From the Departments of Molecular Pharmacology and
§ Biochemistry, Albert Einstein College of Medicine,
Bronx, New York 10461
Received for publication, December 9, 2002
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ABSTRACT |
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Protein-tyrosine phosphatase 1B
(PTP1B) has been implicated as an important regulator in several
signaling pathways including those initiated by insulin and leptin.
Potent and specific PTP1B inhibitors could serve as useful tools in
elucidating the physiological functions of PTP1B and may constitute
valuable therapeutics in the treatment of several human diseases. We
have determined the crystal structure of PTP1B in complex with compound
2, the most potent and selective PTP1B inhibitor reported to
date. The structure at 2.15-Å resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal
noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural
data are further corroborated by results from kinetic analyses of the
interactions of PTP1B and its site-directed mutants with compound 2 and
several of its variants. Although many of the residues important for
interactions between PTP1B and compound 2 are not unique to PTP1B, the
combinations of all contact residues differ between PTP isozymes, which
provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by
residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.
Protein-tyrosine phosphatases
(PTPs)1 are signaling enzymes
that regulate a wide variety of cellular processes including cell growth, differentiation, metabolism, progression through the cell cycle, cell-cell communication, cell migration, gene transcription, ion
channel activity, the immune response, and apoptosis/survival decisions. Evidence suggests that PTPs can exert both positive and
negative effects on signaling pathways and play crucial physiological roles in a variety of mammalian tissues and cells. Defective or inappropriate regulation of PTP activity can lead to aberrant tyrosine
phosphorylation, which contributes to the development of many human
diseases including cancers and diabetes (1, 2).
Given the complexity of cellular signaling and the large number of
phosphoproteins in the cell, it is likely that a single PTP may be
involved in the regulation of multiple signaling pathways, and that
multiple PTPs may act cooperatively to regulate a particular pathway.
For example, PTP1B is regarded as a major negative regulator of both
insulin- and leptin-stimulated signal transduction pathways (3-6),
which suggests that specific PTP1B inhibitors may enhance insulin and
leptin sensitivity and act as effective therapeutics for the treatment
of type II diabetes, insulin resistance, and obesity. In addition to a
role in insulin and leptin signaling, PTP1B is also implicated in
several other physiological and pathological processes including
transformation by the Neu oncogene (7), Src kinase
activation (8), antagonizing signaling by the EGFR (9, 10), and the
oncoprotein p210bcr-abl (11), and the
negative regulation of integrin-mediated adhesion and signaling by
binding to and dephosphorylating A major goal in the PTP field is to establish the precise functional
roles for individual PTPs, both in normal cellular physiology and in
pathogenic conditions. This is an important prerequisite for PTP-based
drug discovery in order to minimize unwanted side effects. Potent and
selective PTP inhibitors would be extremely useful in helping to
delineate the physiological roles of PTPs and to validate them as
therapeutic targets. Furthermore, these inhibitors could provide the
foundation upon which therapeutically useful agents can be devised.
Unfortunately, as a consequence of the conserved nature of the
PTP-active sites (i.e. Tyr(P)-binding sites), there are
currently few PTP inhibitors that exhibit the potency and specificity
required for biological and pharmacological investigation.
Fortunately, PTP substrate specificity studies have shown that Tyr(P)
alone is not sufficient for high affinity binding and that residues
flanking Tyr(P) furnish additional interactions for specific and high
affinity substrate recognition (14-20). These studies, together with
the discovery of a second aryl phosphate-binding site adjacent to the
active site in PTP1B (21), demonstrated the potential for multiple site
recognition of substrates by PTPs. They also suggested a novel paradigm
for the design of potent and specific PTP inhibitors, namely bidentate
ligands that bind to both the active site and a unique adjacent
peripheral site. By using this approach we have acquired a highly
potent and selective PTP1B inhibitor (Fig. 1, compound
1), which displays a Ki value of 2.4 nM for PTP1B and exhibits several orders of magnitude
selectivity in favor of PTP1B against a panel of PTPs (22). Compound
1 is the most potent and selective PTP1B inhibitor reported
to date. This result serves as a proof-of-concept in PTP inhibitor
development, as it demonstrates the feasibility of acquiring potent,
yet highly selective, PTP inhibitory agents. In order to gain insight
into the structural basis of the potency and selectivity of compound
1 for PTP1B, and to lay the groundwork for the future design
of more potent and specific inhibitors that can interfere with the
processes controlled by PTP1B and other PTPs, we have determined the
crystal structure of PTP1B in complex with compound 2 (Fig.
1). Compounds 1 and 2 exhibit identical potency
and selectivity profiles. In addition, we used a combination of kinetic
studies and site-directed mutagenesis to probe the binding interactions
between compound 1 (and several analogs thereof) and
PTP1B.
