage plates (1.2 µL, 0.5 mM in DMF),
the reaction plates were placed at rt for 1 day. The reaction wells from aniline were monitored by LCꢀ
MS to show that reactions occurred well in great conversions. Thus a library of 192 compounds was
obtained with estimated concentration at 4 mM (assuming 80% product yield), which was screened
against LMWꢀPTP and other PTPs. All compounds were prepared as racemic mixtures.
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Representative Procedure for the Synthesis of Products. To αꢀSulfophenyl acetyl chloride (0.234
g, 1 mmol) and DIEA (0.522 mL, 3 mmol) in DMF (2 mL) was added propyl amine (0.09 mL,
1
.1mmol), the mixture was stirred at rt for 1 h. After quenching with water, it was subjected to HPLC
purification, and product 11 was obtained as colorless oil (93% yield, >95% purity).
Characterizations of Compounds 1 to 33.
1
1: H NMR (500 MHz, DMSO) δ 10.44 (s, 1H), 7.89 (s, 2H), 7.59ꢀ7.58 (m, 2H), 7.28ꢀ7.26 (m, 3H), 4.78
13
(
s, 1H), 2.43 (s, 3H); C NMR (500 MHz, DMSO) δ 166.5, 139.0, 134.8, 130.6, 130.0, 127.4, 127.0,
+
1
24.2, 121.8, 71.6, 22.6. ESIꢀHRMS Cacld. for C15H12Br2NO4S (MꢀH ): 459.8859; found 459.8866.
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9
1
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
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1
2
: H NMR (500 MHz, DMSO) δ 10.29 (s, 1H), 7.83 (d, J = 1.7 Hz, 1H), 7.57ꢀ7.56 (m, 2H), 7.46ꢀ7.17
13
(
m, 10H), 5.15 (s, 2H), 4.73 (s, 1 H); C NMR (DMSO) δ 165.9, 149.4, 136.7, 135.1, 133.2, 129.9,
1
28.5, 127.9, 127.5, 127.4, 126.9, 121.3, 120.5, 118.7, 114.7, 71.7, 70.3. ESIꢀHRMS Cacld.for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C21H17ClNO5S (MꢀH ): 430.0521, found 430.0519.
1
3
: H NMR (500 MHz, DMSO) δ 10.48 (s, 1H), 8.23 (s, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 7.3
1
3
Hz, 2H), 7.35ꢀ7.25 (m, 4H), 4.82 (s, 1H); C NMR (DMSO) δ 166.1, 139.92, 139.6, 136.4, 136.2,
1
35.5, 135.2, 129.9, 127.4, 127.0, 120.1, 118.2, 118.0, 116.9, 104.1, 71.8. ESIꢀHRMS Cacld.for
+
C16H11F3N3O4S (MꢀH ): 398.0428; found 398.0434.
1
4
: H NMR (500 MHz, DMSO) δ 10.60 (s, 1H), 9.65 (t, J = 1.3 Hz, 1H), 8.26 (t, J = 1.7 Hz, 1H), 7.90 (t,
J = 1.6 Hz, 1H), 7.84ꢀ7.83 (m, 2H), 7.76ꢀ7.73 (m, 2H), 7.59ꢀ7.58 (m, 2H), 7.30ꢀ7.24 (m, 3H), 4.80 (s,
13
1
H); C NMR (DMSO) δ 166.5, 140.2, 135.1, 134.3, 130.0, 129.6, 127.3, 127.0, 122.6, 120.8, 120.7,
+
1
19.7, 71.8. ESIꢀHRMS Cacld. for C17H14N3O4S (MꢀH ): 356.0711; found 356.0706.
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1
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1
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2
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
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4
4
4
4
4
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5
5
5
5
5
5
5
5
6
0
1
2
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5
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8
9
0
1
2
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4
5
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7
8
9
0
1
2
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4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
: H NMR (500 MHz, DMSO) δ 10.43 (s, 1H), 7.64ꢀ7.58 (m, 4H), 7.41 (d, J = 8.4 Hz, 2H), 7.30ꢀ7.25
13
(
m, 3H), 7.40ꢀ7.34 (m, 3H), 4.82 (s, 1H), 3.90 (t, J = 4.9 Hz, 4H), 3.43 (s, 4H); C NMR (DMSO) δ
166.1, 135.0, 129.9, 127.4, 127.0, 120.0, 71.6, 64.5, 52.8. ESIꢀHRMS Cacld. for C18H19N2O5S (Mꢀ
+
H ): 375.1020; found 375.1020.
