Synthesis of (glycopyranosyl-triazolyl)-purines and their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B)

Development of novel purine derivatives has attracted considerable interest, since both purine and purine-based nucleosides display a wide range of crucial biological activities in nature. We report here a novel expansion of these studies by introducing gluco- or galactopyranosyl scaffold to the N- or 9-position (or both) of 6-Cl purine moiety via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. By such an efficient reaction, a series of glycosyl-triazolyl-purines were successfully synthesized in good yields. Biological evaluation showed that the majority of these glycoconjugates were good PTP1B inhibitors with IC(50) values in low micromolar range (1.5-11.1 μM). The benzylated sugar derivatives displayed better inhibitory potency than that of the acetylated ones. Replacement of Cl by MeO at C(6) of the purine moiety decreased the inhibition in the case of benzylated (glycosyl-mono-triazolyl)-purines 11 and 12 (IC(50) >80 μM), whereas MeO-substituted benzylated bis[galactosyl-triazolyl]-purine 16 possessed the best inhibitory activity with an IC(50) value of 1.5 μM. Additionally, these compounds exhibited 2- to 57-fold selectivity over other PTPs (TCPTP, SHP1, SHP2, and LAR).     

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.     

Molecular modeling of protein tyrosine phosphatase 1B (PTP 1B) inhibitors

Binding modes of a series of aryloxymethylphosphonates and monoanionic biosteres of phosphate group from a series of benzylic alpha,alpha-diflluoro phosphate and its biosteres as protein tyrosine phosphatase 1B (PTP 1B) inhibitors have been identified by molecular modeling techniques. We have performed docking and molecular dynamics simulations of these inhibitors with PTP 1B enzyme. The initial conformation of the inhibitors for docking was obtained from simulated annealing technique. Solvent accessible surface area calculations suggested that active site of PTP 1B is highly hydrophobic. The results indicate that for aryloxymethylphosphonates, in addition to hydrogen bonding interactions, Tyr46, Arg47, Asp48, Val49, Glu115, Lys116, Lys120 amino acid residues of PTP 1B are responsible for governing inhibitor potency of the compounds. The sulfonate and tetrazole functional groups have been identified as effective monoanionic biosteres of phosphate group and biphenyl ring system due to its favorable interactions with Glu115, Lys116, Lys120 residues of PTP 1B found to be more suitable aromatic functionality than naphthalene ring system for benzylic alpha,alpha-diflluoro phosphate and its biosteres. The information generated from the present study should be useful in the design of more potent PTP 1B inhibitors as anti diabetic agents.     

New prenylated isoflavonoids as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Erythrina addisoniae

Bioassay-guided fractionation of the EtOAc extract of the root of Erythrina addisoniae (Leguminosae) resulted in the isolation of four new (1–4), along with 2 known prenylated isoflavonoids (5–6). The structures of the isolates were assigned on the basis of spectroscopic data analysis, focusing on interpretation of 1D and 2D NMR, and MS data. All the isolates were evaluated for their inhibitory effects on protein tyrosine phosphatase 1B (PTP1B), as well as their growth inhibition on MCF7, adriamycin-resistant MCF7 (MCF7/ADR), and MDA-MB-231 breast cancer cell lines. Compounds which exhibited PTP1B inhibitory activity (IC₅₀ values ranging from 4.6 ± 0.3 to 24.2 ± 2.1 μM) showed potential cytotoxic activity (IC₅₀ values ranging from 3.97 ± 0.17 to 11.4 ± 1.9 μM). Taken together, our data suggest that prenylated isoflavonoids, especially the isoflavone-type skeleton could be considered as new lead compounds against breast cancer via PTP1B inhibition.     

Isoxazole carboxylic acids as protein tyrosine phosphatase 1B (PTP1B) inhibitors

Guided by X-ray crystallography, we have extended the structure-activity relationship (SAR) study on an isoxazole carboxylic acid-based PTP1B inhibitor (1) and more potent and equally selective (>20-fold selectivity over the highly homologous T-cell PTPase, TCPTP) PTP1B inhibitors were identified. Inhibitor 7 demonstrated good cellular activity against PTP1B in COS 7 cells.     

