Allele-specific inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are critical cell-signaling molecules. Inhibitors that are selective for individual PTPs would be valuable tools for dissecting complicated phosphorylation networks. However, the common architecture of PTP active sites impedes the discovery of such compounds. To achieve target selectivity, we have redesigned a PTP/inhibitor interface. Site-directed mutagenesis of a prototypical phosphatase, PTP1B, was used to generate "inhibitor-sensitized" PTPs. The PTP1B mutants were targeted by modifying a broad specificity PTP inhibitor with chemical groups that are sterically incompatible with wild-type PTP active sites. From a small panel of putative inhibitors, compounds that selectively inhibit Ile219Ala PTP1B over the wild-type enzyme were identified. Importantly, the corresponding mutation also conferred novel inhibitor sensitivity to T-cell PTP, suggesting that a readily identifiable point mutation can be used to generate a variety of inhibitor-sensitive PTPs.     

Structural basis for inhibition of protein tyrosine phosphatases by Keggin compounds phosphomolybdate and phosphotungstate

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.     

Hypervalent organochalcogenanes as inhibitors of protein tyrosine phosphatases

A series of organochalcogenanes was synthesized and evaluated as protein tyrosine phosphatases (PTPs) inhibitors. The results indicate that organochalcogenanes inactivate the PTPs in a time- and concentration-dependent fashion, most likely through covalent modification of the active site sulfur-moiety by the chalcogen atom. Consequently, organochalcogenanes represent a new class of mechanism-based probes to modulate the PTP-mediated cellular processes.     

Potent inhibition of protein tyrosine phosphatases by quinquedentate binuclear copper complexes: synthesis, characterization and biological activities

Three phosphono-containing multidentate ligands were employed to synthesize quinquedentate binuclear copper complexes, [Cu(2)L(2)] (1-3) (H(2)L1 = diethyl(propane-1,3-diylbis(azanediyl))bis((2-hydroxyphenyl)methylene)bis(hydrogen phosphonate), H(2)L2 = diethyl(ethane-1,2-diylbis(azanediyl))bis((2-hydroxyphenyl)methylene)bis(hydrogen phosphonate), H(2)L3 = diethyl(hexane-1,6-diylbis(azanediyl))bis((2-hydroxyphenyl)methylene)bis(hydrogen phosphonate)), which were characterized by elemental analysis, IR, X-ray diffraction analysis, electrospray ionization mass spectra. Complexes 1 and 2 crystallized in the triclinic system with space group P ?1. The speciation of the Cu-H(2)L1 system in aqueous solution was investigated by potentiometric pH titrations. The three dicopper complexes exhibited potent and almost the same inhibitory effects against protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP) with IC(50) of 0.16-0.24 μM, about 10-fold stronger inhibition than against Src homology phosphatase 1 (SHP-1), 30-fold than against Src homology phosphatase 2 (SHP-2) and more than 100-fold than against megakaryocyte protein-tyrosine phosphatase 2 (PTP-MEG2). Fluorescence titrations revealed complex 1 bond to the five PTPs with molar ratio of 1:1 and binding constants of 1.62 × 10(6), 3.09 × 10(6), 1.95 × 10(5), 2.24 × 10(5), 1.55 × 10(4) M(-1) for PTP1B, TCPTP, SHP-1, SHP-2 and PTP-MEG2, respectively, consistent with the inhibitory abilities from IC(50) and K(i) values. Also, the three copper complexes could inhibit phosphatase activity of cell extracts from C6 rat glioma cells. The results suggested the structures of copper complexes influence selectivity over different PTPs.     

Synthesis of 3,5-difluorotyrosine-containing peptides: application in substrate profiling of protein tyrosine phosphatases

Fully protected 3,5-difluorotyrosine (F2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The F2Y-containing peptides display kinetic properties toward protein tyrosine phosphatases (PTPs) similar to their corresponding tyrosine-containing counterparts but are resistant to tyrosinase action. These properties make F2Y a useful tyrosine surrogate during peptide library screening for optimal PTP substrates.     

