Tapping the therapeutic potential of protein tyrosine phosphatase 4A with small molecule inhibitors

Protein tyrosine phosphatases (PTPs) are emerging new targets for drug discovery. PTPs and protein tyrosine kinases (PTKs) maintain cellular homeostasis through opposing roles: tyrosine O-dephosphorylation and -phosphorylation, respectively. An imbalance in the phosphorylation equilibrium results in aberrant protein signaling and pathophysiological conditions. PTPs have historically been considered ‘undruggable’, in part due to a lack of evidence defining their relationship to disease causality and a focus on purely competitive inhibitors. However, a better understanding of protein–protein interfaces and shallow active sites has recently renewed interest in the pursuit of allosteric and orthosteric modulators of targets outside the major druggable protein families. While their biological mechanism of action still remains to be clarified, PTP4A1–3 (also referred to as PRL1-3) are validated oncology targets and play an important role in cell proliferation, metastasis, and tumor angiogenesis. In this Digest, recent syntheses and structure-activity relationships (SAR) of small molecule inhibitors (SMIs) of PTP4A1–3 are summarized, and enzyme docking studies of the most potent chemotype are highlighted. In particular, the thienopyridone scaffold has emerged as a potent lead structure to interrogate the function and druggability of this dual-specificity PTP.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.     

Regulation of Signaling by Protein-Tyrosine Phosphatases: Potential Roles in the Nervous System

During neuronal development, cells respond to a variety of environmental cues through cell surface receptors that are coupled to a signaling transduction machinery based on protein tyrosine phosphorylation and dephosphorylation. Receptor and non-receptor tyrosine kinases have received a great deal of attention; however, in the last few years, receptor (plasma membrane associated) and non-receptor protein-tyrosine phosphatases (PTPs) have also been shown to play important roles in development of the nervous system. In many cases PTPs have provocative distribution patterns or have been shown to be associated with specific cell adhesion and growth factor receptors. Additionally, altering PTP expression levels or activity impairs neuronal behavior. In this review we outline what is currently known about the role of PTPs in development, differentiation and neuronal physiology.     

Receptor type protein tyrosine phosphatases (RPTPs) – roles in signal transduction and human disease

Protein tyrosine phosphorylation is a fundamental regulatory mechanism controlling cell proliferation, differentiation, communication, and adhesion. Disruption of this key regulatory mechanism contributes to a variety of human diseases including cancer, diabetes, and auto-immune diseases. Net protein tyrosine phosphorylation is determined by the dynamic balance of the activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Mammals express many distinct PTKs and PTPs. Both of these families can be sub-divided into non-receptor and receptor subtypes. Receptor protein tyrosine kinases (RPTKs) comprise a large family of cell surface proteins that initiate intracellular tyrosine phosphorylation-dependent signal transduction in response to binding of extracellular ligands, such as growth factors and cytokines. Receptor-type protein tyrosine phosphatases (RPTPs) are enzymatic and functional counterparts of RPTKs. RPTPs are a family of integral cell surface proteins that possess intracellular PTP activity, and extracellular domains that have sequence homology to cell adhesion molecules. In comparison to extensively studied RPTKs, much less is known about RPTPs, especially regarding their substrate specificities, regulatory mechanisms, biological functions, and their roles in human diseases. Based on the structure of their extracellular domains, the RPTP family can be grouped into eight sub-families. This article will review one representative member from each RPTP sub-family.     

Protein tyrosine phosphatase profiling analysis of HIB-1B cells during brown adipogenesis

A number of evidence have been accumulated that the regulation of reversible tyrosine phosphorylation, which can be regulated by the combinatorial activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays crucial roles in various biological processes including differentiation. There are a total of 107 PTP genes in the human genome, collectively referred to as the "PTPome." In this study, we performed PTP profiling analysis of the HIB-1B cell line, a brown preadipocyte cell line, during brown adipogenesis. Through RT-PCR and real-time PCR, several PTPs showing differential expression pattern during brown adipogenesis were identified. In the case of PTP-RE, it was shown to decrease significantly until 4 days after brown adipogenic differentiation, followed by a dramatic increase at 6 days. The overexpression of PTP-RE led to decreased brown adipogenic differentiation via reducing the tyrosine phosphorylation of the insulin receptor, indicating that PTP-RE functions as a negative regulator at the early stage of brown adipogenesis.     