Materials--
p-Nitrophenyl phosphate
(pNPP) was purchased from Fluka. All mutant forms of PTP1B
were generated using the QuickChange kit from Stratagene (23). The
oligonucleotide primers used for mutagenesis were from Sigma. All of
the mutations were confirmed by DNA sequencing. The catalytic domain of
PTP1B (residues 1-321) and its mutants were expressed in
Escherichia coli and purified to homogeneity as described
previously (21, 23). The synthesis of compound 1 was
reported in Ref. 22. The preparation of compounds 3 and
5 was described in Refs. 24 and 23, respectively.
Solid-phase Synthesis of Compounds 2 and
4--
Compounds 2 and 4 were synthesized
on the Rink amide resin using a standard protocol for
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N-hydroxybenzotriazole/N-methylmorpholine activation of carboxylic acids. The coupling reaction was
performed in N,N-dimethylformamide for 1.5 h
using a 3-fold excess of acid relative to resin-bound amine. The
following compounds were sequentially coupled to the Rink resin to
furnish compound 2:
N-
The following compounds were sequentially coupled to the Rink resin to
furnish compound 4:
Fmoc-4-phosphonodifluoromethyl-L-phenyalanine with an
unprotected side chain, Fmoc-L-tyrosine with a
tert-butyl protected side chain, and
4-phosphonodifluoromethylphenylacetic acid (23). Fmoc removal was
effected by 20% piperidine in
N,N-dimethylformamide. Final cleavage and side
chain deprotection was achieved by treatment with a mixture of 2.5%
triisopropylsilane, 2.5% water, and 95% trifluoroacetic acid at room
temperature for 2.5 h. The resin was removed by filtration, and
the remaining solution was concentrated. The residue was triturated
with ether, dissolved in water, and purified by semi-preparative
reverse-phase high performance liquid chromatography (0.1%
trifluoroacetic acid in water and acetonitrile) to afford the desired
compound. The NMR and mass spectroscopy data for compound 4:
1H NMR (300 MHz, D2O): Kinetic and Inhibition Studies--
Kinetic parameters for
PTP1B-catalyzed hydrolysis of pNPP and inhibition constants
for PTP1B inhibitors were determined as described previously (23). All
experiments were performed at 30 °C in 50 mM
3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM EDTA
with an ionic strength of 0.15 M adjusted with NaCl.
Co-crystallization of PTP1B with the Inhibitors--
All
crystallization experiments were carried out at 4 °C using either
the hanging drop or the sitting drop vapor diffusion methods. Native
PTP1B crystals were grown according to published conditions (25). In
soaking experiments, PTP1B crystals were immersed in crystallization
solutions containing PTP1B inhibitors over a range of concentrations
(from 0.3 to 1 mM) overnight. For co-crystallization, stock
solutions of PTP1B in 100 mM MES (pH 6.5), 25 mM NaCl, 0.2 mM EDTA, and 3.0 mM
dithiothreitol were prepared with 1 mM PTP1B inhibitor. In
each well, 2 µl of 10 mg/ml PTP1B solution was mixed with an equal
volume of the precipitating solution (90 mM sodium
cacodylate (pH 6.0-7.5), 90 mM magnesium acetate, 5%
Jeffamine 600, and 23.3% (w/v) polyethylene glycol 8000), and the
mixture was equilibrated against 0.5 ml of the precipitating solution.
Data Collection and Processing--
A single crystal was washed
several times in immersion oil type B (Cargille Laboratories) and
flash-frozen in a stream of nitrogen gas at Structure Solution--
Diffraction from the PTP1B/compound
2 co-crystal is consistent with the space group
P212121 (a = 52.84 Å, b = 85.62 Å, and c = 88.68 Å).
Although similar to crystals of PTP1B bound to the consensus peptide
ELEFpYMDYE-NH2 (where pY is Tyr(P)) (20), a 3.5-Å
difference in the b axis (~4%) precluded structure
solution by difference Fourier method. Accordingly, the structure was
solved by molecular replacement using the structure of PTP1B/C215S with the consensus peptide as the search model (20), excluding solvent molecules, ions, and the peptide. Rotation and translation searches with program CNS (27) using data in the 30-4-Å resolution
range yielded an unambiguous solution with a correlation coefficient of
0.623 and a packing value of 50.22%, respectively. After one cycle of
rigid body refinement and simulated annealing, both
2Fo Crystallization and Crystal Packing--
Initial crystallization
experiments were performed with compound 1 (Fig.
1). After the stock of compound
1 was depleted, subsequent crystallization trials were
conducted with compound 2 (Fig. 1), a synthetic intermediate
used to prepare fluorescently labeled or cell-permeable analogs of
compound 1. Compound 2 displays the same potency
(Ki = 1.8 nM) and selectivity toward
PTP1B as compound 1 (Ki = 2.4 nM). This is consistent with our previous observations that attachment of an ethyl thiol group to the C-terminal carboxyamide of
Tyr(P) did not affect the affinity of substrates for PTP1B (22).