1
6
: H NMR (500 MHz, DMSO) δ 11.05 (s, 1H), 9.29 (d, J = 2.3 Hz, 1H), 9.01 (d, J = 2.1 Hz, 1H), 8.14
(d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.85ꢀ7.84 (m, 1H), 7.84ꢀ7.82 (m, 1H), 7.76ꢀ7.63 (m,
13
2
1
H), 7.32ꢀ7.27 (m, 3H), 4.89 (s, 1H); C NMR (DMSO) δ 167.2, 141.3, 138.9, 134.7, 133.3, 130.4,
30.0, 128.6, 128.3, 128.2, 127.4, 127.0, 124.2, 99.5, 71.6. ESIꢀHRMS Cacld. for C17H13N2O4S (Mꢀ
+
H ): 341.0602; found 341.0602.
1
7: H NMR (500 MHz, DMSO) δ 7.97 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.1 Hz,
13
2
H), 7.43 (t, J = 7.5 Hz, 1H), 7.33ꢀ7.28 (m, 4H), 5.09 (s, 1H); C NMR (DMSO) δ 167.0, 157.6, 148.5,
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34.3, 131.5, 130.0, 127.6, 127.3, 126.2, 123.6, 121.8, 120.6, 70.3. ESIꢀHRMS Cacld.for
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
C15H11N2O4S2 (MꢀH ): 347.0166; found 347.0166.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
: H NMR (500 MHz, DMSO) δ 10.90 (s, 1H), 8.27 (s, 2H), 7.71 (s, 1H), 7.63ꢀ7.61(m, 2H), 7.30ꢀ7.24
1
3
(
s, 5H), 4.85 (s, 1H); C NMR (DMSO) δ 167.2, 158.4 (q, J = 38.6 Hz), 141.1, 134.6, 130.8 (q, J = 32.4
Hz), 127.4, 127.1, 123.2 (q, J = 271.0 Hz), 120.0, 115.0 (q, J = 286.2 Hz), 71.8. ESIꢀHRMS Cacld. for
+
C16H10F6NO4S (MꢀH ): 426.0240; found 426.0237.
1
9
: H NMR (500 MHz, DMSO) δ 10.23 (s, 1H), 7.56ꢀ7.54 (m, 4H), 7.30ꢀ7.25 (m, 5H), 7.03 (t, J = 7.4
13
Hz, 1H), 4.76 (s, 1H); C NMR (DMSO) δ 166.2, 139.2, 135.2, 130.1, 129.1, 127.8, 127.4, 123.7,
+
1
19.3, 72.2. ESIꢀHRMS Cacld.for C14H13NO4S (MꢀH ): 292.0638; found 292.0645.
1
1
0: H NMR (500 MHz, DMSO) δ 12.0 (s, 1H), 7.49ꢀ7.23 (m, 7H), 4.93 (s, 1H). ESIꢀHRMS Cacld. for
+
C11H9N2O4S2 (MꢀH ): 297.0009; found 296.9999.
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SO H
3
H
N
O
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11
1
11: H NMR (500 MHz, CDCl ) δ 8.23 (s, 1H), 7.45ꢀ7.44 (m, 2H), 7.27ꢀ7.20 (m, 3H), 4.42 (s, 1H), 3.11ꢀ
3
13
3
1
.02 (m, 2 H), 1.46ꢀ1.39 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H); C NMR (500 MHz, CDCl ) δ 167.3, 135.7,
3
+
29.6, 127.4, 126.8, 71.5, 40.4, 22.3, 11.4. ESIꢀHRMS Cacld.for C11H16NO4S (M+H ): m/z 258.0795;
found 258.0799.
SO H
F
3
H
N
S
O
N
12
1
1
2: H NMR (500 MHz, DMSO) δ 12.43 (s, 1H), 8.08ꢀ8.05 (m, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.76 (d, J
1
3
=
8.1 Hz, 1H), 7.45ꢀ7.42 (m, 1H), 7.33ꢀ7.29 (m, 2H), 7.19ꢀ7.12 (m, 2H), 5.46 (s, 1H). C NMR
(DMSO) δ 166.3, 160.2 (d, J = 243.9 Hz, 1H), 157.6, 148.5, 131.9 (d, J = 3.3 Hz), 131.5, 129.0 (d, J =
8
.1 Hz), 126.1, 123.5, 123.4 (d, J = 3.5 Hz), 121.7, 121.4 (d, J = 13.2 Hz), 114.4 (d, J = 22.4 Hz), 61.6.