Proteomic approaches to studying protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that play key roles in cell signaling. Deregulation of PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. Since phosphate removal by the PTPs can both enhance and antagonize cellular signaling, it is essential to elucidate the physiological context in which PTPs operate. Two powerful proteomic approaches have been developed to rapidly establish the exact functional roles for every PTP, both in normal cellular physiology and in pathogenic conditions. In the first, an affinity-based substrate-trapping approach has been employed for PTP substrate identification. Identification and characterization of specific PTP-substrate interactions will associate functions with PTP as well as implicate PTP to specific signaling pathways. In the second, a number of activity-based PTP probes have been developed that can provide a direct readout of the functional state of the PTPs in complex proteomes. The ability to profile the entire PTP family on the basis of changes in their activity is expected to yield new functional insights into pathways regulated by the PTPs and contribute to the discovery of PTPs as novel therapeutic targets. Effective application of these proteomic techniques will accelerate the functional characterization of PTPs, thereby facilitating our understanding of PTPs in cell signaling and in diseases.     

Modulation of protein tyrosine phosphatase 1B by erythropoietin in UT-7 cell line.

BACKGROUND/ AIMS: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. METHODS: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. RESULTS: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). CONCLUSION: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.     

Regulation of the catalytic activity of PTP1B: roles for cell adhesion, tyrosine residue 66, and proline residues 309 and 310

The reversible phosphorylation of proteins on tyrosine residues is fundamental to a variety of intracellular signaling pathways and is controlled by the actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). While much progress has been made in understanding the regulation of PTKs, there is still relatively little known concerning the regulation of PTPs. Using immune complex phosphatase assays, we demonstrated that the enzymatic activity of the nonreceptor type PTP, PTP1B, is regulated by cell adhesion. Placing primary human foreskin fibroblasts (HFFs) in suspension leads to a distinct increase in PTP1B activity, whereas the readhesion of suspended HFFs onto fibronectin or collagen I inhibited activity. To gain insight into the mechanisms involved, we analyzed recombinant forms of PTP1B mutated at potential regulatory sites. Our results indicated that tyrosine residue 66 is essential for maintaining activity at 37 degrees C. We also found that the C-terminal region of PTP1B and localization to the endoplasmic reticulum are not required for the inhibition of activity by cell adhesion. However, analysis of PA-PTP1B, in which alanines are substituted for prolines 309 and 310, revealed an important role for these residues as the catalytic activity of this mutant did not decrease following readhesion onto collagen I. Since the binding of p130cas and Src to PTP1B is dependent upon these proline residues, we assayed the regulation of PTP1B in mouse embryo fibroblasts deficient in these proteins. We found that neither p130cas nor Src is required for the inhibition of PTP1B activity by adhesion to extracellular matrix proteins. Additionally, pretreatment with cytochalasin D did not prevent the reduction of PTP1B activity when cells adhered to collagen I, indicating that cell spreading is not required for this regulation. The control of the catalytic activity of PTP1B by cell adhesion demonstrated in this study is likely to have important implications for growth factor and insulin signaling.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.     