Soaking native PTP1B crystals with either compound 1 or
2 significantly changed the diffraction patterns, indicating that both inhibitors could bind PTP1B in the crystalline state; however, these data were difficult to process due to high mosaicity of
the crystals. PTP1B could be crystallized as very thin needles in the
presence of the inhibitor at a 1:3 ratio in the same crystallization conditions of the native protein (25). Microseeding or macroseeding produced larger needles, although the best crystal of this form exhibited diffraction to only 3.2 Å. After an extensive search of
crystallization conditions, the additive Jeffamine 600 (90 mM sodium cacodylate, pH 6.0, 90 mM magnesium
acetate, 5% Jeffamine 600, and 23.3% (w/v) polyethylene glycol 8000)
resulted in high quality crystals for the complex of PTP1B with
compound 2 that diffracted to 2.15-Å resolution.
The Overall Structure of PTP1B·2 Complex--
The
final model for the PTP1B·2 complex included PTP1B
residues 2-298 and all atoms in compound 2 except the 1-carbamoyl-2-mercaptoethylcarbamoyl portion in the C-terminal extension. The structure was refined to an R-factor of 20.2%
(Rfree = 24.0%) for data from 30 to 2.15 Å (Table
I). Compound 2 was
unambiguously fitted to well defined electron density (Fig. 2). The overall structure of PTP1B in the
complex is very similar to those observed in the complexes between
PTP1B and Tyr(P)-containing peptides (16, 20, 29). As observed in other
complexes, the WPD loop (residues 179-189) adopts a closed
conformation in the PTP1B·2 structure. Compound
2 extensively interacts with PTP1B in a defined manner such
that phosphonodifluoromethylphenylalanine (F2Pmp) occupies
the active site, the linker Asp forms interactions with residues Arg-47
and Asp-48, and the distal 4-phosphonodifluoromethyl phenylacetyl group
makes both van der Waals and ionic contacts with Arg-47 and Lys-41
(Fig. 3). These assignments are in
complete agreement with predictions based on biochemical and mutational analyses (23). Although the first five methylene units extending from
the carboxyamide of F2Pmp are visible in the electron
density map, they make no significant interactions with PTP1B,
consistent with the finding that compounds 1 and
2 exhibit the same potency and selectivity against
PTP1B.
Interactions between F2Pmp and the Active
Site--
The F2Pmp in compound 2 assumes the
same position in the active site of PTP1B as that observed for Tyr(P)
in PTP1B-phosphopeptide complexes. The three terminal phosphonate
oxygens form an extensive array of hydrogen bonds with the main chain
nitrogens of the phosphate-binding loop (residues 215-221) and the
guanidinium side chain of Arg-221 (Fig.
4a). The phenyl ring of
F2Pmp is sandwiched between the side chains of Tyr-46 and
Phe-182 and makes van der Waals contacts with the side chains of
Ala-217, Ile-219, Val-49, and Gln-262 (Fig. 4b). These
interactions are observed in almost all PTP1B complexes containing
Tyr(P) in the active site.
Unique to F2Pmp, which is a nonhydrolyzable mimic of
Tyr(P), is the difluoromethylene group that replaces the phenolic
oxygen in Tyr(P). The two fluorine atoms at the benzylic position are known to be important for high affinity binding to PTP1B. For example,
when Tyr(P) in the hexapeptide DADEpYL-NH2 is replaced with
F2Pmp, the Ki for the resulting peptide
(200 nM for PTP1B) is over 1000 times more potent than the
same peptide containing phosphonomethylphenylalanine (Pmp) (30-32).
The structure of PTP1B·2 provides direct evidence for the
proposed specific interactions between the fluorine atoms and PTP1B
active site residues (31). As shown in Fig. 4c, the fluorine
atoms and one of the terminal oxygens of the phosphonate are
hydrogen-bonded to an enzyme-bound water molecule that is coordinated
by the main chain nitrogen of Phe-182 and the amide side chain of
Gln-266. A similar water molecule is also observed in several
PTP1B-Tyr(P) peptide and PTP1B-difluorophosphonate complexes (20,
33-35). In addition to the H-bonds, the fluorine atoms are also
involved in van der Waals interactions (17 total, 8 of which are in the range of 3.5-4.0 Å, the remainders are within 4.5 Å) with the side
chains of Phe-182, Asp-181, and Gln-262.
Interactions between Compound 2 and PTP1B beyond the
Active Site--
In addition to the interactions between
F2Pmp and the active site, there are extensive and specific
interactions between several PTP1B surface residues beyond the active
site with the remainder of compound 2. Fig. 4a
shows specific polar interactions between compound 2 and
PTP1B. Most notably, Asp-48 forms two hydrogen bonds to the main chain
nitrogen and the C-terminal amide of F2Pmp. In addition,
Arg-47 plays a critical role for high affinity binding of compound
2. The side chain of Arg-47 is highly flexible and can adopt
different conformations depending on the nature of the residues
immediately N-terminal to Tyr(P) (20). In the structure of
PTP1B·2, Arg-47 is found in a conformation that is
distinct from those observed in previous studies, enabling Arg-47 to
make contacts with both the Asp linker and the distal aryl
difluorophosphonate group. Specifically, the main chain nitrogen of
Arg-47 makes a hydrogen bond with the carbonyl of the
4-phosphonodifluoromethylphenylacetyl group, whereas the guanidinium
group of Arg-47 forms two hydrogen bonds with the Asp side chain of
compound 2. A polar interaction between the N
In addition to electrostatic interactions, there are also van der Waals
contacts between PTP1B and the Asp and 4-phosphonodifluoromethyl phenylacetyl group in compound 2 (Fig. 4b). Thus,
C Mutational Analysis of the Interactions between PTP1B and Its
Inhibitors--
The contributions of several surface residues to
ligand binding were evaluated by site-directed mutagenesis with several
PTP1B inhibitors including compounds 1, 3, and
4 (Fig. 1). The wild-type and mutant PTP1Bs were expressed
in E. coli and purified to near-homogeneity as described
(23). All kinetic measurements were performed at 30 °C at pH 7.0, 50 mM 3,3-dimethylglutarate buffer, containing 1 mM EDTA and an ionic strength of 0.15 M. As
shown in Table II, with the exception of Y46A and F182A, no significant differences in the kinetic parameters (kcat and Km) for
pNPP hydrolysis and the Ki values for
vanadate were observed for the wild-type and mutant PTP1Bs, indicating
no structural perturbations in the active site by the mutations.
Because Tyr-46 and Phe-182 are involved in hydrophobic stacking with
Tyr(P), drastic alteration of the side chain may affect the precise
positioning of active site residues important for catalysis (18,
23).
This ensemble of mutants was also examined for the binding of compounds
1, 3, and 4. Compound 3 is
a previously characterized competitive inhibitor of PTP1B with a
Ki value of 1 µM (24). Compound
4 differs from compound 1 in that the linker is
Tyr instead of Asp. All compounds inhibited the reaction catalyzed by
wild-type and mutant PTP1Bs reversibly and competitively with respect
to the substrate. For comparison, the Ki values of
1, 3, and 4 for PTP1B and the mutants
are listed in Table II. The selectivity profile of compound
4 for PTP1B against a panel of protein phosphatases is shown
in Table III.
Several years ago we identified a second aryl phosphate-binding
site in PTP1B and proposed that this region of the protein could be
targeted for the design of potent and selective PTP1B inhibitors (21).
This concept has galvanized substantial efforts in academic
laboratories and pharmaceutical companies to develop PTP1B inhibitors
by tethering together two small ligands that could address both the
active site and the second aryl phosphate-binding site. Recent results
from Novo Nordisk (36) and Abbott Laboratories (37) demonstrated that
this is indeed a viable approach to obtaining potent and selective
PTP1B inhibitors.
Other efforts aimed at targeting both the active site and the second
aryl phosphate-binding site have yielded numerous bis-aryl difluorophosphonate inhibitors (e.g. compound 3)
that display low µM affinity and reasonable selectivity
for PTP1B (24, 38, 39). The structure of PTP1B in complex with a
bis-aryl difluorophosphonate indicates that the distal phosphonate does
not bind to the second aryl phosphate-binding site but rather extends
into the solvent and makes water-mediated ionic interactions with the
guanidinium group of Arg-47 (34). The central F2Pmp group
in the tripeptide inhibitor
benzoyl-Glu-F2Pmp-F2Pmp-NH2 also
binds in a solvent-exposed area close to Arg-47 (35). Unfortunately, no
specific atomic interactions between this inhibitor and PTP1B could be
defined due to a high degree of flexibility in that area.
Benzoyl-Glu-F2Pmp-F2Pmp-NH2 was
reported to exhibit an IC50 of 6 nM for both
PTP1B and TC-PTP (40); however, this apparent affinity may have
been overestimated as the measurements were made under no or low salt
conditions. We noted earlier that the Ki values for
PTPs are highly sensitive to the ionic strength of assay buffers (22).
Accordingly, the IC50 of
benzoyl-Glu-F2Pmp-F2Pmp-NH2 for
PTP1B under physiological conditions (ionic strength of 0.15 M) is estimated to be around 60 nM. This
affinity is nearly 30-fold lower than those of compounds 1 and 2, which may explain the lack of strong interactions between the central F2Pmp and PTP1B in the structure of
PTP1B/benzoyl-Glu-F2Pmp-F2Pmp-NH2. Consistent with the higher affinity of compounds 1 and 2 for PTP1B, the electron density corresponding to compound 2 and its contact residues in PTP1B are well defined in the
structure of PTP1B·2 and highlight extensive interactions involving both the Asp linker and the distal aryl difluorophosphonate, in addition to those observed between the active site and the F2Pmp group.
