Receptor protein tyrosine phosphatase zeta as a therapeutic target for glioblastoma therapy

Astrocytomas are the most frequent brain tumour type in adults. The most common astrocytoma is the glioblastoma (GBM), which is also the most malignant and refractory to treatment--ultimately leading to the patient's death within a year of diagnosis. Neither the classical nor more experimental therapeutic approaches have significantly improved the clinical outcome of this disease. Expression profile analysis of primary tumours has provided recent insight into the identification of new GBM therapeutic targets. These proteins serve as excellent candidates to either inhibit the target molecule's functions (e.g., angiogenesis, migration or proliferation) or, coupled with a toxin or radionucleotide, to bind and exterminate the tumour cells. The receptor protein tyrosine phosphatase zeta (RPTPzeta) and one of its main ligands, pleiotropin (Ptn), are overexpressed in GBMs, thus making them potentially very good targets for the development of new immunotherapeutics. This review will summarise recent advances in GBM therapies focusing on RPTPzeta as a target for immunotherapeutics.     

Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs^III) and its intermediate metabolites such as monomethylarsonous acid (MMA^III) and dimethylarsinous acid (DMA^III) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA^III and DMA^III) but not by iAs^III. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA^III directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation.     

Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors

A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors.     

An overview of the protein tyrosine phosphatase superfamily

The human genome encodes approximately 100 phosphatases that belong to the protein tyrosine phosphatase (PTP) superfamily, whose substrates range from proteins to phosphoinositides and mRNAs. The hallmark for this superfamily is the active site sequence C(X)5R, also known as the PTP signature motif. The PTPs are key regulatory components in signal transduction pathways and the importance of PTPs in the control of cellular signaling is well established. Furthermore, there are compelling reasons to believe that PTP inhibitors may serve as novel medicinal agents for the treatment of various diseases. Based on structure and substrate specificity, the PTP super-family is divided into four distinct subfamilies: 1). pTyr specific PTPs, 2). dual specificity phosphatases, 3). Cdc25 phosphatases, and 4). LMW PTPs. The PTPs have similar core structures made of a central parallel beta-sheet with flanking alpha-helices containing a beta-loop-alpha loop that encompasses the PTP signature motif. Not surprisingly, they employ a common chemical mechanism for phosphate hydrolysis despite the differences in substrate specificity. Despite the conserved structural and catalytic properties, there are also sufficient differences in the active site pockets and its immediate surrounding environment among different PTPs. Further structural and mechanistic study will continue to be of considerable importance, providing a solid basis for inhibitor design.     

Using yeast to screen for inhibitors of protein tyrosine phosphatase 1B

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been proposed as a novel therapy to treat type 2 diabetes and obesity. In order to identify novel PTP1B inhibitors, we have developed a robust screen in Saccharomyces cerevisiae where growth is dependent on PTP1B catalytic activity. This was based on the observation that overexpression of v-Src, a tyrosine kinase, in yeast leads to lethality through mitotic dysfunction and this lethality can be reversed by co-expression of PTP1B. The expression levels of v-Src and PTP1B were optimized to obtain a balance between robust growth and sensitivity to inhibitors. Screening was carried out in 96-well plates and growth of the liquid culture measured by absorbance at 600 nm. Initial characterization was performed using vanadate as well as some novel PTP1B inhibitors. Vanadate specifically inhibited PTP1B-dependent growth in a dose dependent manner with an EC50 of 0.92 +/- 0.07 mM. This simple yeast growth interference assay has the potential for use as a high throughput screen for PTP1B inhibitors in sample collections or crude mixtures.     

Ouabain antagonism of noradrenaline inhibitions of cerebellar purkinje cells and dopamine inhibitions of caudate neurones

As a test of the hypothesis that catecholamines exert their inhibitory effects on neuronal activity via stimulation of an electrogenic ion pump, the ability of iontophoretically applied ouabain, a known inhibitor of neuronal Na-K adenosine triphosphatase, to antagonize noradrenaline and γ-aminobutyric acid inhibitions of Purkinje cells and dopamine and γ-aminobutyric acid inhibitions of caudate cells was examined. Ouabain exhibited antagonistic activity toward amine inhibitions on both types of cells while leaving the amino acid inhibition intact. The results are interpreted as being consistent with a “pump” hypothesis of catecholamine inhibition of neuronal excitability.     

