Protein Tyrosine Phosphatase Inhibitor Research Articles

Structure-Based Virtual Screening of Protein Tyrosine Phosphatase Inhibitors: Significance, Challenges, and Solutions.

Phosphorylation, a critical mechanism in biological systems, is estimated to be indispensable for about 30% of key biological activities, such as cell cycle progression, migration, and division. It is synergistically balanced by kinases and phosphatases, and any deviation from this balance leads to disease conditions. Pathway or biological activity-based abnormalities in phosphorylation and the type of involved phosphatase influence the outcome, and cause diverse diseases ranging from diabetes, rheumatoid arthritis, and numerous cancers. Protein tyrosine phosphatases (PTPs) are of prime importance in the process of dephosphorylation and catalyze several biological functions. Abnormal PTP activities are reported to result in several human diseases. Consequently, there is an increased demand for potential PTP inhibitory small molecules. Several strategies in structure-based drug designing techniques for potential inhibitory small molecules of PTPs have been explored along with traditional drug designing methods in order to overcome the hurdles in PTP inhibitor discovery. In this review, we discuss druggable PTPs and structure-based virtual screening efforts for successful PTP inhibitor design.

Ginkgolic Acid C 17:1, Derived from Ginkgo biloba Leaves, Suppresses Constitutive and Inducible STAT3 Activation through Induction of PTEN and SHP-1 Tyrosine Phosphatase.

Ginkgolic acid C 17:1 (GAC 17:1) extracted from Ginkgo biloba leaves, has been previously reported to exhibit diverse antitumor effect(s) through modulation of several molecular targets in tumor cells, however the detailed mechanism(s) of its actions still remains to be elucidated. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that regulates various critical functions involved in progression of diverse hematological malignancies, including multiple myeloma, therefore attenuating STAT3 activation may have a potential in cancer therapy. We determined the anti-tumor mechanism of GAC 17:1 with respect to its effect on STAT3 signaling pathway in multiple myeloma cell lines. We found that GAC 17:1 can inhibit constitutive activation of STAT3 through the abrogation of upstream JAK2, Src but not of JAK1 kinases in U266 cells and also found that GAC can suppress IL-6-induced STAT3 phosphorylation in MM.1S cells. Treatment of protein tyrosine phosphatase (PTP) inhibitor blocked suppression of STAT3 phosphorylation by GAC 17:1, thereby indicating a critical role for a PTP. We also demonstrate that GAC 17:1 can induce the substantial expression of PTEN and SHP-1 at both protein and mRNA level. Further, deletion of PTEN and SHP-1 genes by siRNA can repress the induction of PTEN and SHP-1, as well as abolished the inhibitory effect of drug on STAT3 phosphorylation. GAC 17:1 down-regulated the expression of STAT3 regulated gene products and induced apoptosis of tumor cells. Overall, GAC 17:1 was found to abrogate STAT3 signaling pathway and thus exert its anticancer effects against multiple myeloma cells.

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

The ultra-rapidly activating delayed rectifier K+ current IKur (encoded by Kv 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial IKur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial IKur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of IKur induced by 30μM genistein or 10μM AG556 was significantly reversed by 1mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hKv 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1μM) slightly enhanced IKur and hKv 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hKv 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial IKur and cloned hKv 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases IKur and hKv 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial IKur .

Optimized logic rules reveal interferon-γ-induced modes regulated by histone deacetylases and protein tyrosine phosphatases.

The pro-inflammatory cytokine interferon-γ (IFN-γ) is critical for activating innate and adaptive immunity against tumours and intracellular pathogens. Interferon-γ is secreted at the fetal-maternal interface in pregnant women and mice. The outer layer of the placenta in contact with maternal blood is composed of semi-allogeneic trophoblast cells, which constitute the fetal component of the fetal-maternal interface. The simultaneous presence of pro-inflammatory IFN-γ and trophoblast cells at the fetal-maternal interface appears to represent an immunological paradox, for trophoblastic responses to IFN-γ could potentially lead to activation of maternal immunity and subsequent attack of the placenta. However, our previous studies demonstrate that IFN-γ responsive gene (IRG) expression is negatively regulated in human and mouse trophoblast cells. In human cytotrophoblast and trophoblast-derived choriocarcinoma cells, janus kinase signalling is blocked by protein tyrosine phosphatases (PTPs), whereas in mouse trophoblast, histone deacetylases (HDACs) inhibit IRG expression. Here, we used genome-wide transcriptional profiling to investigate the collective roles of PTPs and HDACs on regulation of IRG expression in human choriocarcinoma cells. Logic-rules were optimized to derive regulatory modes governing gene expression patterns observed upon different combinations of treatment with PTP and HDAC inhibitors. The results demonstrate that IRGs can be divided into several categories in human choriocarcinoma cells, each of which is subject to distinct mechanisms of repression. Hence, the regulatory modes identified in this study suggest that human trophoblast and choriocarcinoma cells may evade the potentially deleterious consequences of exposure to IFN-γ by using several overlapping mechanisms to block IRG expression.