Computational analysis of the regulation of EGFR by protein tyrosine phosphatases

The tyrosine phosphorylated epidermal growth factor receptor (EGFR) initiates numerous cell signaling pathways. Although EGFR phosphorylation levels are ultimately determined by the balance of receptor kinase and protein tyrosine phosphatase (PTP) activities, the kinetics of EGFR dephosphorylation are not well understood. Previous models of EGFR signaling have generally neglected PTP activity or computed PTP activity by considering data that do not fully reveal the kinetics and compartmentalization of EGFR dephosphorylation. We developed a compartmentalized, mechanistic model to elucidate the kinetics of EGFR dephosphorylation and the coupling of this process to phosphorylation-dependent EGFR endocytosis. Model regression against data from HeLa cells for EGFR phosphorylation response to EGFR activation, PTP inhibition, and EGFR kinase inhibition led to the conclusion that EGFR dephosphorylation occurs at the plasma membrane and in the cell interior with a timescale that is smaller than that for ligand-mediated EGFR endocytosis. The model further predicted that sufficiently rapid dephosphorylation of EGFR at the plasma membrane could potentially impede EGFR endocytosis, consistent with recent experimental findings. Overall, our results suggest that PTPs regulate multiple receptor-level phenomena via their action at the plasma membrane and cell interior and point to new possibilities for targeting PTPs for modulation of EGFR dynamics.     

Protein tyrosine phosphatases in osteoclast differentiation, adhesion, and bone resorption

Osteoclasts are large cells derived from the monocyte-macrophage hematopoietic cell lineage. Their primary function is to degrade bone in various physiological contexts. Osteoclasts adhere to bone via podosomes, specialized adhesion structures whose structure and subcellular organization are affected by mechanical contact of the cell with bone matrix. Ample evidence indicates that reversible tyrosine phosphorylation of podosomal proteins plays a major role in determining the organization and dynamics of podosomes. Although roles of several tyrosine kinases are known in detail in this respect, little is known concerning the roles of protein tyrosine phosphatases (PTPs) in regulating osteoclast adhesion. Here we summarize available information concerning the known and hypothesized roles of the best-researched PTPs in osteoclasts - PTPRO, PTP epsilon, SHP-1, and PTP-PEST. Of these, PTPRO, PTP epsilon, and PTP-PEST appear to support osteoclast activity while SHP-1 inhibits it. Additional studies are required to provide full molecular details of the roles of these PTPs in regulating osteoclast adhesion, and to uncover additional PTPs that participate in this process.     

An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases

Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H(2)O(2) production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.     

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.     

Protein tyrosine phosphatases and the immune response

Reversible tyrosine phosphorylation of proteins is a key regulatory mechanism for numerous important aspects of eukaryotic physiology and is catalysed by kinases and phosphatases. Together, cells of the immune system express at least half of the 107 protein tyrosine phosphatase (PTP) genes in the human genome, most of which encode multidomain proteins that contain protein- and phospholipid-interaction domains. Here, we discuss the diverse but specific, and important, roles that PTPs have in immune cells, focusing mainly on T and B cells, and we highlight recent evidence that even subtle alterations in PTPs can cause immune dysfunction and human disease.     

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain–containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal–regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.     

Bicyclic benzofuran and indole-based salicylic acids as protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) constitute a large and structurally diverse family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. Malfunction of PTP activity has significant implications in many human diseases, and the PTP protein family provides an exciting array of validated diabetes/obesity (PTP1B), oncology (SHP2), autoimmunity (Lyp), and infectious disease (mPTPB) targets. However, despite the fact that PTPs have been garnering attention as novel therapeutic targets, they remain largely an untapped resource. The main challenges facing drug developers by the PTPs are inhibitor specificity and bioavailability. Work over the last ten years has demonstrated that it is feasible to develop potent and selective inhibitors for individual members of the PTP family by tethering together small ligands that can simultaneously occupy both the active site and unique nearby peripheral binding sites. Recent results with the bicyclic salicylic acid pharmacophores indicate that the new chemistry platform may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Structural analysis of PTP-inhibitor complexes reveals molecular determinants important for the development of more potent and selective PTP inhibitors, thus offering hope in the medicinal chemistry of a largely unexploited protein class with a wealth of attractive drug targets.     

Recent advances in PTP1B signaling in metabolism and cancer

Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanating from receptor tyrosine kinases. However, recent evidence demonstrates that misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.     

Identification of tyrosine phosphatases that dephosphorylate the insulin receptor. A brute force approach based on "substrate-trapping" mutants

Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substrate-trapping" PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate.