Human Protein Tyrosine Phosphatase 1B Research Articles

Benzoquinones from Ardisia japonica with Inhibitory Activity towards Human Protein Tyrosine Phosphatase 1B (PTP1B)

From the whole plant of Ardisia japonica, four [1,4]benzoquinones were isolated by means of bioassay-directed fractionation of the EtOH extract. Apart from the two known compounds maesanin (1) and its congener 2, two new benzoquinones, i.e., 5-ethoxy-2-hydroxy-3-[(10Z)-pentadec-10-en-1-yl][1,4]benzoquinone (3) and 5-ethoxy-2-hydroxy-3-[(8Z)-tridec-8-en-1-yl][1,4]benzoquinone (4), were identified. All compounds showed significant in vitro bioactivities against the PTP1B enzyme, with IC50 values in the range of ca. 3-19 microM.

Expression of Catalytic Domain of Protein Tyrosine Phosphatase 1B and Preparation of Its Polyclonal Antibody

This study focuses on the expression of human protein tyrosine phosphatase 1B(PTP1B) catalytic domain (ΔPTP1B) and preparation of polyclonal antibody against ΔPTP1B. ΔPTP1B gene was PCR amplified with the cDNA of human PTP1B as the template, and cloned into the pT7 expression vector. The recombinant pT7-ΔPTP1B was expressed in E. coli Rosetta (DE3) host cells and purified. The antiserum was prepared by immunizing rabbit with purified recombinant ΔPTP1B. The polyclonal antibody against ΔPTP1B was purified by PVDF immobilized antigen affinity chromatography. ΔPTP1B was correctly cloned, expressed, and purified as confirmed by PCR, DNA sequence analysis, SDS-PAGE western blotting, and HPLC. The titer and sensitivity of the antibody were 1: 2500(volume ratio) and 0.1 ng, respectively. This study provides an important basis for further studying the biological function of PTP1B and its relationship with human diseases.

The Role of the C-terminal Domain of Protein Tyrosine Phosphatase-1B in Phosphatase Activity and Substrate Binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.

Human protein tyrosine phosphatase 1B inhibitors: QSAR by genetic function approximation

Human protein tyrosine phosphatase 1B inhibitors: QSAR by genetic function approximation

Human protein tyrosine phosphatase 1B inhibitors: QSAR by genetic function approximation

Protein tyrosine phosphatase 1B (PTP 1B), a negative regulator of insulin receptor signaling system, has emerged as a highly validated, attractive target for the treatment of non-insulin dependent diabetes mellitus (NIDDM) and obesity. As a result there is a growing interest in the development of potent and specific inhibitors for this enzyme. This quantitative structure-activity relationship (QSAR) study for a series of formylchromone derivatives as PTP 1B inhibitors was performed using genetic function approximation (GFA) technique. The QSAR models were developed using a training set of 29 compounds and the predictive ability of the QSAR model was evaluated against a test set of 7 compounds. The internal and external consistency of the final QSAR model was 0.766 and 0.785. The statistical quality of QSAR models was assessed by statistical parameters r2, (crossvalidated r2), (predictive r2) and lack of fit (LOF) measure. The results indicate that PTP 1B inhibitory activity of the formylchromone derivatives is strongly dependent on electronic, thermodynamic and shape related parameters.

Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Leishmania parasites are eukaryotic protozoans responsible for a variety of human diseases known as leishmaniasis, which ranges from skin lesions to fatal visceral infections. Leishmania is transmitted by the bite of an infected sandfly where it exists as promastigotes and, upon entry into a mammalian host, differentiates into amastigotes, which replicate exclusively in macro-phages. The biochemical pathways enabling Leishmania to differentiate and survive in the mammalian host are poorly defined. We have therefore examined the role of protein-tyrosine phosphorylation, which is essential in regulating cell function in higher eukaryotes. Using the recently completed Leishmania genome, we have identified and cloned a Leishmania protein-tyrosine phosphatase (PTP) gene (LPTP1) by virtue of its homology with the human protein-tyrosine phosphatase 1B gene (hPTP1B). The enzyme activity of recombinant LPTP1 was confirmed using a combination of PTP-specific substrates and inhibitors. We further demonstrate, by creating LPTP1 null mutants through gene targeting, that LPTP1 is necessary for survival as amastigotes in mice, but it is dispensable for survival as promastigotes in culture. Human PTPs, including the PTP1B enzyme, are actively pursued drug targets for a variety of diseases. The observations with the LPTP1 mutants in mice suggest that it may also represent a drug target against the mammalian amastigote stage. However, in silico structure analysis of LPTP1 revealed a striking similarity with hPTP1B in the active site suggesting that, although this is an attractive drug target, it may be difficult to develop an inhibitor specific for the Leishmania LPTP1.