Expression and alteration of BKCa channels in the sphincter of Oddi's from rabbits with hypercholesterolemia

ABSTRACTThis study aimed to investigate the expression and function of BKCa channels in the Sphincter of Oddi (SO) in a rabbit model of hypercholesterolemia (HC). New Zealand white rabbits were randomly divided into 2 groups: the control group was fed standard chow (n = 18) whereas the high-cholesterol group was fed cholesterol-enriched chow containing 1.5% cholesterol (n = 18). The serum cholesterol level was significantly greater in the HC groups than in the control group, but there was no significant difference in body weight between the control and HC groups. Although the total protein expression of BKCa α- and β1-subunit was not significantly different between the control and HC groups, the Tyr-phosphorylation of BKCa α-subunit was significantly decreased in the HC group than in the control group. In addition, hypercholesterolemia significantly increased Acetylcholine (ACh)-induced contraction of the SO rings. Pretreatment with 30 μM NS1619, a BKCa channel agonist, significantly reduced ACh-induced contraction of the SO rings in HC rabbits. Moreover, pretreatment with 100 μM Na3OV4, a protein tyrosine phosphatase inhibitor, significantly reduced ACh-induced contraction of the SO rings in HC rabbits, whereas it significantly increased upon pretreating with 10 μM Genistein, a tyrosine kinase inhibitor. Whole-cell patch clamp recordings showed that BKCa current density was significantly lower in SOSMCs from HC group than that from control group. Our findings suggest that hypercholesterolemia-induced downregulation of BKCa channel, and Tyr-phosphorylation of BKCa α-subunit may contribute to the hyperresponsiveness of the SO ring in HC rabbits.

An epigenetic modifier induces production of (10′S)-verruculide B, an inhibitor of protein tyrosine phosphatases by Phoma sp. nov. LG0217, a fungal endophyte of Parkinsonia microphylla

Incorporation of the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), to a culture broth of the endophytic fungus Phoma sp. nov. LG0217 isolated from Parkinsonia microphylla changed its metabolite profile and resulted in the production of (10′S)-verruculide B (1), vermistatin (2) and dihydrovermistatin (3). When cultured in the absence of the epigenetic modifier, it produced a new metabolite, (S,Z)-5-(3′,4′-dihydroxybutyldiene)-3-propylfuran-2(5H)-one (4) together with nafuredin (5). The structure of 4 was elucidated by spectroscopic analyses and its absolute configuration was determined by application of the modified Mosher’s ester method. The absolute structure of (10′S)-verruculide B was determined as 5-[(10′S,2′E,6′E)-10′,11′-dihydroxy-3′,7′,11′-trimethyldodeca-2′,6′-dien-1′-yl]-(3R)-6,8-dihydroxy-3-methylisochroman-1-one (1) with the help of CD and NOE data. Compound 1 inhibited the activity of protein tyrosine phosphatases (PTPs) 1B (PTP1B), Src homology 2-containing PTP 1 (SHP1) and T-cell PTP (TCPTP) with IC50 values of 13.7±3.4, 8.8±0.6, and 16.6±3.8μM, respectively. Significance of these activities and observed modest selectivity of 1 for SHP1 over PTP1B and TCPTP is discussed.

Phloroglucinol Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activities from Eugenia jambolana Seeds.

Fifteen new phloroglucinol derivatives, jamunones A-O (1-8 and 10-16, respectively), along with one known analogue spiralisone C (9), were isolated from Eugenia jambolana seeds. Their structures were elucidated by detailed nuclear magnetic resonance and mass spectrometry spectroscopic data interpretation. Compounds 1-9, 11, 12, and 14-16 inhibited protein tyrosine phosphatase 1B activity with IC50 values ranging from 0.42 to 3.2 μM.