Squaric acids: a new motif for designing inhibitors of protein tyrosine phosphatases

[structure: see text] Protein tyrosine phosphatases (PTPases) are important targets in medicinal chemistry. These enzymes play a role in a number of human diseases, including type II diabetes and infection by Yersinia pestis, the causative agent of bubonic plague. Derivatives of squaric acids such as 2-aryl-1-hydroxycyclobut-1-ene-3,4-diones represent a new class of monoanionic inhibitors for PTPases.     

Peptidic alpha-ketocarboxylic acids and sulfonamides as inhibitors of protein tyrosine phosphatases

One common approach for designing protein tyrosine phosphatase (PTPase) inhibitors is to incorporate a nonhydrolyzable phosphotyrosine (pTyr) mimic into a peptide substrate for PTPases. This report describes the synthesis of three such nonhydrolyzable pTyr mimics that contain alpha-ketoacid, alpha-hydroxyacid, and methylenesulfonamide functional groups in place of the phosphate. These pTyr mimics were incorporated into the peptide sequence Ac-Asp-Ala-Asp-Glu-X-Leu-NH(2), where X is the pTyr mimic, and analyzed for activity against the Yersinia PTPase and PTP1B.     

Rapid assembly and in situ screening of bidentate inhibitors of protein tyrosine phosphatases

[reaction: see text] We have successfully designed and synthesized a small library of protein tyrosine phosphatase (PTP) inhibitors, in which the so-called "click chemistry" or Cu(I)-catalyzed 1,3-dipolar alkyne-azide coupling reaction was carried out for rapid assembly of 66 different bidentate compounds. Subsequent in situ enzymatic screening revealed a potential PTP1B inhibitor (IC(50) = 4.7 microM) which is 10-100 fold more potent than other PTPs.     

Ugi reaction-assisted rapid assembly of affinity-based probes against potential protein tyrosine phosphatases

The multi-component Ugi reaction has been employed to assemble a small library of affinity-based probes (AfBPs) that target potential protein tyrosine phosphatases. The probes showed good labelling of PTP1B and MptpB, and were subsequently used to label endogenous PTP1B in MCF-7 cell lysates.     

Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg²⁺. The chimeric proteins'' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

Protein tyrosine phosphatases have a key catalytic residue on a mobile loop (the WPD-loop), making the connections between this loop sequence and its dynamics, together with the dynamics of other mobile loops, particularly important.     

Mononuclear copper(II) complexes with 3,5-substituted-4-salicylidene-amino-3,5-dimethyl-1,2,4-triazole: synthesis, structure and potent inhibition of protein tyrosine phosphatases

Six copper complexes of Schiff base ligands containing 3,5-substituted-4-salicylideneamino-3,5-dimethyl-1,2,4-triazole have been synthesized and well characterized. The structures of complexes 1 and 2 were determined by X-ray crystal analysis. Fluorescence and potentiometric study indicated that in the physiological pH range, one ligand was dissociated from the complexes to form 1:1 mononucleus copper complexes. The complexes potently inhibit protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2) and Src homology phosphatase 1 (SHP-1) with 3-4 fold selectivity against PTP1B over TCPTP and PTP-MEG2, and 3-9 fold over SHP-1, but display almost no inhibition against Src homology phosphatase 2 (SHP-2). Complex 1 inhibits PTP1B with a competitive model with K(i) of 30 nM. Substitution with small groups at the phenyl of the ligand does not obviously influence the inhibitory ability of the complexes.     

Aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) play key roles in the regulation of normal and pathological processes ranging from cell proliferation, differentiation, metabolism, and survival to many human diseases including cancer and diabetes. Functional studies of PTP can be greatly facilitated by small molecule probes that covalently label the active site of a PTP through an activity-dependent chemical reaction. In this article, we characterize phenyl vinyl sulfonate (PVSN) and phenyl vinyl sulfone (PVS) as a new class of mechanism-based PTP probes. PVSN and PVS inactivate a broad range of PTPs in a time- and concentration-dependent fashion. The PVSN- and PVS-mediated PTP inactivation is active site-directed and irreversible, resulting from a Michael addition of the active-site Cys Sgamma onto the terminal carbon of the vinyl group. Structural and mechanistic analyses reveal the molecular basis for the preference of PVSN/PVS toward the PTPs, which lies in the ability of PVSN and PVS to engage the conserved structural and catalytic machinery of the PTP active site. In contrast to early alpha-bromobenzyl phosphonate-based probes, PVSN and PVS are resistant to solvolysis and are cell-permeable and thus hold promise for in vivo applications. Collectively, these properties bode well for the development of aryl vinyl sulfonate/sulfone-based PTP probes to interrogate PTP activity in complex proteomes.     