Determinants for Potent and Specific Binding of Compound
1 to PTP1B--
The remarkable potency and selectivity of
compounds 1 and 2 for PTP1B are the results of
numerous specific interactions with PTP1B. It is clear from the
structure of PTP1B·2 that F2Pmp occupies the
active site in a manner similar to that of Tyr(P). Common interactions
for both F2Pmp and Tyr(P) with the active site include
hydrophobic interactions between the phenyl ring and the side chains of
Tyr-46, Val-49, Phe-182, Ala-217, Ile-219, and Gln-262 and polar
interactions between the phosphonate/phosphate and the backbone amides
of the phosphate-binding loop (residues 215-221) and the side chain of
Arg-221. Unique to F2Pmp are additional hydrogen bonding
interactions involving an ordered water molecule that links the main
chain nitrogen of Phe-182 and the amide side chain of Gln-266 with the
two benzylic fluorines and a terminal phosphonate oxygen. In addition,
the fluorine atoms are also involved in van der Waals interactions with
the side chains of Phe-182, Asp-181, and Gln-262 (Fig.
4c). All of these interactions contribute to the high
affinity of compound 1 for PTP1B, and the interactions with
the two fluorine atoms in particular may explain why F2Pmp is superior to Tyr(P) or Pmp for binding to the PTP active site. However, these residues that interact with F2Pmp are
unlikely major contributors to the extraordinary selectivity exhibited by compound 1 because they are highly conserved among PTPs. Phe-182 may be an exception as both His and Gln residues can be found
at this position.
Other residues adjacent to the active site, including Lys-41, Arg-47,
and Asp-48, are important for both potency and selectivity of compounds
1 and 2. The carboxylate of Asp-48 makes two
hydrogen bonds to the amide nitrogens on both sides of
F2Pmp in compound 2. In addition, the aliphatic
portion of Asp-48 is also within van der Waals contacts of the C
Residues Lys-41 and Arg-47 are found in a unique arrangement of
hydrogen-bonding network and hydrophobic interactions with the Asp
linker and the distal 4-phosphonodifluoromethylphenylacetyl moiety.
These residues are highly variable among the PTPs and are likely
responsible for the potency and selectivity displayed by compound
1. Extensive interactions are observed between Lys-41 and
the distal difluorophosphonate including electrostatic (between the
amino group of Lys-41 and a terminal oxygen and a fluorine of the
distal difluorophosphonate) and nonpolar (between the fluorine atoms of
the distal difluorophosphonate and the C
Arg-47 plays an important role in binding both the Asp linker and the
distal aryl difluorophosphonate group in compound 2. As
shown in Fig. 4, the main chain nitrogen of Arg-47 makes a hydrogen
bond with the carbonyl of 4-phosphonodifluoromethylphenylacetyl group, whereas the guanidinium group of Arg-47 participates in two
hydrogen bonds with the carboxylate of the Asp linker in compound 2. Furthermore, there are also hydrophobic interactions between the aliphatic portion of the side chain of Arg-47 and the
phenyl ring and the fluorine atoms of the 4-phosphonodifluoromethyl phenylacetyl group in compound 2. Understandably,
replacement of Arg-47 by an Ala, Val, Glu, or Lys, residues commonly
found at position 47 in other PTPs, results in 56-, 80-, 280-, and
12-fold decrease in binding affinity, respectively, for compound
1 (Table II). These results suggest that Arg-47 is probably
the most important determinant for PTP1B potency and selectivity. Compound 1 binds to the Y46A mutant 3,400-fold less strongly than the wild-type enzyme. This may be due to compounded effects on
both ablation of the
Much smaller effects (<4-fold) on the binding of compound 3 are observed for the Arg-47 mutants (Table II). This is consistent with
the weak interactions between the guanidinium group and the distal
phosphonate (34). Interestingly, when Arg-24, a residue situated in the
second aryl phosphate-binding pocket of PTP1B, is changed to an Ala, a
more than 3-fold reduction in the affinity of compound 3 for
PTP1B is observed, even though it does not have any effect on the
PTP1B-catalyzed pNPP hydrolysis and vanadate binding (Table
II). In contrast, no change in binding affinity of compound
2 is observed for R24A, in accordance with the finding that
the 4-phosphonodifluoromethylphenylacetic acid moiety in compound
2 does not interact with Arg-24. Together with the lack of
discrete electron density outside the active site of the
PTP1B·3
structure,2 the results raise
the possibility that the second difluorophosphonate moiety in compound
3 may be capable of binding in more than one orientation and
may be loosely associated with positively charged residues
(e.g. Arg-24 and Arg-47) on the surface of the enzyme.