High-Fat-Diet-Induced Deficits in Dopamine Terminal Function Are Reversed by Restoring Insulin Signaling.

Systemically released insulin crosses the blood-brain barrier and binds to insulin receptors on several neural cell types, including dopaminergic neurons. Insulin has been shown to decrease dopamine neuron firing in the ventral tegmental area (VTA), but potentiate release and reuptake at dopamine terminals in the nucleus accumbens (NAc). Here we show that prolonged consumption of a high fat diet blocks insulin's effects in the NAc, but insulin's effects are restored by inhibiting protein tyrosine phosphatase 1B, which supports insulin receptor signaling. Mice fed a high fat diet (60% kcals from fat) displayed significantly higher fasting blood glucose 160 mg/dL, compared to 101 mg/dL for control-diet-fed mice, and high-fat-diet-fed mice showed reduced blood glucose clearance after an intraperitoneal glucose tolerance test. Using fast scan cyclic voltammetry to measure electrically evoked dopamine in brain slices containing the NAc core, high-fat-diet-fed mice exhibited slower dopamine reuptake compared to control-diet-fed mice (2.2 ± 0.1 and 2.67 ± 0.15 μM/s, respectively). Moreover, glucose clearance rate was negatively correlated with Vmax. Insulin (10 nM to 1 μM) dose dependently increased reuptake rates in control-diet-fed mice compared with in the high-fat-diet group; however, the small molecule insulin receptor sensitizing agent, TCS 401 (300 nM), restored reuptake in high-fat-diet-fed mice to control-diet levels, and a small molecule inhibitor of the insulin receptor, BMS 536924 (300 nM), attenuated reuptake, similar to high-fat-diet-fed mice. These data show that a high-fat diet impairs dopamine reuptake by attenuating insulin signaling at dopamine terminals.

Sulfonyl-bridged Calix[4]arene as an Inhibitor of Protein Tyrosine Phosphatases

Previously, phosphonic acid derivatives of calix[4]arene and thiacalix[4]arene were found to be potential inhibitors of protein tyrosine phosphatase 1B. In the present paper, the inhibitory activity of unsubstituted sulfonyl-bridget calix[4]arene towards some of the therapeutically important protein tyrosine phosphatases has been established. The obtained results showed that the sulfonylcalix[4]arene is able to inhibit protein tyrosine phosphatase MEG2 with IC50 value in the micromolar range. At the same time, the inhibitor demonstrated lower activity in case of other protein tyrosine phosphatases such as PTP1B, MEG1, TC-PTP, SHP2, and PTPβ. The performed molecular docking indicated that the inhibitor binds to the active site region of MEG2 and PTP1B with WPD-loop in the open conformation.

Covalent inhibition of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are a large family of 107 signaling enzymes that catalyze the hydrolytic removal of phosphate groups from tyrosine residues in a target protein. The phosphorylation status of tyrosine residues on proteins serve as a ubiquitous mechanism for cellular signal transduction. Aberrant function of PTPs can lead to many human diseases, such as diabetes, obesity, cancer, and autoimmune diseases. As the number of disease relevant PTPs increases, there is urgency in developing highly potent inhibitors that are selective towards specific PTPs. Most current efforts have been devoted to the development of active site-directed and reversible inhibitors for PTPs. This review summarizes recent progress made in the field of covalent inhibitors to target PTPs. Here, we discuss the in vivo and in vitro inactivation of various PTPs by small molecule-containing electrophiles, such as Michael acceptors, α-halo ketones, epoxides, and isothiocyanates, etc. as well as oxidizing agents. We also suggest potential strategies to transform these electrophiles into isozyme selective covalent PTP inhibitors.

Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa

Protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase are important targets to treat obesity and diabetes, due to their deep correlation with insulin and leptin signalling, and glucose regulation. The methanol extract of Paulownia tomentosa fruits showed potent inhibition against both enzymes. Purification of this extract led to eight geranylated flavonoids (1–8) displaying dual inhibition of PTP1B and α-glucosidase. The isolated compounds were identified as flavanones (1–5) and dihydroflavonols (6–8). Inhibitory potencies of these compounds varied accordingly, but most of the compounds were highly effective against PTP1B (IC50 = 1.9–8.2 μM) than α-glucosidase (IC50 = 2.2–78.9 μM). Mimulone (1) was the most effective against PTP1B with IC50 = 1.9 μM, whereas 6-geranyl-3,3′,5,5′,7-pentahydroxy-4′-methoxyflavane (8) displayed potent inhibition against α-glucosidase (IC50 = 2.2 μM). All inhibitors showed mixed type Ι inhibition toward PTP1B, and were noncompetitive inhibitors of α-glucosidase. This mixed type behavior against PTP1B was fully demonstrated by showing a decrease in Vmax, an increase of Km, and Kik/Kiv ratio ranging between 2.66 and 3.69.

Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

AbstractThe main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes ofLeishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinantLmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinantLmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in differentLeishmaniaspecies; theLmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

Protein Tyrosine Phosphatase-Prospective Target against Cancer: A Mini Review

Contemporary quantum leap on functional characterization of Protein Tyrosine Phosphatase (PTP) superfamily provides an incipient perspective on regulating signal transduction. PTPs are required in the regulation of several cellular processes; especially under stressed and pathogenic conditions leading to sundry human diseases. Concrete inhibition of PTP by oxyanions and active-site directed inhibitors (alkylating agents) may provide implements for human disease treatment involving them. The physiological paramountcy for the advancement of Protein Tyrosine Phosphatase, Non-receptor Type 1 (PTP1B) -predicated therapeutics is a prominent target for diabetes and inordinate corpulence treatment. PTPs are exhilarating quarry for active-site-mediated inhibitors generation. There, proneness to oxidation often create problem on high throughput screens, further the propensity for highly charged potent inhibitors, like non-hydrolysable pTyr mimetics, test with reverence to bioavailability. Subsequent preliminary concerns about specificity and quandaries with deference to hydrophilicity of phosphormimetics, promising successes attained by structure-predicated drug design, mainly the one exploit identical surface topology circumventing the catalytic pocket of each PTP. In PTP1B, it was found that a particular pTyr binding site could be habituated to succeed highly concrete bidentate inhibitors that bind both sites. This conventional approach can avail us to target highly categorical and efficacious inhibitors. Tyrosine phosphorylation increases 1-2% of total protein phosphorylation in tumorigenic transformation or magnification factor simulation essential for a controlled cellular event. This event is controlled by two molecular switches of enzymes protein tyrosine kinase and protein tyrosine phosphatase. Eccentric tyrosine phosphorylation is considered as one of the hallmarks of cancer. PTP has been recommended as next generation drug targets and a sum of PTP have been embroiled in sundry human disease, like cancer. The catalytic mechanism of PTP was demystified by site-directed mutagenesis then kinetic analyses with structural information. They have loss/ gain of function in cancer signaling events leading to dearth of inhibitors to control gain of function including modification of loss of function of PTP cognate genes. This review is about the consequentiality of tyrosine phosphatase enzyme and its role in the mundane cellular event and how it modifies the active site to agonise substrate and alter its action ultimately leading to tumorigenesis.

Tyrosine kinase/phosphatase inhibitors decrease dengue virus production in HepG2 cells

Dengue virus is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. High rates of dengue virus replication and virion production are related to disease severity. To identify anti-DENV compounds, we performed cell-based ELISA testing to detect the level of DENV E protein expression. Among a total of 83 inhibitors, eight were identified as inhibitors with antiviral activity. Epidermal growth factor receptor inhibitor II (EGFR/ErbB-2/ErbB-4 inhibitor II) and protein tyrosine phosphatase inhibitor IV (PTP inhibitor IV) significantly inhibited dengue virus production and demonstrated low toxicity in hepatocyte cell lines. Our results suggest the efficacy of tyrosine kinase/phosphatase inhibitors in decreasing dengue virus production in HepG2 cells.