Mechanism of action of pyridazine analogues on protein tyrosine phosphatase 1B (PTP1B)

The inhibitory effect on PTP1B caused by the addition of pyridazine analogues has been investigated. Biophysical techniques, that is, mass spectrometry (MS), nuclear magnetic resonance (NMR), and isothermal titration calorimetry (ITC) were used for the characterization. Pyridazine analogues cause catalytic oxidation of the reducing agent, generating hydrogen peroxide that oxidizes the active site cysteine on the enzyme, leading to enzyme inactivation. Two additional compound classes show the same effect. We found one common structural feature in these molecules that allows the reaction with triplet molecular oxygen to be less endothermic. A proposed mechanism for the catalytic redox cycle is described.     

Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense

Four new caged xanthones (1–4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1–6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1–6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC₅₀ values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining K_m, V_max, and the ratio of K_ik and K_iv, which revealed that all the compounds behaved as competitive inhibitors.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Molecular docking and virtual screening for novel protein tyrosine phosphatase 1B (PTP1B) inhibitors

Protein tyrosine phosphatase 1B (PTP1B) functions as major negative regulator of insulin and leptin signaling pathways. In view ofthis, PTP1B is an significant target for drug development against cancer, diabetes and obesity. The aim of the current study is toidentify PTP1B inhibitors by means of virtual screening with docking. 523,366 molecules from ZINC database have been screenedand based on DOCK grid scores and hydrogen bonding interactions five new potential inhibitors were identified. ZINC12502589,ZINC13213457, ZINC25721858, ZINC31392733 and ZINC04096400 were identified as potential lead molecules for inhibition ofPTP1B. The identified molecules were subjected to Lipinski''s rule of five parameters and found that they did not violate any rule.More specific analysis of pharmacological parameters may be scrutinized through a complete ADME/Tox evaluation. Pharmaalgorithm was used to Calculate ADME–Tox profiles for such molecules. In general, all the molecules presented advantages and aswell as disadvantages when compared to each other. No marked difference in health effects and toxicity profiles were observedamong these molecules.     

Identification and characterization of potent and selective inhibitors targeting protein tyrosine phosphatase 1B (PTP1B)

Protein tyrosine phosphatase 1B (PTP1B) plays an important role in the negative regulation of insulin and leptin signaling. The development of small molecular inhibitors targeting PTP1B has been validated as a potential therapeutic strategy for Type 2 diabetes (T2D). In this work, we have identified a series of compounds containing dihydropyridine thione and particular chiral structure as novel PTP1B inhibitors. Among those, compound 4b showed moderate activity with IC₅₀ value of 3.33 μM and meanwhile with good selectivity (>30-fold) against TCPTP. The further MOA study of PTP1B demonstrated that compounds 4b is a substrate-competitive inhibitor. The binding mode analysis suggested that compound 4b simultaneously occupies the active site and the second phosphotyrosine (pTyr) binding site of PTP1B. Furthermore, the cell viability assay of compound 4b showed tolerable cytotoxicity in L02 cells, thus 4b may be prospectively used to further in vivo study.     

Specific inhibition of sensitized protein tyrosine phosphatase 1B (PTP1B) with a biarsenical probe

Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of the insulin-receptor and leptin-receptor signaling pathways, and it has therefore emerged as a critical antitype-II-diabetes and antiobesity drug target. Toward the goal of generating a covalent modulator of PTP1B activity that can be used for investigating its roles in cell signaling and disease progression, we report that the biarsenical probe FlAsH-EDT(2) can be used to inhibit PTP1B variants that contain cysteine point mutations in a key catalytic loop of the enzyme. The site-specific cysteine mutations have little effect on the catalytic activity of the enzyme in the absence of FlAsH-EDT(2). Upon addition of FlAsH-EDT(2), however, the activity of the engineered PTP1B is strongly inhibited, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that the cysteine-rich PTP1B variants can be targeted with the biarsenical probe in either whole-cell lysates or intact cells. Together, our data provide an example of a biarsenical probe controlling the activity of a protein that does not contain the canonical tetra-cysteine biarsenical-labeling sequence CCXXCC. The targeting of "incomplete" cysteine-rich motifs could provide a general means for controlling protein activity by targeting biarsenical compounds to catalytically important loops in conserved protein domains.     

Spirobiflavonoid stereoisomers from the endangered conifer Glyptostrobus pensilis and their protein tyrosine phosphatase 1B inhibitory activity

Six spirobiflavonoid stereoisomers including two new ones, spiropensilisols A (1) and B (2), were isolated from a mass-limited trunk barks of Glyptostrobus pensilis, an endangered conifer endemic to China. The new structures featuring a benzofuran-containing spirolactone and their absolute configurations were determined by extensive spectroscopic methods. All the isolates showed significant inhibitory activities against the human protein tyrosine phosphatase 1B (PTP1B) enzyme, a potential therapeutic target for diabetes and obesity, with IC₅₀ values ranging from 3.3 to 17.1 µM. A preliminary SAR analysis with assistance of the molecular modeling approach was performed for the most potent compound (i.e., 1), to understand the nature of interactions governing the binding mode of spirobiflavonids within the active site of the PTP1B enzyme.     