The Flexibility of Arg-47--
PTP1B is somewhat promiscuous in
its substrate preference as it dephosphorylates a wide variety of
protein/peptide substrates with almost equal efficiency (42). The
structural basis for the plasticity in PTP1B substrate recognition
appears to be largely conferred by a single residue, Arg-47, which can
accommodate both acidic and hydrophobic residues (20). As depicted in
Fig. 5 and summarized in Table
IV, depending on the nature of the
residues immediately N-terminal to Tyr(P), the side chain of Arg-47 can adopt different conformations, generating distinct ligand binding surfaces. For example, when a Glu residue is positioned at the
The combination of an Asp at the Conclusions and Implications for PTP Inhibitor Design--
We have
determined the crystal structure of PTP1B in complex with the most
potent and selective PTP1B inhibitor identified to date. This
structure, together with results from kinetic analyses of the
interactions of PTP1B and its site-directed mutants with the inhibitor
and several of its variants, provides a molecular explanation for the
extraordinary potency and selectivity of compound 1. The
results show that whereas the nonhydrolyzable Tyr(P) mimic
F2Pmp occupies the active site, the rest of the molecule interacts with a distinct area involving residues Lys-41, Arg-47, and
Asp-48. Thus, in addition to the second aryl phosphate-binding pocket,
we have identified another area, positioned within the vicinity of the
active site, which can also be targeted to enhance potency and
selectivity for PTP1B. Furthermore, we predict that inhibitors with
even higher potency and selectivity can be devised by simultaneously
addressing both the new area (defined by Lys-41, Arg-47, and Asp-48)
and the second aryl phosphate-binding pocket (Fig. 3b).
Finally, the flexibility of Arg-47 should be exploited to incorporate
hydrophobic elements in the design strategy in order to improve the
physical properties of PTP1B inhibitors (e.g. cell
permeability). In principle, an identical approach used for PTP1B
(i.e. to develop inhibitors that target both the active site
as well as unique adjacent peripheral sites) could also be employed to
produce specific small molecule inhibitors for all members of the PTP
family that would enable the pharmacological modulation of selected
signaling pathways for treatment of various diseases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin (12) and
p130cas (Crk-associated substrate) (13).
These results, taken together, suggest that PTP1B may be a participant
in several signaling pathways. Conversely, in addition to PTP1B, both
leukocyte common antigen-related phosphate and PTP
have been
implicated as negative regulators of insulin signaling (2).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-[(9H-fluoren-9-yl methoxy)carbonyl]-S-trityl-L-cysteine,
6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid,
4-(difluorophosphonomethyl)-N-[(9H-fluoren-9-yl
methoxy)carbonyl]-L-phenylalanine, N-
-[(9H-fluoren-9-yl
methoxy)carbonyl]-L-aspartic acid-
-t-butyl ester, and 4-(phosphonodifluoromethyl)phenylacetic acid. Fmoc removal after each coupling was effected by 20% piperidine in N,N-dimethylformamide. Final cleavage and side
chain deprotection was achieved by treatment with trifluoroacetic acid
with 5% 1,2-ethanedithiol and 1% m-cresol at room
temperature for 3 h. Removal of the solvent in vacuo
gave a crude oil that was triturated with cold ether. The crude mixture
thus obtained was centrifuged; the ether was removed by decantation,
and the resulting white solid was purified by semipreparative
reverse-phase high performance liquid chromatography (H2O/CH3CN in 0.1% trifluoroacetic acid).
Compound 2 was obtained as a white solid by lyophilization.
1H NMR (300 MHz, CD3OD):
1.05 (m, 2H), 1.24 (m, 2H), 1.43 (m, 2H), 2.17 (t, 2H, J = 7.3 Hz),
2.58-3.00 (m, 10H), 3.52 (s, 2H), 4.32-4.53 (m, 3H), 7.18 (d, 2H, J = 8.0 Hz), 7.24 (d, 2H, J = 8.0 Hz), 7.44 (d, 2H, J = 7.3 Hz), 7.46 (d, 2H,
J = 7.3 Hz); 31P NMR (121.5 MHz,
CD3OD):
5.32 (t, J = 107.0 Hz);
19F NMR (282 MHz, CD3OD)
108.6 (d,
J = 107.0 Hz),
108.8 (d, J = 107.0 Hz); electrospray ionization-mass spectroscopy was a calculated mass of
873.2 and found at [M + H]+ 874.1.
7.63 (d,
J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz,
2H), 7.4 (d, J = 8.0 Hz, 2H), 7.2 (d, J = 8.0 Hz, 2H), 7.0 (d, J = 8.3 Hz, 2H), 6.7 (d,
J = 8.3 Hz, 2H), 4.7 (dd, J = 6.0, 9.3 Hz, 1H), 4.6 (dd, J = 6.0, 9.3 Hz, 1H), 3.63 (m, 1H), 3.56 (m, 1H), 3.3 (dd, J = 6.0, 14 Hz, 1H), 3.07 (dd,
J = 9.3, 14 Hz, 1H), 3.02 (dd, J = 6.0, 14 Hz, 1H), 2.8 (dd, J = 9.3, 14 Hz, 1H);
13C NMR (75 MHz, D2O):
175, 174, 173, 155, 139, 137, 133 (m), 130.6, 129.6, 129.2, 128.2, 126(m), 120(m), 116, 55.2, 54.6, 42, 37, 36; 31P NMR (121.5 MHz,
D2O):
5.40 (t, J = 106 Hz), 5.33 (t,
J = 106 Hz); electrospray ionization-mass spectroscopy
was calculated at 705 and found at 706.1.