Drugging the Undruggable: Therapeutic Potential of Targeting Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are essential signaling enzymes that, together with protein tyrosine kinases, regulate tyrosine phosphorylation inside the cell. Proper level of tyrosine phosphorylation is important for a diverse array of cellular processes, such as proliferation, metabolism, motility, and survival. Aberrant tyrosine phosphorylation, resulting from alteration of PTP expression, misregulation, and mutation, has been linked to the etiology of many human ailments including cancer, diabetes/obesity, autoimmune disorders, and infectious diseases. However, despite the fact that PTPs have been garnering attention as compelling drug targets, they remain a largely underexploited resource for therapeutic intervention. Indeed, PTPs have been widely dismissed as "undruggable", due to concerns that (1) the highly conserved active site (i.e., pTyr-binding pocket) makes it difficult to achieve inhibitor selectivity among closely related family members, and (2) the positive-charged active site prefers negatively charged molecules, which usually lack cell permeability. To address the issue of selectivity, we advanced a novel paradigm for the acquisition of highly potent and selective PTP inhibitors through generation of bivalent ligands that interact with both PTP active site and adjacent unique peripheral pockets. To overcome the bioavailability issue, we have identified nonhydrolyzable pTyr mimetics that are sufficiently polar to bind the PTP active site, yet still capable of efficiently penetrating cell membranes. We show that these pTyr mimetics interact in the desired inhibitory fashion with the PTP active site and tethering them to appropriate molecular fragments to engage less conserved interactions outside of PTP active site can increase PTP inhibitor potency and selectivity. We demonstrate through three pTyr mimetics fragment-based approaches that it is completely feasible to obtain highly potent and selective PTP inhibitors with robust in vivo efficacy in animal models of oncology, diabetes/obesity, autoimmune disorders, and tuberculosis. We hope that these results will help dispel concerns about the druggability of PTPs and entice further effort in fostering a PTP-based drug discovery enterprise. Well-characterized, potent, selective and bioactive inhibitors are essential tools for functional interrogation of PTPs in disease biology and target validation. They will also play a critical role in illuminating the druggability of PTPs and provide the groundwork for new therapies for the treatment of human diseases.

Inhibition of protein tyrosine phosphatases unmasks vasoconstriction and potentiates calcium signaling in rat aorta smooth muscle cells in response to an agonist of 5-HT2B receptors BW723C86

In blood vessels, serotonin 5-HT2B receptors mainly mediate relaxation, although their activation by the selective agonist BW723C86 is known to exert contraction of aorta in deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-l-arginine (l-NAME) hypertensive rats [Russel et al., 2002; Banes et al., 2003] and in mice with type 2 diabetes [Nelson et al., 2012]. The unmasking effect on vasoconstriction can be caused by a shift in the balance of tyrosine phosphorylation in smooth muscle cells (SMC) due to oxidative stress induced inhibition of protein tyrosine phosphatases (PTP). We have demonstrated that BW723C86 which does not cause contraction of rat aorta and mesenteric artery rings, evoked a vasoconstrictor effect in the presence of PTP inhibitors sodium orthovanadate (Na3VO4) or BVT948. BW723C86 induced a weak rise of [Ca2+]i in the SMC isolated from rat aorta; however, after pre-incubation with Na3VO4 the response to BW723C86 increased more than 5-fold. This effect was diminished by protein tyrosine kinase (PTK) inhibitor genistein, inhibitor of Src-family kinases PP2, inhibitor of NADPH-oxidase VAS2870 and completely suppressed by N-acetylcysteine and 5-HT2B receptor antagonist RS127445. Using fluorescent probe DCFH-DA we have shown that Na3VO4 induces oxidative stress in SMC. In the presence of Na3VO4 BW723C86 considerably increased formation of reactive oxygen species while alone had no appreciable effect on DCFH oxidation. We suggest that oxidative stress causes inhibition of PTP and unmasking of 5-HT2B receptors functional activity.

Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures.

Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF). Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB), have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3), we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated) at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1) protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.

Effects of hPTPβ inhibitor on microcirculation of rat cremaster muscle flap following ischemia-reperfusion injury.

