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.     

Purification and characterization of T cell protein tyrosine phosphatase reveals significant functional homology to protein tyrosine phosphatase-1B

We have developed a protocol for rapid purification of T cell protein tyrosine phosphatase (TCPTP) and the structurally related protein tyrosine phosphatase-1B (PTP-1B) from bacterial cells. The pH profile for TCPTP was bell-shaped with an optimum of 5.5. The catalytic domain and full-length versions of TCPTP bound a potent inhibitor with affinities similar to those of PTP-1B. The K(m) values for the catalytic domains of TCPTP and PTP-1B increased with increasing ionic strength, whereas the k(cat) values remained unchanged. Arrhenius plots revealed that TCPTP and PTP-1B possess similar activation energies of 25.3+/-1.2 and 18.4+/-3.0 kJ/mol, respectively. Increasing solvent microviscosity (up to 40% (w/v) sucrose) did not affect k(cat)/K(m) of either enzyme. However, high sucrose concentrations protected both enzymes from thermal inactivation. These studies show that, although they share a 72% amino acid sequence identity within their catalytic domains, TCPTP and PTP-1B are functionally very similar in vitro.     

Crystal structure of human protein tyrosine phosphatase SHP-1 in the open conformation

SHP‐1 belongs to the family of non‐receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail‐truncated SHP‐1 and SHP‐2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP‐1, we have determined the crystal structure of the full‐length SHP‐1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N‐SH2 domain and the incorporation of sulfate ions into the ligand‐binding site of each domain indicate that the SHP‐1 is in the open conformation. The N‐SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C‐SH2 domain, resulting in exposure of the active site. Meanwhile, the C‐SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N‐SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP‐1 in the open conformation. Based on the structural comparison, a model for the activation of SHP‐1 is proposed. J. Cell. Biochem. 112: 2062–2071, 2011. © 2011 Wiley‐Liss, Inc.     

Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical

Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H₂O₂ include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H₂O₂ abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H₂O₂ is poorly reactive in chemical terms and the second order rate constants for the H₂O₂-mediated PTP inactivation are ~ 10 M^− 1 s^− 1, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H₂O₂. We find that hydroxyl radical is produced from H₂O₂ solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H₂O₂ for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H₂O₂ under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Temporal variations in protein tyrosine phosphatase activity during cell proliferation and differentiation

Cellular oscillations in total extractable protein content and protein tyrosine phosphatase activity were analysed in proliferating and differentiating human acute promyelocytic leukaemic (HL-60) cell extracts. Differentiation was induced along the granulocytic pathway using all-trans retinoic acid (ATRA). High frequency rhythms with distinct, well defined waveforms of varying amplitude were observed for both the total protein content and for the enzyme activity of protein tyrosine phosphatase (PTP). Linear correlation analysis showed that there was no obvious relationship between the protein content and the corresponding PTP activity, suggesting that the periodic variations between these two components are relatively independent of each other. ATRA significantly altered the characteristics of the rhythms with respect to the period and amplitude and had a dampening effect on the oscillatory pattern of PTP activity. Modulation of such characteristics may be of significance with respect to the regulation of the differentiation processes and the possible reversal of transformation.     

Targeted mutagenesis and genetic analysis of a Drosophila receptor-linked protein tyrosine phosphatase gene

Several Drosophila receptor-linked protein tyrosine phosphatases (R-PTPs) are selectively expressed on axons of the developing embryonic central nervous system. The extracellular domains of these axonal R-PTPs are homologous to neural adhesion molecules. Thus, R-PTPs may directly couple cell recognition to signal transduction via control of tyrosine phosphorylation. To examine the function of these molecules during nervous system development, we wished to generate mutations in R-PTP genes. It was unclear whether a mutation in a single R-PTP gene would confer lethality, however, because the similarities in sequence and expression pattern between the axonal R-PTPs suggest that they may have partially redundant functions. To circumvent this problem, we developed a directed mutagenesis strategy based on local transposition of P elements, and used this approach to isolate a null mutation in the DPTP99A gene. This strategy, which we describe in detail here, should be applicable to any Drosophila gene within a lettered division of an appropriately marked P element. Flies lacking DPTP99A expression are viable and fertile, and we have been unable to detect any alterations in the embryonic nervous system of DPTP99A embryos using a variety of antibody markers.     

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration.METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel.RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes.CONCLUSION: These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.     