Modification of the N-terminus of peptidomimetic protein tyrosine phosphatase 1B (PTP1B) inhibitors: identification of analogues with cellular activity

Low molecular weight peptidomimetic compounds based on O-malonyl tyrosine and O-carboxymethyl salicylic acid are potent inhibitors of PTP1B. Modifications of the N-terminal Boc-Phe moiety were undertaken in an effort to improve physical chemical properties and to achieve cellular activity. Although Phe ultimately proved to be the optimal N-terminal amino acid, several viable replacements for the Boc group were identified, two of which afforded analogues that were effective at enhancing the insulin-stimulated uptake of 2-deoxyglucose by L6 myocytes.     

Redox regulation of the protein tyrosine phosphatase PTP1B in cancer cells

The oxidation and inactivation of protein tyrosine phosphatases is one mechanism by which reactive oxygen species influence tyrosine phosphorylation-dependent signaling events and exert their biological functions. In the present study, we determined the redox status of endogenous protein tyrosine phosphatases in HepG2 and A431 human cancer cells, in which reactive oxygen species are produced constitutively. We used mass spectrometry to assess the state of oxidation of the catalytic cysteine residue of endogenous PTP1B and show that this residue underwent both reversible and irreversible oxidation to high stoichiometry in response to intrinsic reactive oxygen species production. In addition, our data show that the oxidation of PTP1B is specific to the active site Cys, with the other Cys residues in the protein remaining in a reduced state. Treatment of these cells with diphenyleniodonium, an inhibitor of NADPH oxidases, decreased reactive oxygen species levels. This resulted in inhibition of protein tyrosine phosphatase oxidation, concomitant with decreased tyrosine phosphorylation of cellular proteins and inhibition of anchorage-independent cell growth. Therefore, our data also suggest that the high level of intrinsic reactive oxygen species may contribute to the transformed phenotype of HepG2 and A431 cells via constitutive inactivation of cellular protein tyrosine phosphatases.     

Synthesis and biological activity of a novel class of pyridazine analogues as non-competitive reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B)

A series of novel pyridazine analogues were prepared and the structure-activity relationship of their behavior as inhibitors of PTP1B was evaluated. Most of the analogues had potencies in the low micromolar range. The in vitro kinetics of this compound series demonstrated that they were reversible non-competitive binders. This indicates that there may exist another site in the enzyme through which enzyme activity can be inhibited, which is not a recognized interaction domain. Some of the analogues exhibited high selectivity for other PTPases, for example, compound 12 mp showed 20-fold selectivity for PTP1B (IC50=5.6 microM) versus both TCPTP and LAR (>100 microM, respectively). In contrast to many tyrosine phosphatase mimetic inhibitors, this compound class lacks negative charge and thus showed high permeability across cell membranes. Selective analogues in the series were analyzed in an in vitro cellular assay, which showed increased insulin-stimulated insulin receptor phosphorylation.     

Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.     

Tea contains potent inhibitors of tyrosine phosphatase PTP1B

Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 value of 0.4–4 g dry tea leaves per liter of water. Black tea shows the strongest inhibition activities, followed by oolong and then by green tea. Biochemical fractionations demonstrated that the major effective components in tea corresponded to oxidized polyphenolic compounds. This was further verified by the fact that tea catechins became potent inhibitors of PTP1B upon oxidation catalyzed by tyrosinases. When applied to cultured cells, tea extracts induced tyrosine phosphorylation of cellular proteins. Our study suggests that some beneficial effects of tea may be attributed to the inhibition of PTP1B.     

Protein tyrosine phosphatase PTP1B in cell adhesion and migration

Cell migration requires a highly coordinated interplay between specialized plasma membrane adhesion complexes and the cytoskeleton. Protein phosphorylation/dephosphorylation modifications regulate many aspects of the integrin-cytoskeleton interdependence, including their coupling, dynamics, and organization to support cell movement. The endoplasmic reticulum-bound protein tyrosine phosphatase PTP1B has been implicated as a regulator of cell adhesion and migration. Recent results from our laboratory shed light on potential mechanisms, such as Src/FAK signaling through Rho GTPases and integrin-cytoskeletal coupling.