Inhibition of protein tyrosine phosphatases (PTP) enhances endothelial receptor tyrosine kinases activation and may have beneficial effects on vessel growth and improve blood flow to ischemic tissue. The purpose of this study is to determine influence of hPTPß inhibitors on ischemia-reperfusion injury in muscle flap. Following cremaster muscle dissection, 60 rats divided into 10 experimental groups (placebo and treatment groups following 0, 1, 2, 3, and 4 h of ischemia). Following group-specific treatment (placebo/hPTPß inhibitor, 15 mg/kg), 2 h of reperfusion is initiated. Observations are performed at 4 h after completion of reperfusion and microcirculatory hemodynamics and leukocyte-endothelial activation were recorded. Administration of hPTPß inhibitor showed preservation of capillary perfusion in group subjected to 2 h of ischemia when compared with placebo (P < .05). The effect of hPTPβ inhibitor on mean venule diameter was found to be altered by duration of ischemia and this effect was statistically significant (P < .05). Treated ischemic groups (1 h, 2 h, and 3 h) showed decreased activation of rolling, sticking, and transmigrating leukocytes compared to respective placebo groups at all time points. The differences were significant for transmigrating leukocytes after 2 h and 3 h of ischemia (P < .05). Endothelial edema index was also significantly reduced in 2 h ischemia group (P < .05). Administration of hPTP inhibitors after submission of tissue to subcritical ischemia (1-2 h) improved functional capillary perfusion and decreased leukocyte-endothelial activation after 4 h after reperfusion. These results indicate that hPTP inhibitor has a potential postischemic therapeutic effect applied after tissue ischemia just before the reperfusion injury.

317 - PTP1B Regulates Argonaute 2 Activity, MED 13 Expression and Cardiac Hypertrophy

Cardiac hypertrophy involves the expression of a gene program that occurs when cardiomyocytes are continuously exposed to stresses. The initial determinant is biomechanical stretch, with the concomitant increased expression and release of growth factors, Ras activation and reactive oxygen species (ROS) signaling. Given the importance of ROS in the transition from cardiac hypertrophy to heart failure and the documented inhibition of Protein Tyrosine Phosphatases (PTPs) by ROS, we hypothesized and explored whether specific PTPs could act as checkpoints in this process. We have identified PTP1B as a target of ROS in hearts undergoing hypertrophy. To understand the role of PTP1B in cardiac hypertrophy, we generated cardiomyocyte-specific PTP1B knockout (PTP1B cKO) mice. Subjecting PTP1B cKO mice to pressure overload (PO) caused a dramatic left ventricular dilation and several features of heart failure when compared to control mice subjected to PO for the same period. Characterization of mRNAs expressed in hypertrophy revealed that the increased expression of b-MHC observed in control mice subjected to PO was compromised in PTP1B cKO-PO mice. We identified a member of the RNA-Induced Silencing Complex, Argonaute 2 (AGO2), as a substrate of PTP1B in PO hearts and observed increased AGO2 phosphotyrosine-393 levels. A miRNA microarray of PO hearts revealed an uncoupling between miR abundance and gene silencing in PTP1B cKO hearts: in hypertrophic PTP1B cKO hearts, the regulation of Mediator 13 (MED13) by AGO2-miR208 was compromised, leading to the activation of Thyroid Receptor b1 (TRb1) and to the suppression of b-MHC expression. Preventing TRb1 activation by inhibiting thyroid hormone synthesis resulted in up-regulation of b-MHC, and recovered the phenotype. We propose a model in which ROS inhibition of PTP1B regulates AGO2 activity, MED13 expression and contributes to cardiac hypertrophy and heart failure.

Prunin is a highly potent flavonoid from Prunus davidiana stems that inhibits protein tyrosine phosphatase 1B and stimulates glucose uptake in insulin-resistant HepG2 cells.

Prunin is the main flavonoid in Prunus davidiana stems and improves hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. The aim of this study was to investigate the in vitro anti-diabetic potential of prunin via the inhibition of protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, peroxynitrite (ONOO-)-mediated tyrosine nitration, and stimulation of glucose uptake in insulin-resistant hepatocytes. In addition, a molecular docking simulation was performed to predict specific prunin binding modes during PTP1B inhibition. Prunin showed strong inhibitory activity against PTP1B, with an IC50 value of 5.5±0.29µM, and significant inhibitory activity against α-glucosidase, with an IC50 value of 317±2.12µM. Moreover, a kinetics study revealed that prunin inhibited PTP1B (K i=8.66) and α-glucosidase (K i=189.56) with characteristics typical of competitive and mixed type inhibitors, respectively. Docking simulations showed that prunin selectively inhibited PTP1B by targeting its active site and exhibited good binding affinity, with a docking score of -9kcal/mol. Furthermore, prunin exhibited dose-dependent inhibitory activity against ONOO--mediated tyrosine nitration and stimulated glucose uptake by decreasing PTP1B expression level in insulin-resistant HepG2 cells. These results indicate that prunin has significant potential as a selective PTP1B inhibitor and may possess anti-diabetic properties by improving insulin resistance.