Discovery of new potent human protein tyrosine phosphatase inhibitors via pharmacophore and QSAR analysis followed by in silico screening

A pharmacophoric model was developed for human protein tyrosine phosphatase 1B (h-PTP 1B) inhibitors utilizing the HipHop-REFINE module of CATALYST software. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of physicochemical descriptors and pharmacophore hypothesis that yield consistent QSAR equation of good predictive potential ( r = 0.87 , F -statistic = 69.13 , r BS 2 = 0.76 , r LOO 2 = 0.68 ) . The validity of the QSAR equation and the associated pharmacophoric hypothesis was experimentally established by the identification of five new h-PTP 1B inhibitors retrieved from the National Cancer Institute (NCI) database.

Berberine potently inhibits protein tyrosine phosphatase 1B: Investigation by docking simulation and experimental validation

Berberine was investigated as an inhibitor of human protein tyrosine phosphatase 1B (h-PTP 1B) in an attempt to explain its anti-hyperglycemic activitiy. The investigation included simulated docking experiments to fit berberine within the binding pocket of h-PTP 1B. Berberine was found to readily fit within the binding pocket of h-PTP 1B in a low energy orientation characterized with optimal electrostatic attractive interactions bridging the isoquinolinium positively charged nitrogen atom of berberine and the negatively charged acidic residue of ASP 48 of h-PTP 1B. Experimentally, berberine was found to potently competitively inhibit recombinant h-PTP 1B in vitro (Ki value = 91.3 nM). Our findings strongly suggest that h-PTP 1B inhibition is at least one of the reasons for the reported anti-hyperglycemic activities of berberine.

Effects of Variable Docking Conditions and Scoring Functions on Corresponding Protein-Aligned Comparative Molecular Field Analysis Models Constructed from Diverse Human Protein Tyrosine Phosphatase 1B Inhibitors

The effects of variable docking conditions and scoring functions on corresponding protein-aligned comparative molecular field analysis (CoMFA) models have been assessed. To this end, a group of diverse inhibitors were docked into the active site of human protein tyrosine phosphatase 1B (h-PTP 1B). The docked structures were utilized to construct corresponding protein-aligned CoMFA models by employing probe-based (H+, OH, CH3) energy grids and genetic partial least squares (G/PLS) statistical analysis. A total of 48 different docking configurations were evaluated, of which some succeeded in producing self-consistent and predictive CoMFA models. However, the best CoMFA model coincided with docking the un-ionized ligands into the hydrated form of the binding site via the PLP1 scoring function and restricted docking settings (r2(LOO) = 0.647, r2(PRESS) against 27 test compounds = 0.617). Interestingly, the most significant CoMFA models were orthogonal and corresponded to significantly different docked conformers/poses. To utilize the predictive potentials of the best CoMFA models collectively, it was decided to combine them in a single quantitative structure-activity relationship (QSAR) model. The combination model illustrated excellent statistical properties (r2(LOO) = 0.890, r2(PRESS) against 27 test compounds = 0.750).

The Role of Protein-tyrosine Phosphatase 1B in Integrin Signaling

Protein-tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. Because PTP1B regulates multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTP1B in these processes. In this study, we utilize potent and selective PTP1B inhibitors to delineate the role of PTP1B in integrin signaling. We show that down-regulation of PTP1B activity with small molecule inhibitors suppresses cell spreading and migration to fibronectin, increases Tyr(527) phosphorylation in Src, and decreases phosphorylation of FAK, p130(Cas), and ERK1/2. In addition, PTP1B "substrate-trapping" mutants bind Tyr(527)-phosphorylated Src and protect it from dephosphorylation by endogenous PTP1B. These results establish that PTP1B promotes integrin-mediated responses in fibroblasts by dephosphorylating the inhibitory pTyr(527) and thereby activating the Src kinase. We also show that PTP1B forms a complex with Src and p130(Cas), and that the proline-rich motif PPRPPK (residues 309-314) in PTP1B is essential for the complex formation. We suggest that the specificity of PTP1B for Src pTyr(527) is mediated by protein-protein interactions involving the docking protein p130(Cas) with both Src and PTP1B in addition to the interactions between the PTP1B active site and the pTyr(527) motif.

Formylchromone derivatives as irreversible and selective inhibitors of human protein tyrosine phosphatase 1B. Kinetic and modeling studies

A series of formylchromone derivatives were synthesized as PTP1B inhibitors and some of them were potent against PTP1B with IC 50 values as low as 1.0 μM. They exhibited remarkable selectivity for PTP1B over other human PTPases. Kinetic studies revealed that formylchromone derivatives are irreversible and active site-directed inhibitors. Molecular modeling study identified the orientation of the inhibitor bound at the active site of PTP1B.

