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.     

BH3 mimetics derived from Bim-BH3 domain core region show PTP1B inhibitory activity

A series of our previously described BH3 peptide mimetics derived from Bim-BH3 domain core region were found to exhibit weak to potent PTP1B binding affinity and inhibitory activities via target-based drug screening. Among these compounds, a 12-aa Bim-BH3 core sequence peptide conjugated to palmitic acid (SM-6) displayed good PTP1B binding affinity (K_D?=?8.38?nmol/L), inhibitory activity (IC₅₀?=?1.20?μmol/L) and selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Furthermore, SM-6 promoted HepG2 cell glucose uptake and inhibited the expression of PTP1B, indicating that SM-6 could improve the insulin resistance effect in the insulin-resistant HepG2 cell model. These results may indicate a new direction for the application of BH3 peptide mimetics and promising PTP1B peptide inhibitors could be designed and developed based on SM-6.     

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.     

Palmitate and inflammatory state additively induce the expression of PTP1B in muscle cells

The factors responsible for up-regulation of PTP1B, a negative regulator of insulin signaling, in insulin resistance state are not well understood. We performed a series of experiments in C2C12 muscle cells to determine the role of palmitate and an inflammatory state in regulation of PTP1B. Palmitate (0.75 mM) induced PTP1B mRNA and protein level only at 16 h. The combination of palmitate and macrophages, accompanied by a great increase of TNF-α and IL-6 in the culture media, additively caused a higher level of PTP1B protein levels in the muscle. Higher concentrations of palmitate reduced insulin stimulated glucose uptake in myotubes. A specific inhibitor of PTP1B partly increased insulin stimulated glucose uptake in palmitate treated cells. In conclusion, our results showing the additive influence of palmitate and the inflammatory state in the expression of PTP1B imply the involvement of these factors in the overexpression of PTP1B in insulin resistance state. We further provided the evidence suggesting the mediatory role for PTP1B in palmitate induced insulin resistance in myotubes.     

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.     

An enzyme-linked immunosorbent assay to measure insulin receptor dephosphorylation by PTP1B

Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.     

Long-term high glucose concentration influences Akt, ERK1/2, and PTP1B protein expression in human aortic smooth muscle cells

Hyperglycemia stimulates a plethora of intracellular signaling pathways within the cells of the vascular wall resulting in dysfunction-associated pathologies. Most of the studies reported so far explored the effect of rather short-time exposure of smooth muscle cells to high glucose concentrations. To mimic situation in Type 2 diabetes in which vascular wall is constantly exposed to circulating hyperglycemia, we report here the long-term (7 days) effect of high glucose concentration on human media artery smooth muscle cells. This consists in up-regulation of PTP1B protein expression, down-regulation of basal Akt phosphorylation, and elevation of basal ERK1/2 activation. Acute stimulation of cells in high glucose with insulin down-regulated PTP1B expression, slightly decreased ERK1/2 activity, and activated Akt, whereas oxidative stress up-regulated Akt and ERK1/2 phosphorylation. In conclusion, long-term high glucose and acute oxidative stress and insulin stimulation imbalance the expression of activated kinases Akt and ERK1/2 and of dephosphorylating PTP1B in the insulin signaling pathway.     

Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs

To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH-PTP2 coupled to glutathione-S-transferase (GST) and insulin receptor substrate-1 (IRS-1)-GST fusion proteins (amino-portion, IRS-1N; carboxyl portion, IRS-1C). GST-SH2 protein of SH-PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl-terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co-immunoprecipitated with IRS-1C, but not with IRS-1N. These results indicate that SH2 domains of SH-PTP2 can directly associate with the Y¹³²²TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS-1, possibly via the V¹¹⁷²IDL motif in vitro.     