178 °C. Data were
collected using a wavelength of 0.98 Å, and 420 frames of 0.5° were
recorded on a MARCCD detector at beamline X9A of the National
Synchrotron Light Source (Brookhaven National Laboratory). The data
were processed with the programs DENZO and SCALEPACK (26). Data
reduction statistics are given in Table I.
Fc and
Fo
Fc electron density maps showed clear density for compound 2 in and adjacent to the
active site of PTP1B. Subsequent refinement consisted of manual rebuilding, simulated annealing, and individual temperature factor refinement using all data from 30 to 2.15 Å. Model building was performed using Program O (28). Simulated annealing OMIT maps, Rfree values, PROCHECK, and WHAT IF were employed to ensure
correct model building and stereochemistry. The final model comprises residues 2-298, one inhibitor molecule, 110 ordered water molecules, 2 magnesium ions, and 2 acetate ions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
The chemical structures of compounds
1-5.
Crystallographic data and refinement statistics
View larger version (45K):
[in a new window]
Fig. 2.
Simulated annealing omit map showing
unbiased density for compound 2. The density shown is a
Fo Fc map contoured at 3.2
,
with the refined models of the inhibitor and PTP1B superimposed. This
figure was generated using SETOR (43).
View larger version (47K):
[in a new window]
Fig. 3.
Overall binding mode of compound 2 with
PTP1B. a, details of the interactions between compound
2 and PTP1B. The contact residues from PTP1B are shown in
pink. Compound 2 is depicted according to atom
type (O, red; N, dark blue; C, gray;
P, yellow; and F, green). The bound water
molecule is represented as a red sphere. b, GRASP
(44) surface representation of PTP1B in complex with compound
2. The blue, white, and red shadings
represent positively charged (+84 kT), neutral, and negatively charged
( 62 kT) surface regions, respectively.
View larger version (17K):
[in a new window]
Fig. 4.
Interactions between 2 and PTP1B.
a, H-bonding (blue dotted lines) and polar
interactions (red dotted lines); b, hydrophobic
interactions (orange dotted lines), and; c,
interactions between the two fluorine atoms in F2Pmp and
PTP1B. The cut-off distance used is 3.2 Å for hydrogen bonds, 4.0 Å for electrostatic or polar interactions, and 4.5 Å for hydrophobic
interactions.
2 of Arg-47
and a fluorine atom of the distal difluorophosphonate group is also
observed. Further away from the active site, the amino group of Lys-41
forms a hydrogen bond with one phosphonate oxygen and engages in a
polar interaction with a fluorine atom of the distal difluorophosphonate.
and C
of Asp-48 are observed to be within van der Waals
contacts of C
of F2Pmp and C
, C
, and C
of the
Asp residue in compound 2. The fluorine atoms at the
benzylic position of the distal difluorophosphonate are engaged in
nonpolar interactions with the C
of both Lys-41 and Arg-47. Finally,
the aliphatic portion of the side chain of Arg-47 is involved in
extensive hydrophobic interactions with the phenyl ring of the
4-phosphonodifluoromethylphenylacetyl group.
Kinetic parameters of pNPP hydrolysis and Ki values of
vanadate, compounds 1, 3, and 4 for
the wild-type and mutant PTP1Bs
Selectivity of compound 4 against a panel of protein
phosphatases
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of
F2Pmp and C
, C
, and C
of the Asp linker in
compound 2. Substitution of Asp-48 by an Ala reduced the
affinity of PTP1B for compound 1 by 40-fold, which is
consistent with the removal of two hydrogen bonds (Table II). Because
these interactions do not exist between simple bis-aryl
difluorophosphonates and PTP1B (34), removal of the side chain of
Asp-48 does not change the binding affinity for compound 3 (Table II). Interestingly, the affinity of compound 1 for
D48N is enhanced by more than 6-fold, which may result from a reduction
of electrostatic repulsion between PTP1B and compound 1.
This supports the notion that residue 48 could be exploited as a
determinant of both affinity and selectivity (41). However, residue 48 is not the sole determinant for the affinity and selectivity of
compound 1 for PTP1B, because other Asn-48-containing PTPs
(e.g. SHP1, SHP2, leukocyte common antigen-related
phosphate, and PTP
) display more than 3 orders of magnitude lower
affinities toward compound 1 than does PTP1B (22).
of Lys-41) interactions.
Accordingly, substitution of Lys-41 by an Ala reduces the
Ki of compound 1 for PTP1B by 9-fold
(Table II). Moreover, changing the CF2 in the distal difluorophosphonate in compound 1 to an oxygen in compound 5 results in an 11-fold loss of inhibitory activity (23). Minor modifications (e.g. addition or deletion of a single
methylene group from phosphorylated phenylacetic acid) of the distal
element also result in a significant decrease in affinity (22).