Potent reversible inhibitors of the protein tyrosine phosphatase CD45

The cytosolic portion of CD45, a major transmembrane glycoprotein found on nucleated hematopoietic cells, contains protein tyrosine phosphatase activity and is critical for T-cell receptor-mediated T-cell activation. CD45 inhibitors could have utility in the treatment of autoimmune disorders and organ graft rejection. A number of 9,10-phenanthrenediones were identified that reversibly inhibited CD45-mediated p-nitrophenyl phosphate (pNPP) hydrolysis. Chemistry efforts around the 9,10-phenanthrenedione core led to the most potent inhibitors known to date. In a functional assay, the compounds were also potent inhibitors of T-cell receptor-mediated proliferation, with activities in the low micromolar range paralleling their enzyme inhibition. It was also discovered that the nature of modification to the phenanthrenedione pharmacophore could affect selectivity for CD45 over PTP1B (protein tyrosine phosphatase 1B) or vice versa.     

Discovery of a novel shp2 protein tyrosine phosphatase inhibitor

Shp2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It is involved in growth factorinduced activation of mitogen-activated protein (MAP) kinases Erk1 and Erk2 (Erk1/2) and has been implicated in the pathogenicity of the oncogenic bacterium Helicobacter pylori. Moreover, gain-of-function Shp2 mutations have been found in childhood leukemias and Noonan syndrome. Thus, small molecule Shp2 PTP inhibitors are much needed reagents for evaluation of Shp2 as a therapeutic target and for chemical biology studies of Shp2 function. By screening the National Cancer Institute (NCI) Diversity Set chemical library, we identified 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877) as a potent Shp2 PTP inhibitor. Molecular modeling and site-directed mutagenesis studies suggested that NSC-87877 binds to the catalytic cleft of Shp2 PTP. NSC-87877 cross-inhibited Shp1 in vitro, but it was selective for Shp2 over other PTPs (PTP1B, HePTP, DEP1, CD45, and LAR). It is noteworthy that NSC-87877 inhibited epidermal growth factor (EGF)-induced activation of Shp2 PTP, Ras, and Erk1/2 in cell cultures but did not block EGF-induced Gab1 tyrosine phosphorylation or Gab1-Shp2 association. Furthermore, NSC-87877 inhibited Erk1/2 activation by a Gab1-Shp2 chimera but did not affect the Shp2-independent Erk1/2 activation by phorbol 12-myristate 13-acetate. These results identified NSC-87877 as the first PTP inhibitor capable of inhibiting Shp2 PTP in cell cultures without a detectable off-target effect. Our study also provides the first pharmacological evidence that Shp2 mediates EGF-induced Erk1/2 MAP kinase activation.     

Effects of alprazolam on the activity of rat cerebellar purkinje neurons: Evidence for mediation by norepinephrine

Acute administration of alprazolam causes a dose-dependent slowing of the spontaneous discharge of cerebellar Purkinje neurons. This effect is reversed by treatment with propranolol. There is also significantly less slowing in animals in which cerebellar neuronal circuitry has been destroyed by pretreatment with 6-hydroxydopamine. These data suggest that some of the depressant effects of alprazolam may be mediated by an interaction with norepinephrine.     

Therapeutic implications for striatal-enriched protein tyrosine phosphatase (STEP) in neuropsychiatric disorders

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that modulates key signaling molecules involved in synaptic plasticity and neuronal function. Targets include extracellular-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase p38 (p38), the Src family tyrosine kinase Fyn, N-methyl-D-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). STEP-mediated dephosphorylation of ERK1/2, p38, and Fyn leads to inactivation of these enzymes, whereas STEP-mediated dephosphorylation of surface NMDARs and AMPARs promotes their endocytosis. Accordingly, the current model of STEP function posits that it opposes long-term potentiation and promotes long-term depression. Phosphorylation, cleavage, dimerization, ubiquitination, and local translation all converge to maintain an appropriate balance of STEP in the central nervous system. Accumulating evidence over the past decade indicates that STEP dysregulation contributes to the pathophysiology of several neuropsychiatric disorders, including Alzheimer's disease, schizophrenia, fragile X syndrome, epileptogenesis, alcohol-induced memory loss, Huntington's disease, drug abuse, stroke/ischemia, and inflammatory pain. This comprehensive review discusses STEP expression and regulation and highlights how disrupted STEP function contributes to the pathophysiology of diverse neuropsychiatric disorders.     