Transgenic Overexpression of Protein-tyrosine Phosphatase 1B in Muscle Causes Insulin Resistance, but Overexpression with Leukocyte Antigen-related Phosphatase Does Not Additively Impair Insulin Action

Previous studies implicate protein-tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related phosphatase (LAR) as negative regulators of insulin signaling. The expression and/or activity of PTP1B and LAR are increased in muscle of insulin-resistant rodents and humans. Overexpression of LAR selectively in muscle of transgenic mice causes whole body insulin resistance. To determine whether overexpression of PTP1B also causes insulin resistance, we generated transgenic mice overexpressing human PTP1B selectively in muscle at levels similar to those observed in insulin-resistant humans. Insulin-stimulated insulin receptor (IR) tyrosyl phosphorylation and phosphatidylinositol 3'-kinase activity were impaired by 35% and 40-60% in muscle of PTP1B-overexpressing mice compared with controls. Insulin stimulation of protein kinase C (PKC)lambda/zeta activity, which is required for glucose transport, was impaired in muscle of PTP1B-overexpressing mice compared with controls, showing that PTP1B overexpression impairs activation of these PKC isoforms. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed that whole body glucose disposal and muscle glucose uptake were decreased by 40-50% in PTP1B-overexpressing mice. Overexpression of PTP1B or LAR alone in muscle caused similar impairments in insulin action; however, compound overexpression achieved by crossing PTP1B- and LAR-overexpressing mice was not additive. Antibodies against specific IR phosphotyrosines indicated overlapping sites of action of PTP1B and LAR. Thus, overexpression of PTP1B in vivo impairs insulin sensitivity, suggesting that overexpression of PTP1B in muscle of obese humans and rodents may contribute to their insulin resistance. Lack of additive impairment of insulin signaling by PTP1B and LAR suggests that these PTPs have overlapping actions in causing insulin resistance in vivo.

Metformin (Glucophage) inhibits tyrosine phosphatase activity to stimulate the insulin receptor tyrosine kinase

Metformin is a commonly used anti-diabetic but whether its mechanism involves action on the insulin receptor or on downstream events is still controversial. With a time course that was slow compared with insulin action, metformin increased tyrosine phosphorylation of the regulatory domain of the insulin receptor (specifically, tyrosine residues 1150 and 1151). In a direct action, therapeutic levels of metformin stimulated the tyrosine kinase activity of the soluble intracellular portion of the beta subunit of the human insulin receptor toward a substrate derived from the insulin receptor regulatory domain. However, metformin did not alter the order of substrate phosphorylation by the insulin receptor kinase. Using a Xenopus oocyte preparation, we simultaneously recorded tyrosine kinase and phosphatase activities that regulate the insulin receptor by measuring the tyrosine phosphorylation and dephosphorylation of peptides derived from the regulatory domain of the human insulin receptor. In an indirect stimulation of the insulin receptor, metformin inhibited endogenous tyrosine phosphatases and purified human protein tyrosine phosphatase 1B that dephosphorylate and inhibit the insulin receptor kinase. Thus, there was evidence that metformin acted directly upon the insulin receptor and indirectly through inhibition of tyrosine phosphatases.

Discovery of a novel protein tyrosine phosphatase-1B inhibitor, KR61639: potential development as an antihyperglycemic agent

Protein tyrosine phosphatase-1B (PTP-1B), a negative regulator of insulin signaling, may be an attractive therapeutic target for type 2 diabetes mellitus. High throughput screening (HTS) for PTP-1B inhibitors using compounds from the Korea Chemical Bank identified several hits (active compounds). Among them, a hit with 1,2-naphthoquinone scaffold was chosen for lead development. KR61639, {4-[1-(1 H-indol-3-yl)-3,4-dioxo-3,4-dihydro-naphthalen-2-ylmethyl]-phenoxy}-acetic acid tert-butyl ester, inhibited human recombinant PTP-1B with an IC 50 value of 0.65 μM in a noncompetitive manner. KR61639 showed modest selectivity over several phosphatases and increased insulin-stimulated glycogen synthesis in HepG2 cells and stimulated 2-deoxyglucose uptake in 3T3/L1 adipocytes. In addition, in vivo study using ob/ob mouse demonstrated that KR61639 exerted a hypoglycemic action when given orally. Thus, KR61639 may be a good starting point for lead optimization in developing a novel antidiabetic agent.

Finding discriminating structural features by reassembling common building blocks.

We present a new method for constructing discriminating substructures by reassembling common medicinal chemistry building blocks. The algorithm can be parametrized to meet differing objectives: (1) to build features that discriminate for biological activity in a local structural neighborhood, (2) to build scaffolds for R-group analysis, (3) to construct cluster signatures that discriminate for membership in the cluster and provide a graphical representation for its members, and (4) to identify substructures that characterize major classes in a heterogeneous compound set. We illustrated the results of the algorithm on a literature dataset is of 118 compounds with in vitro inhibition data against recombinant human protein tyrosine phosphatase 1B (PTP-1B).