Collectively, these results suggest that the interactions of the distal
difluorophosphonate with Lys-41 are very specific. As controls, removal
of side chains from adjacent residues, Leu-37, Lys-39, Asn-40, and
Asn-42, causes little change in the Ki values for
vanadate, compound 1, or compound 3 (Ref. 23 and
data not shown). Interestingly, although PTP1B and TC-PTP display more
than 70% sequence identity, an Arg, instead of a Lys, occupies
position 41 in TC-PTP. When Lys-41 was changed to an Arg, a 3-fold
lower affinity for compound 1 was observed for PTP1B/K41R,
suggesting that the structural difference at position 41 may be
partially responsible for the modest selectivity (10-fold) of compound
1 in favor of PTP1B.
-
interaction between the side chain of
Tyr-46 and the phenyl ring of F2Pmp and disruption of
interactions of Arg-47 and Asp-48 with the rest of compound
1. Indeed, the
-
interaction between the side chain of
Tyr-46 and the phenyl ring of the aryl difluorophosphonate only
contributes 20-fold to the overall binding affinity between PTP1B and
compound 3 (Table II).
1-position of the EGFR peptide DADEpYL-NH2, a preference
for a second acidic residue is observed at the
2-position (16). However, when a Phe residue occupies the
1-position in the PTP1B consensus peptide ELEFpYMDYE-NH2, Arg-47 can reposition
itself to participate in hydrophobic interactions with both the
1-
and
3-positions (20).
View larger version (22K):
[in a new window]
Fig. 5.
Interactions of Arg-47 and Lys-41 with the
EGFR peptide (a), the PTP1B
consensus peptide (b), compound
2 (c), and compound 4 (d). Hydrophobic interactions
are indicated by orange dotted lines, H-bonds by blue
dotted lines, and polar interactions by red dotted
lines.
Torsion angles of Arg-47 in the complexes of PTP1B with the EGFR
peptide (DADEpYL-NH2), the consensus peptide
(ELEFpYMDYE-NH2), and compound 2
1-position and a
4-phosphonodifluoromethyl phenylacetyl group at the
2-position
induces a distinct conformation of Arg-47 in PTP1B·2 that
bears some resemblance to that observed in the PTP1B·EGFR peptide
complex with a major difference (52°) in the
4 value
(Fig. 5 and Table IV). This rotamer permits optimal interactions
between Arg-47 with both the Asp linker and the distal aryl
difluorophosphonate group. Interestingly, when the Asp linker in
compound 1 is replaced by a Tyr, the affinity of the
resulting compound 4 for PTP1B is only 2-fold less potent
than that of compound 1 (Table II). Moreover, compound
4 is also highly selective for PTP1B, exhibiting 4-fold
selectivity over TC-PTP and displaying a greater than 3 orders of
magnitude preference for PTP1B versus nearly all
phosphatases examined (Table III). In the absence of a crystal
structure for PTP1B·4, we manually docked compound 4 to the structure of PTP1B in the consensus peptide
complex. The model suggests that the interactions between Arg-47 and
compound 4 are mostly hydrophobic in nature involving the
phenyl rings of both the Tyr linker and
4-phosphonodifluoromethylphenylacetyl group (Fig. 5d). The
model also predicts a polar interaction between a fluorine atom of the
distal difluorophosphonate group and the amino group of Lys-41, which
is supported by the 2- and 4-fold decrease in the binding affinity of
compound 4 for K41R and K41A, respectively (Table II). The
more moderate effects on binding of compound 4 (as compared
with compound 1) observed for the Arg-47 mutants (Table II)
suggest that most substitutions at Arg-47 can retain some of the
hydrophobic interactions. The results once again highlight the
plasticity of Arg-47 in PTP1B ligand binding.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Waxen Shi for help with preparation of figures and discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM55242 and the G. Harold and Leila Y. Mathers Charitable Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N6W) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Present address: Dept. of Pharmacology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205.
To whom correspondence may be addressed: Dept. of
Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park
Ave., Bronx, NY 10461. Tel.: 718-430-2746; Fax: 718-430-8565; E-mail: almo@aecom.yu.edu.
** Irma T. Hirschl Career Scientist. To whom correspondence may be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4288; Fax: 718-430-8922; E-mail: zyzhang@aecom.yu.edu.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M212491200
2 M. Sarmiento, S. C. Almo, and Z.-Y. Zhang, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTP, protein-tyrosine phosphatase; EGFR, epidermal growth factor receptor; NMR, nuclear magnetic resonance; Tyr(P), phosphotyrosine; PTP1B, protein-tyrosine phosphatase 1B; pNPP, p-nitrophenyl phosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; F2Pmp, phosphonodifluoromethylphenylalanine; MES, 4-morpholineethanesulfonic acid; TC, T cell.
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