+
ESI Cacld. for C15H10FN2O4S2 (MꢀH ): 365.0071; found 365.0081.
SO H
Cl
3
H
N
S
O
N
13
1
1
3: H NMR (500 MHz, DMSO) δ 8.19 (d, J = 1.8 Hz, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.97 (d, J = 7.9
13
Hz, 1H), 7.77ꢀ7.41 (m, 2H), 7.35ꢀ7.28 (m, 3H), 5.65 (s, 1H). C NMR (DMSO) δ 166.2, 157.6, 148.5,
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Journal of Medicinal Chemistry
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33.9, 132.0, 132.0, 131.5, 128.8, 128.5, 126.2, 126.1, 123.5, 121.7, 120.6, 65.9. ESI Cacld. for
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
C15H10ClN2O4S2 (MꢀH ): 380.9776; found 380.9773.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SO H
Me
3
H
N
S
O
N
14
1
1
4: H NMR (500 MHz, DMSO) δ 12.46 (br, 1H), 7.98ꢀ7.96 (m, 2H), 7.75 (d, J = 8.1 Hz, 1H), 7.43 (t, J
13
=
7.3 Hz, 1H), 7.30 (t, J = 7.3 Hz, 1H), 7.15ꢀ7.14 (m, 3H), 5.34 (s, 1H). C NMR (DMSO) δ 167.1,
157.7, 148.5, 137.1, 132.7, 131.4, 129.6, 129.5, 126.9, 126.1, 125.0, 123.5, 121.7, 120.5, 65.7, 19.8. ESI
+
Cacld. for C16H13N2O4S2 (MꢀH ): 361.0322; found 361.0328.
SO H
3
H
F
N
S
O
N
15
1
1
5: H NMR (500 MHz, DMSO) δ 12.28 (s, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.51
(d, J = 10.0 Hz, 1H), 7.43 (t, J = 7.4 Hz, 1H), 7.35ꢀ7.29 (m, 3H), 7.12 (t, J = 7.1 Hz, 1H), 5.15 (s, 1H);
13
C NMR (DMSO) δ 166.6, 161.5 (d, J = 240.0 Hz, 1H), 157.5, 148.5, 136.9 (d, J = 7.9 Hz), 131.5,
1
29.2 (d, J = 7.9 Hz), 126.2, 126.1, 123.5, 121.7, 120.6, 116.5 (d, J = 22.3 Hz), 114.0 (d, J = 21.0 Hz),
+
69.6. ESIꢀHRMS Cacld.for C15H10FN2O4S2 (MꢀH ): 365.0071; found 365.0071.
SO H
3
H
Me
N
S
O
N
16
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Page 20 of 48
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
6: H NMR (500 MHz, DMSO) δ 7.97 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.45ꢀ7.36 (m, 3H),
13
7
.30 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H), 5.02 (s, 1H), 2.30 (s, 3H). C
NMR (DMSO) δ 167.7, 157.5, 148.4, 136.4, 134.2, 131.4, 130.4, 127.9, 127.5, 127.0, 126.1, 123.5,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
21.7, 120.5, 70.3, 21.1. ESI Cacld. for C16H13N2O4S2 (MꢀH ): 361.0322; found 361.0333.
SO H
3
H
NC
N
S
O
N
17
1
17: H NMR (500 MHz, DMSO) δ 8.06 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.78ꢀ
13
7
.75 (m, 2H), 7.55 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.3 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 5.25 (s, 1H). C
NMR (DMSO) δ 166.4, 157.5, 148.5, 135.9, 135.1, 133.4, 131.5, 130.9, 128.9, 126.2, 123.6, 121.7,
+
120.6, 118.9, 110.5, 69.3. ESI Cacld. for C16H10N3O4S2 (MꢀH ): 372.0118; found 372.0129.
SO H
3
H
O N
N
S
2
O
N
18
1
1
8: H NMR (500 MHz, DMSO) δ 8.58 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.99ꢀ7.94 (m, 2H), 7.77 (d, J =
13
8.1 Hz, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 5.37 (s, 1H). C
NMR (DMSO) δ 166.3, 157.5, 148.5, 147.2, 137.0, 136.4, 131.5, 129.0, 126.2, 124.6, 123.6, 122.2,
+
1
21.7, 120.6, 69.2. ESI Cacld. for C15H10N3O6S2 (MꢀH ): 392.0017; found 392.0026.