地塞米松诱导产生耐受性树突状细胞的机制

糖皮质激素地塞米松是临床广泛应用的一类免疫抑制剂。近年来研究发现,在免疫应答反应的早期,地塞米松是通过影响体内的树突状细胞（dendritic cell,DC）而发挥作用的。地塞米松可通过抑制DC的分化和成熟、改变DC的表型和DC的某些重要生物学功能,抑制特异性免疫应答反应的启动并减少炎性细胞因子的产生。利用地塞米松对DC的影响来治疗自身免疫病、肿瘤、移植排斥反应等疾病可能会成为基础和临床医学研究的新热点。     

Classification of binding site conformations of protein tyrosine phosphatase 1B

Hundred and two binding sites from 91 Protein Data Bank files for protein tyrosine phosphatase 1B with different ligands have been compared. It was found that they can be divided into five clusters. Additional clusters were formed by the unliganded and oxidized enzyme. The centroids of the clusters can be used as starting points for further studies of enzyme-inhibitor interaction by computer simulations. A special software tool has been created for the investigation of protein tyrosine phosphatase 1B and other enzymes. It performs multiple comparisons of selected parts of Protein Data Bank files, as well as further clustering, and determines mobility of separate residues.     

Development of molecular probes for second-site screening and design of protein tyrosine phosphatase inhibitors

We report on the design, synthesis, and evaluation of a series of furanyl-salicyl-nitroxide derivatives as effective chemical probes for second-site screening against phosphotyrosine phosphatases (PTPs) using NMR-based techniques. The compounds have been tested against a panel of PTPs to assess their ability to inhibit a broad spectrum of these phosphatases. The utility of the derived compounds is illustrated with the phosphatase YopH, a bacterial toxin from Yersinia pestis. Novel chemical fragments were identified during an NMR-based screen for compounds that are capable of binding on the surface of YopH in regions adjacent the catalytic site in the presence of the spin-labeled compounds. Our data demonstrate the value of the derived chemical probes for NMR-based second-site screening in PTPs.     

Maternal marginal iodine deficiency limits dendritic growth of cerebellar purkinje cells in rat offspring by NF‐κB signaling and MAP1B

Iodine deficiency (ID) during early pregnancy had an adverse effect on children's psychomotor and motor function. It is worth noting that maternal marginal ID tends to be a common public health problem. Whether marginal ID potentially had adverse effects on the development of cerebellum and the underlying mechanisms remain unclear. Therefore, our aim was to study the effects of marginal ID on the dendritic growth in filial cerebellar Purkinje cells (PCs) and the underlying mechanism. In the present study, we established Wistar rat models by feeding dam rats with a diet deficient in iodine and deionized water supplemented with potassium iodide. We examined the total dendritic length using immunofluorescence, and Western blot analysis was conducted to investigate the activity of nuclear factor‐κB (NF‐κB) signaling and microtubule‐associated protein 1B (MAP1B). Our results showed that marginal ID reduced the total dendritic length of cerebellar PCs, slightly down‐regulated the activity of NF‐κB signaling and decreased MAP1B in cerebellar PCs on postnatal day (PN) 7, PN14, and PN21. Our study may support the hypothesis that decreased T₄ induced by marginal ID limits PCs dendritic growth, which may involve in the disturbance of NF‐κB signaling and MAP1B on the cerebellum. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1241–1251, 2017.     

High-throughput methods in identification of protein tyrosine phosphatase inhibitors and activators

Reversible protein tyrosine phosphorylation, catalysed by the counter-actors protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs), is a fundamentally important regulatory mechanism of proteins in living cells, controlling cell communication, proliferation, differentiation, motility, and molecular trafficking. The activities of PTPs and PTKs are derailed in several diseases such as cancer and type II diabetes, making them attractive drug targets. Developing drugs against PTKs has started a decade earlier than that on PTPs, and at present there are several molecules targeting PTKs on the market. PTPs in turn are of raising interest, with PTP1B on the lead for its effects on type II diabetes and obesity. In the search for modulators of PTP activity, high-throughput methods are important as the initial step to find suitable lead compounds for drug development. Also, high-throughput methods are very useful in elucidating the specific function of different PTPs. In this review, the different high-throughput studies performed to find inhibitors and activators of classical PTPs are discussed.     

人源蛋白酪氨酸磷酸酶(PTP1B)抑制剂的高通量筛选

本研究拟建立体外人源蛋白酪氨酸磷酸酶(PTP1B)抑制剂高通量筛选模型,用于PTP1B抑制剂的发现。利用大肠杆菌重组表达PTP1B,以对硝基苯磷酸二钠(PNPP)为特异性的底物,建立了基于酶反应速率的384孔微板为载体的PTP1B抑制剂高通量筛选模型(Z'=0.78)。选择24 240个样品进行筛选,对抑制率大于70%的80个样品作为活性样品进行复筛,最终确定6个具有较强的抑制活性的化合物J5753、J10550、J10551、J10583、J10585和J11101,其IC50值分别为21.58、18.39、15.37、11.92、37.27和36.61μg·mL-1。结果表明,所建立的PTP1B抑制剂高通量筛选模型具有快速、灵敏、稳定、重复性好的特点。