Aspirin inhibits lipopolysaccharide-induced COX-2 expression and PGE2 production in porcine alveolar macrophages by modulating protein kinase C and protein tyrosine phosphatase activity

Aspirin has been demonstrated to be effective in inhibiting COX-2 and PGE₂ in Alveolar macrophages (AMs). However, the mechanisms have not been fully understood. In the present study, we found that pretreatment with aspirin inhibited LPS-induced COX-2 and PGE₂ upregulation, IκBα degradation, NFκB activation and the increase of PKC activity, but elevated LPS-induced the decrease of PTP activity. The PKC inhibitor calphostin C dramatically reduced the COX-2 mRNA and PGE₂ levels, but the PTP inhibitor peroxovanadium (POV) significantly increased the COX-2 mRNA and PGE₂ levels. Furthermore, the PTP inhibitor mitigated the inhibitory effect of aspirin on COX-2 and PGE₂ upregulation and NF-κB activation, whereas the PKC inhibitor enhanced the inhibitory effects of aspirin on the production of COX-2 and PGE₂. Our data indicate a novel mechanism by which aspirin acts as a potent anti-inflammatory agent in alveolus macrophages and ALI. [BMB Reports 2014; 47(1): 45-50]     

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.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

The protein tyrosine phosphatase alpha modifies insulin secretion in INS-1E cells

Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.     

Identification of novel imidazole flavonoids as potent and selective inhibitors of protein tyrosine phosphatase

A series of imidazole flavonoids as new type of protein tyrosine phosphatase inhibitors were synthesized and characterized. Most of them gave potent protein phosphatase 1B (PTP1B) inhibitory activities. Especially, compound 11a could effectively inhibit PTP1B with an IC₅₀ value of 0.63 μM accompanied with high selectivity ratio (9.5-fold) over T-cell protein tyrosine phosphatase (TCPTP). This compound is cell permeable with relatively low cytotoxicity. The high binding affinity and selectivity was disclosed by molecular modeling and dynamics studies. The structural features essential for activity were confirmed by quantum chemical studies.     

Design, synthesis, and evaluation of bromo-retrochalcone derivatives as protein tyrosine phosphatase 1B inhibitors

A series of bromo-retrochalcones was designed, synthesized and evaluated as PTP1B inhibitors based on licochalcone A and E. Compounds 6, 12, 13, 14, 25, 36, 37, 39, and 41 showed potent inhibitory effects against PTP1B, and compound 37, the most potent among the series, had an IC₅₀ value of 1.9 μM, about two-fold better than that of the positive control, ursolic acid.     

Nuclear localization of non-receptor protein tyrosine phosphatase epsilon is regulated by its unique N-terminal domain

Precise subcellular localization is an important factor in regulation of the functions of protein tyrosine phosphatases. The non-receptor form of protein tyrosine phosphatase epsilon (cyt-PTP(epsilon)) can be found in cell nuclei, among other cellular locations, while p67 PTP(epsilon), a naturally occurring isoform which lacks the 27 N terminal residues of cyt-PTP(epsilon), is exclusively cytosolic. Using deletion and scanning mutagenesis we report that the first 10 amino acid residues of cyt-PTP(epsilon), in particular residues R4, K5, and R9, are critical components for its nuclear localization. We also establish that increased oxidative stress enhances accumulation of cyt-PTP(epsilon) in cell nuclei. Of the four known protein forms of PTP(epsilon), cyt-PTP(epsilon) is the only one which includes the extreme N-terminal sequence containing R4, K5, and R9. The role of the unique N terminus of cyt-PTP(epsilon) is therefore to regulate its subcellular localization. The existence of naturally occurring forms of PTP(epsilon) which lack this sequence and which are generated by translational and posttranslational mechanisms, suggests that nuclear localization of cyt-PTP(epsilon) can be actively regulated by cells.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

Modification of oscillatory behaviour of protein tyrosine kinase and phosphatase during all-trans retinoic acid-induced differentiation of leukaemic cells

Granulocytic maturation of human acute promyelocytic leukaemic (HL-60) cells was induced using all-trans retinoic acid (ATRA). Time-dependent changes in the enzyme activities of protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK), and the total extractable protein content were monitored in proliferating and differentiating cells. The existence of periodicity was demonstrated clearly in both PTP and PTK enzyme activities and in the amount of protein extracted from the cells. Following ATRA treatment, differentiation-induced changes in rhythmic characteristics such as period and amplitude were evident. A noticeable effect was that of ATRA on the enzyme activity of PTP, for which four distinct patterns of oscillatory behaviour were identified. This study examines these changes, in an attempt to gain insight into the role which biochemical oscillators may play in the regulation of molecular control mechanisms.     

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.