Development of a colorimetric inhibition assay for microcystin-LR detection: comparison of the sensitivity of different protein phosphatases

A colorimetric protein phosphatase (PP) inhibition test for the detection of microcystin-LR (MC-LR) has been developed. Three PP2As, one recombinant and two natural versions, as well as one PP1 produced by molecular engineering, were tested. First, assays were performed using the enzymes in solution to compare their sensitivity to MC-LR. The PP2A purchased from ZEU Immunotec and PP1 appeared more sensitive to the toxin than the other enzymes. With PP2A from ZEU Immunotec, the colorimetric test showed a detection limit of 0.0039 μg L⁻¹ and an IC₅₀ value of 0.21 μg L⁻¹. With PP1, the assay gave a detection limit of 0.05 μg L⁻¹ and an IC₅₀ value of 0.56 μg L⁻¹. Therefore, this assay allowed the detection of lower microcystin-LR (MC-LR) concentrations than the maximum level (1 μg L⁻¹) recommended by the World Health Organisation (WHO).The main drawback of this PP-based approach in solution is poor enzyme stabilisation. To overcome this problem, enzymes were entrapped within either a photopolymer or an agarose gel. PP2A from ZEU Immunotec and PP1 were immobilised at the bottom of microwells. The agarose-based tests performed better than the photopolymer-based assay for all of the enzymes. Therefore, the agarose gel is a good candidate to replace the photopolymer, which is generally used in PP-immobilising membranes. The assays based on enzyme-entrapping agarose gels showed detection limits equal to 0.17 μg L⁻¹ and 0.29 μg L⁻¹ with immobilised PP2A from ZEU and PP1, respectively. In view of these performances, these tests can potentially be used for monitoring water quality.     

Probing the phosphopeptide specificities of protein tyrosine phosphatases,SH2 and PTB domains with combinatorial library methods

Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains. This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed.     

The biological buffer bicarbonate/CO2 potentiates H2O2-mediated inactivation of protein tyrosine phosphatases

Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H(2)O(2)-mediated cellular signal transduction processes, but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer bicarbonate/CO(2) potentiates the ability of H(2)O(2) to inactivate PTPs. The results of biochemical experiments and high-resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H(2)O(2) with HCO(3)(-)/CO(2).     

Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases

A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine/threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine/threonine and tyrosine phosphatases were developed. Specific inhibitor cocktails were added to each assay to limit the activity of other phosphatases. LAR, CD-45, and PTP-1B all rapidly hydrolyze DiFMUP in the tyrosine phosphatase assay. The activity of non-tyrosine phosphatases is less than 6% of the LAR activity. PP-1 and PP-2A are highly active in the serine/threonine phosphatase assay. Inhibition of LAR and PP-2A in these assays is demonstrated using known inhibitors. Both of these assays are sensitive, robust, kinetic assays that can be used to quantify enzyme activity.     

Reversible "irreversible" inhibition of chymotrypsin using nanoparticle receptors

Anionically functionalized amphiphilic nanoparticles efficiently inhibit chymotrypsin through electrostatic binding followed by protein denaturation. We demonstrate the ability to disrupt this "irreversible" inhibition of chymotrypsin through modification of the nanoparticle surface using cationic surfactants. Up to 50% of original chymotrypsin activity is rescued upon long-chain surfactant addition. Dynamic light-scattering studies demonstrate that chymotrypsin is released from the nanoparticle surface. The conformation of the rescued chymotrypsin was characterized by fluorescence and fluorescence anisotropy, indicating that chymotrypsin regains a high degree of native structure upon surfactant addition.