Interaction between chicken protein tyrosine phosphatase 1 (CPTP1)-like rat protein phosphatase 1 (PTP1) and p60(v-src) in v-src-transformed Rat-1 fibroblasts.

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kDa in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60(v-src) in vivo and CPTP1 also can associate with p60(v-src) in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60(v-src) was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60(v-src) in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kDa protein which has a possibility to be tyrosine- phosphorylated by p60(v-src) in v-src-transformed Rat-1 fibroblasts. These results suggest that HPTP1B-type PTPs may play an important role in p60(src) dependent signal pathway in eucaryotic cells.

Structural, kinetic and biochemical characterization of the oxidation states of human protein tyrosine phosphatase 1B: implications for redox regulation of phosphatase activity

Structural, kinetic and biochemical characterization of the oxidation states of human protein tyrosine phosphatase 1B: implications for redox regulation of phosphatase activity

Regulation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B activity by casein kinase II- and p56lck-mediated phosphorylation.

Protein tyrosine phosphorylation and dephosphorylation are important in the regulation of cell proliferation and signaling cascade. In order to examine whether phosphatase activity of CPTP1 and HPTP1B, typical nontransmembrane protein tyrosine phosphatase, could be controlled by phosphorylation, affinity-purified PTPs were phosphorylated by CKII and p56lck in vitro. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII, and tyrosine residue by p56lck. Phosphatase activity of CPTP1 was gradually increased by three-fold concomitant with phosporylation by CKII. Phosphorylation of HPTP1B by CKII resulted in quick two-fold enhancement of its phosphatase activity within 5 min of incubation and remained in that state. In the presence of CKII inhibitor, heparin or poly(Glu.Tyr), both phosphorylation and enhancement of phosphatase activity of CPTP1 and HPTP1B were mostly blocked. p56lck catalyzed tyrosine phosphorylation of CPTP1 and HPTP1B was only observed by inhibiting the intrinsic tyrosine phosphatase activity. Taken together, these results indicate that CPTP1 or HPTP1B possesses a capability to regulate its phosphatase activity through phosphorylation processes and may participate in the cellular signal cascades.

New azolidinediones as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties.

Insulin resistance in the liver and peripheral tissues together with a pancreatic cell defect are the common causes of type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibiton of PTPases may be an effective method in the treatment of type 2 diabetes. A series of azolidinediones has been prepared as protein tyrosine phosphatase 1B (PTP1B) inhibitors. Several compounds were potent inhibitors against the recombinant rat and human PTP1B enzymes with submicromolar IC(50) values. Elongated spacers between the azolidinedione moiety and the central aromatic portion of the molecule as well as hydrophobic groups at the vicinity of this aromatic region were very important to the inhibitory activity. Oxadiazolidinediones 87 and 88 and the corresponding acetic acid analogues 119 and 120 were the best h-PTP1B inhibitors with IC(50) values in the range of 0.12-0.3 microM. Several compounds normalized plasma glucose and insulin levels in the ob/ob and db/db diabetic mouse models.

Potent non-peptidyl inhibitors of protein tyrosine phosphatase 1B

The development of inhibitors of protein tyrosine phosphatases (PTPs) has recently been the subject of intensive investigation due to their potential as chemotherapeutics and as tools for studying signal transduction pathways. Here we report the evaluation of a variety of small molecule, non-peptidyl inhibitors of protein tyrosine phosphatase 1B (PTP1B), bearing the α,α-difluoromethylenephosphonic acid (DFMP) group, a non-hydrolyzable phosphate mimetic. A series of phenyl derivatives bearing a single DFMP group were initially surveyed. In general, these were not significantly more potent inhibitors than the parent compound, α,α-difluorobenzylphosphonic acid, with the exception being the meta-phenyl substituted species which decreased the IC50 by approximately 17-fold relative to α,α-difluorobenzylphosphonic acid. However, certain compounds bearing two DFMP moieties were very potent inhibitors. Some of these are among the most potent small molecule inhibitors of any PTP reported to date with the best one exhibiting a Ki of 1.5μM. The structural basis for these results are discussed. One of the bis-DFMP inhibitors was examined in detail and it was found that the fluorines were essential for potent inhibition. Inhibition was independent of pH between pH 5.5–7.2 suggesting that both the mono and dianionic forms of the individual DFMP groups bind equally well. The trends observed in the inhibitory potency of these compounds with PTP1B were very similar to the trends observed by other workers on the Km’s of the analogous phenylphosphate substrates with rat PTP1. This indicates that studies of non-peptidyl substrates with rat PTP1 can be used as a guide for the development of human PTP1B inhibitors.