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Journal of Medicinal Chemistry
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SO H
3
H
F C
3
N
S
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
1
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
9: H NMR (500 MHz, DMSO) δ 8.03 (s, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.76
(
d, J = 8.1 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.30 (d, J
1
3
= 7.6 Hz, 1H), 5.29 (s, 1H). C NMR (125 MHz, DMSO) δ 166.5, 157.5, 148.5, 135.6, 134.2, 131.5,
1
28.6, 128.3 (q, J = 31.0 Hz), 126.4 (d, J = 3.6 Hz), 126.2, 124.4 (q, J = 270.4 Hz), 124.0 (d, J = 3.4
+
Hz), 123.6, 121.7, 120.6, 69.6. ESI Cacld. for C16H10F3N2O4S2 (MꢀH ): 415.0040; found 415.0046.
SO H
3
H
N
S
O
N
Me
2
0
1
2
0: H NMR (500 MHz, DMSO) δ 7.97 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.47ꢀ7.42 (m, 3H),
13
7
.30 (t, J = 7.6 Hz, 1H), 7.11 (d, J = 7.8 Hz, 2H), 5.02 (s, 1H), 2.28 (s, 3H). C NMR (DMSO) δ 167.2,
157.6, 148.4, 136.4, 131.4, 131.3, 129.7, 128.1, 126.1, 123.5, 121.7, 120.5, 70.0, 20.7. ESI Cacld. for
+
C16H13N2O4S2 (MꢀH ): 361.0322; found 361.0330.
SO H
3
H
N
S
O
N
Br
2
1
1
21: H NMR (500 MHz, DMSO) δ 12.28 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H),
13
7
.55ꢀ7.50 (m, 4H), 7.43 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 7.4 Hz, 1H), 5.12 (s, 1H); C NMR (DMSO) δ
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Page 22 of 48
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
66.6, 157.5, 148.5, 133.7, 132.1, 131.5, 130.4, 126.1, 123.5, 121.7, 120.6, 120.6, 69.5. ESIꢀHRMS
+
Cacld. for C15H10BrN2O4S2 (MꢀH ): 424.9271; found 424.9270.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SO H
3
H
N
S
O
N
22
Cl
1
2
2: H NMR (500 MHz, DMSO) δ 12.57 (s, 1H), 7.92ꢀ7.25 (m, 8H), 5.04 (s, 1H). ESIꢀHRMS Cacld. for
+
C15H10ClN2O4S2 (MꢀH ): 380.9776; found 380.9777.
SO H
3
H
N
S
O
N
23
1
2
3: H NMR (500 MHz, DMSO) δ 12.32 (s, 1H), 7.77ꢀ7.24 (m, 8H), 5.07 (s, 1H). ESIꢀHRMS Cacld. for
+
C16H13N2O4S2 (MꢀH ): 361.0322; found 361.0316.
SO H
3
H
N
S
O
N
2
4 MeO
1
2
4: H NMR (500 MHz, DMSO) δ 12.40 (br, 1H), 7.59ꢀ6.98 (m, 8H), 5.10 (s, 1H), 3.91 (br, 3H). ESIꢀ
+
HRMS Cacld. for C16H13N2O5S2 (MꢀH ): 377.0271; found 377.0266.
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Journal of Medicinal Chemistry
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SO H
3
H
N
S
O
N
2
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
25: H NMR (500 MHz, DMSO) δ 12.24 (br, 1H), 7.99ꢀ7.29 (m, 7H), 4.94 (s, 1H), 2.30 (6H). ESIꢀ
+
HRMS Cacld. for C17H15N2O4S2 (MꢀH ): 375.0479; found 375.0473.
SO H
3
H
N
S
O
N
F
26
1
26: H NMR (500 MHz, DMSO) δ 12.35 (br, 1H), 7.88ꢀ7.28 (m, 8H), 5.07 (s, 1H). ESIꢀHRMS Cacld.
+
for C15H10FN2O4S2 (MꢀH ): m/z 365.0071; found 365.0071.
SO H
3
H
N
S
O
N
7
Cl
2
1
27: H NMR (500 MHz, DMSO) δ 12.35 (br, 1H), 8.10 (s. 1H), 7.72ꢀ7.27 (m, 7H), 5.05 (s, 1H). ESIꢀ
+
HRMS Cacld. for C15H10ClN2O4S2 (MꢀH ): 380.9776; found 380.9768.