Organo-vanadium compounds are potent activators of the protein kinase B signaling pathway and protein tyrosine phosphorylation: mechanism of insulinomimesis

Organo-vanadium compounds (OVC) have been shown to be more effective than inorganic vanadium compounds in ameliorating glucose homeostasis and insulin resistance in rodent models of diabetes mellitus. However, the precise molecular mechanism of OVC efficiency remains poorly defined. Since inorganic vanadium compounds have been found to activate several key components of the insulin signaling cascade, such as protein kinase B (PKB), the objective of the present study was to investigate if stimulation of PKB and its downstream target glycogen synthase kinase-3 (GSK-3), are responsible for the more potent insulinomimetic effects of OVC. Among several vanadium compounds tested, vanadium (IV) oxo bis (acetylacetonate) and vanadium (IV) oxo bis(maltolato) markedly induced the phosphorylation of PKB as well as GSK-3beta compared to vanadyl sulfate (VS), an inorganic vanadium salts in Chinese hamster ovary cells overexpressing the insulin receptor (IR). Furthermore, the OVC were stronger inhibitors of protein tyrosine phosphatase (PTPase) activity than VS. The higher PTPase inhibitory potential of the OVC was associated with more robust tyrosine phosphorylation of several cellular proteins, including the IRbeta subunit and insulin receptor substrate-1 (IRS-1). In addition, greater IRS-1/p85alpha interaction was elicited by the OVC than by VS. These data indicate that the higher PTPase inhibitory potential of OVC translates into greater phosphorylation of PKB and GSK-3beta, which, in turn, may contribute to a more potent effect of OVC on glucose homeostasis.     

Tyrosine phosphatase PTP1B modulates store-operated calcium influx

We have studied modulation of “store-operated calcium influx” by tyrosine phosphatases in the pancreatic acinar cell line AR42J and in HEK 293 cells. We show that inhibition of tyrosine phosphatases by bis-(N,N-dimethyl-hydroxamido) hydrooxovanadate (DMHV) leads to an increase in Ca²⁺ release-activated Ca²⁺ (CRAC) entry. This effect can be blocked in the presence of 2-aminoethyldiphenyl borate (2-APB). Furthermore, transfection of HEK 293 cells with the human wild-type tyrosine phosphatase PTP1B leads to inhibition of CRAC influx, whereas transfection with the substrate-trapping mutant of PTP1B (D181A) slightly increases Ca²⁺ influx. It also decreases enzymatic activity of PTP1B as compared to non-transfected cells. Our data suggest that CRAC influx is modulated by tyrosine phosphorylation and dephosphorylation which involves the tyrosine phosphatase PTP1B.     

4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B

Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC₅₀ values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2–10 fold selectivity over a panel of PTP’s. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.     

PTP1B inhibitory effects of tridepside and related metabolites isolated from the Antarctic lichen Umbilicaria antarctica

The selective inhibition of PTP1B has been widely recognized as a potential drug target for the treatment of type 2 diabetes and obesity. In the course of screening for PTP1B inhibitory natural products, the MeOH extract of the dried sample of the Antarctic lichen Umbilicaria antarctica was found to exhibit significant inhibitory effect, and the bioassay-guided fractionation and purification afforded three related lichen metabolites 1-3. Compounds 1-3 were identified as gyrophoric acid (1), lecanoric acid (2), and methyl orsellinate (3) mainly by analysis of NMR and MS data. These compounds inhibited PTP1B activity with 50% inhibitory concentration values of 3.6 ± 0.04 μM, 31 ± 2.7 μM, and 277 ± 8.6 μM, respectively. Furthermore, the kinetic analysis of PTP1B inhibition by compound 1 suggested that the compound inhibited PTP1B activity in a non-competitive manner.     

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).     

Evaluation of licorice flavonoids as protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator in insulin- and leptin-signaling cascades as well as a positive regulator in tumorigenesis, and much attention has been paid to PTP1B inhibitors as potential therapies for diabetes, obesity, and cancer. In the present study, the screening of a compound library of licorice flavonoids allowed for the discovery of several compounds, including licoagrone (3), licoagrodin (4), licoagroaurone (5), and isobavachalcone (6), as new PTP1B inhibitors. It was revealed that these compounds inhibit the activity of PTP1B in different modes and with different selectivities and that they exhibit different cellular activity in the insulin-signaling pathway. Glycybenzofuran (1), a competitive PTP1B inhibitor, showed both excellent inhibitory selectivity against PTP1B and cellular activity on the insulin-stimulated Akt phosphorylation level. The similarity of its action profiling in the insulin-signaling pathway suggested its potential as a new anti-insulin-resistant drug candidate.     

