Crystal Structure of PTP1B Complexed with a Potent and Selective Bidentate Inhibitor*

Jin-Peng SunDagger , Alexander A. Fedorov§, Seung-Yub LeeDagger , Xiao-Ling GuoDagger , Kui Shen§, David S. Lawrence§, Steven C. Almo§||, and Zhong-Yin ZhangDagger §**

From the Departments of Dagger  Molecular Pharmacology and § Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, December 9, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 PTPalpha have been implicated as negative regulators of insulin signaling (2).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha -[(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-alpha -[(9H-fluoren-9-yl methoxy)carbonyl]-L-aspartic acid-beta -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): delta  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): delta  5.32 (t, J = 107.0 Hz); 19F NMR (282 MHz, CD3OD) delta  -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.

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): delta  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): delta  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): delta  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.

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 -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.

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 - 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   The chemical structures of compounds 1-5.

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.


                              
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Table I
Crystallographic data and refinement statistics
Completeness and Rmerge are given for all data and for all data in the highest resolution shell.


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Fig. 2.   Simulated annealing omit map showing unbiased density for compound 2. The density shown is a Fo - Fc map contoured at 3.2sigma , with the refined models of the inhibitor and PTP1B superimposed. This figure was generated using SETOR (43).


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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.

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.


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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.

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 Neta 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.

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, Cbeta and Cgamma of Asp-48 are observed to be within van der Waals contacts of Calpha of F2Pmp and Calpha , Cbeta , and Cgamma 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 Cdelta 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.

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).


                              
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Table II
Kinetic parameters of pNPP hydrolysis and Ki values of vanadate, compounds 1, 3, and 4 for the wild-type and mutant PTP1Bs
All measurements were made using pNPP as a substrate at pH 7.0, 30 °C, and I = 0.15 M.

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.


                              
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Table III
Selectivity of compound 4 against a panel of protein phosphatases


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Calpha of F2Pmp and Calpha , Cbeta , and Cgamma 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 PTPalpha ) display more than 3 orders of magnitude lower affinities toward compound 1 than does PTP1B (22).

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 Cdelta 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.

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 pi -pi 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 pi -pi 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).

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 -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).


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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.


                              
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Table IV
Torsion angles of Arg-47 in the complexes of PTP1B with the EGFR peptide (DADEpYL-NH2), the consensus peptide (ELEFpYMDYE-NH2), and compound 2

The combination of an Asp at the -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 chi 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.

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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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