SO H
3
H
N
S
O
N
NO2
28
1
2
8: H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 9.00 (s. 1H), 8.21ꢀ7.24 (m, 7H), 5.09 (s, 1H). ESIꢀ
+
HRMS Cacld. for C15H10N3O6S2 (MꢀH ): 392.0017; found 392.0014.
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Page 24 of 48
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
SO H
3
H
N
S
O
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
9
1
2
9: H NMR (500 MHz, DMSO) δ 12.16 (s, 1H), 7.70ꢀ7.23 (m. 8H), 5.00 (s, 1H), 2.35 (s, 3H). ESIꢀ
+
HRMS Cacld. for C16H13N2O4S2 (MꢀH ): 361.0322; found 361.0315.
SO H
3
H
N
S
O
N
OCF3
30
1
3
0: H NMR (500 MHz, DMSO) δ 12.35 (s, 1H), 8.05ꢀ7.25 (m. 8H), 5.04 (s, 1H). ESIꢀHRMS Cacld. for
+
C16H10F3N2O5S2 (MꢀH ): 430.9989; found 430.9993.
SO H
3
H
N
S
O
N
OMe
31
1
3
1: H NMR (500 MHz, DMSO) δ 12.05 (s, 1H), 7.49ꢀ6.93 (m. 8H), 4.94 (s, 1H), 3.70 (s, 3H). ESIꢀ
+
HRMS Cacld. for C16H13N2O5S2 (MꢀH ): 377.0271; found 377.0268.
SO H
3
H
N
S
O
N
3
CO Et
2
2
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Page 25 of 48
Journal of Medicinal Chemistry
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7
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
2: H NMR (500 MHz, DMSO) δ 12.47 (s, 1H), 8.59 (s, 1H), 7.95ꢀ7.25 (m, 8H), 5.10 (s, 1H), 4.26 (br,
+
2
H), 1.27 (br, 3H). ESIꢀHRMS Cacld. for C18H15N2O6S2 (MꢀH ): 419.0377; found 419.0374.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
S
O
N
NO2
33
1
3
3: H NMR (500 MHz, DMSO) δ 8.70 (d, J = 2.3 Hz, 1H), 8.26 (dd, J = 2.4, 9.0 Hz, 1H), 7.73 (d, J =
1
3
9
.0 Hz, 1H), 7.38ꢀ7.31 (m, 5H); C NMR (DMSO) 170.1, 162.4, 153.0, 143.9, 132.5, 129.4, 129.3,
+
128.0, 121.9, 120.7, 118.1, 43.2. ESIꢀHRMS Cacld. for C15H10N3O3S (MꢀH ): 312.0448; found
3
12.0444.
Protein Expression and Purification. Expression and purification of the Hisꢀtagged LMWꢀPTP
3
9
isoform A was performed as described elsewhere. LMWꢀPTPA was expressed in BL21 E coli cells.
Protein expression was induced by 0.6 mM IPTG and harvested after overnight incubation at 25 °C. The
cells were pelleted by centrifugation at 5,000 rpm and resuspended in 30 ml of 100 mM Tris buffer, pH
7
.8, with 500 mM NaCl and 10 mM imidazole. The suspensions were frozen and thawed before physical
lysis by a French Press. The bacterial lysate was centrifuged at 15,000 rpm at 4 °C for 30 min. The
supernatant was collected and incubated with 2 ml of NiꢀNTA resin by endꢀoverꢀend rotation for 1 h at
4
°C. The beads were washed with the same Tris buffer with 20 mM imidazole to remove nonspecific
binding. LMWPTP was eluted with 250 mM imidazole in the same Tris buffer. The protein was
concentrated and further purified by going through a Sephadex G75 column. The other PTPs for
selectivity studies were expressed and purified from E. coli by the same method used for LMWꢀPTP.
These PTPs included SHP2, PTP1B, TCꢀPTP, SHP1, LYP, HePTP, Meg2, PTPH1, FAPꢀ1, PTPꢀPEST,
CD45, LAR, PTPα, PTPβ, PTPγ, PTPε, PTPσ, PTPꢁ, MKP3, VHR, VHZ, CDC14A, SSu72, and PP5.