Biological activity of a fragment of insulin

Insulin controls or alters glucose, protein, and fat metabolism as well as other cellular functions. Insulin binds to a specific receptor on the cell membrane initiating a protein phosphorylation cascade that controls glucose uptake and metabolism and long-term effects such as mitogenesis. This process also initiates insulin uptake and ultimate cellular metabolism in all insulin sensitive cells. The effects of insulin on other cellular metabolic properties have not been clearly related to this mechanism. Here we show that intracellular metabolism of insulin may be related to some aspects of insulin actions, specifically control of fat metabolism. A normal intracellular degradation product of insulin has been synthesized and tested for actions on fat turnover in cultured adipocytes. This 7-peptide, B-chain fragment (HLVEALY) inhibits both basal and stimulated lipolysis as measured by glycerol release, but does not inhibit FFA release because of a lack of effect on FFA reesterification in the adipocyte. HLVEALY also enhances insulin's effects on lipogenesis. This study shows that a fragment of insulin produced by the action of the insulin-degrading enzyme has both independent biological effects and interactions with insulin. This supports a biologically important effect of insulin metabolism and insulin degradation products on insulin action on non-glucose pathways.     

Eudesmanolide sesquiterpenes and protein tyrosine phosphatase 1B inhibitory ent-kaurene diterpenes from aerial parts of Indonesian Wedelia prostata

Seven eudesmanolide sesquiterpenes (1–7) and two ent-kaurene diterpenes (8 and 9) including two new (9R)-eudesman-9,12-olides, named wedelolides I and J (1 and 2), were isolated from the aerial parts of Indonesian Wedelia prostata. The structures of 1 and 2 were assigned based on their spectroscopic data. Diterpenes 8 and 9 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC₅₀ values of 8.3 and 28 μM, respectively. Among sesquiterpenes 1–7, compound 4, wedelolide D, exhibited 32% inhibitory activity against PTP1B at 20 μM.     

Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B

Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B.     

The two faces of PTP1B in cancer

PTP1B is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and is a promising drug target for type 2 diabetes and obesity. Accumulating evidence also indicates that PTP1B is involved in cancer, but contrasting findings suggest that it can exert both tumor suppressing and tumor promoting effects depending on the substrate involved and the cellular context. In this review, we will discuss the diverse mechanisms by which PTP1B may influence tumorigenesis as well as recent in vivo data on the impact of PTP1B deficiency in murine cancer models. Together, these results highlight not only the great potential of PTP1B inhibitors in cancer therapy but also the need for a better understanding of PTP1B function prior to use of these compounds in human patients.     

PTP1B inhibitory activity of kaurane diterpenes isolated from Siegesbeckia glabrescens

Protein tyrosine phosphatase 1B (PTP1B) is considered as a therapeutic target for the treatment of diabetes and obesity. In our preliminary screening study, a MeOH extract of the aerial part of Siegesbeckia glabrescens was found to inhibit PTP1B activity at 30 μg/mL. Bioassay‐guided fractionation led to the isolation of two active diterpenes, ent-16βH,17-isobutyryloxy-kauran-19-oic acid (1) and ent-16βH,17-acetoxy-18-isobutyryloxy-kauran-19-oic acid (2), along with ent-16βH,17-hydroxy-kauran-19-oic acid (3). Compounds 1 and 2 inhibited the PTP1B activity with IC₅₀ values of 8.7 ± 0.9 and 30.6 ± 2.1 μM, respectively. Kinetic studies suggest that both 1 and 2 are non-competitive inhibitors of PTP1B. However, compound 3 substituted with a hydroxyl group at C-17 in kaurane-type showed no inhibitory effects towards PTP1B.