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5
5
5
5
6
Crystallization, Data Collection, and Structure Determination. Protein crystals were first grown
in 100 mM MES buffer, 30% PEGME 5,000 and 0.2 M Ammonium sulfate, the same buffer used
3
9
elsewhere in the presence of 1 mM compound 7. Crystallization conditions were systematically
scanned against a number of different buffers instead of MES and eventually the optimal condition was
finalized for crystal growth of the LMWPTP•7 complex. The buffer grid is 25ꢀ30% PEGME 5,000 in
0
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00 mM BisꢀTris, pH 6.0ꢀ6.5. The data was collected at the APS 19BM or 19ID beamlines (Argonne,
IL). Initial phases of the LMWPTP complexes were determined by molecular replacement with the
CCP4 software package. The published LMWPTP structure (5PNT) was used as the initial search model.
After one round of refinement by Refmac in the CCP4 package, further refinement was carried out in
Phenix.
Determination of Inhibition Constant and IC . PTP activity was measured using pꢀnitrophenyl
50
phosphate (pNPP) as a substrate in 50 mM 3,3ꢀdimethylglutarate (DMG) buffer, pH 7.0 at 25 °C. The
reaction was initiated by the addition of enzyme to a reaction solution containing 2ꢀ5 mM pNPP with
different concentrations of inhibitors. The reactions were incubated at room temperature for 5ꢀ10 min
and quenched with 5N NaOH. The nonenzymatic hydrolysis of pNPP was corrected by measuring the
control without the addition of enzyme. The amount of product pꢀnitrophenol was determined from the
absorbance at 405 nm detected by a Spectra MAX340 microplate spectrophotometer (Molecular
ꢀ1
ꢀ1
Devices) using a molar extinction coefficient of 18,000 M cm . IC was calculated by fitting the data
5
0
using SigmaPlot. To determine the mode of inhibition, the reactions were initiated by addition of
enzyme to the reaction solution containing different concentrations of pNPP with different
concentrations of inhibitor. Similarly, K was obtained by fitting the data using SigmaPlot Enzyme
i
Kinetics Module. In addition to LMWꢀPTP, other PTPs evaluated included the receptorꢀlike PTPs,
CD45, PTPα, PTPβ, PTPꢁ, PTPε, LAR, PTPσ and PTPγ, cytosolic PTPs, PTP1B, TCꢀPTP, Lyp, SHP1,
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SHP2, PTPH1, HePTP, PTPꢀMeg2, PTPꢀFAP1, and PTPꢀPEST, the dual specificity phosphatase VHR,
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VHZ, MKP3, CDC14A, RNA Polymerase II CTD Phosphatase, and protein phosphatase 5. The IC50
determination for these PTPs was performed under the same conditions as used for LMWꢀPTP except
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that the pNPP concentrations used corresponded to the K values of the PTPs studied and the enzyme
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concentrations ranged from 5ꢀ20 nM.
Cell culture and immunoblot analysis. HepG2 cells were cultured at 37 °C and 5% CO in
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Dulbecco’s modified Eagle’s medium (Corning) supplemented with 10% fetal bovine serum (HyClone).
The 60ꢀ80% confluent HepG2 cells were starved overnight in serum free medium, followed by
treatment with vehicle or compound for 2h, and then either left unstimulated or stimulated with 50 nM
insulin (Sigma) for 30 min. Cells were lysed and the lysates were electrophoresed on a 10%
polyacrylamide gel, transferred to a nitrocellulose membrane, and blotted with antiꢀphosphoꢀAKT, antiꢀ
AKT antibodies (Cell Signaling). The blots were developed by the enhanced chemiluminescence
technique using the SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific). The
protein levels of phosphoꢀAKT and AKT were quantified by Image J software, and the ratios of
pAKT/AKT were normalized to the vehicle control.
Acknowledgement: This work was supported in part by National Institutes of Health Grants CA69202
and P30CA023168.
Ancillary Information
Data deposition: The coordinates for the structures of LMWꢀPTP
ꢀ7 (accession number 5KQG), LMWꢀ
PTP 9 (accession number 5KQL), LMWꢀPTP MES (accession number 5KQM), and the apoꢀform
ꢀ
ꢀ
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LMWꢀPTP (accession number 5KQP) have been deposited in the Protein Data Bank. Authors will
release the atomic coordinates and experimental data upon article publication.
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*
To whom correspondence should be addressed. Phone: (765) 494ꢀ1403; Eꢀmail: zhangꢀzy@purdue.edu
These authors contributed equally to the work.
#
Abbreviations footnote: LMWꢀPTP, low molecular weight protein tyrosine phosphatase; PTP, protein
tyrosine phosphatase; SPAA, SulfoPhenyl Acetic Amide.
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References
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