Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) - Volume 11, Issue 1, 2011

Signal transduction pathways play crucial role in number of cellular processes including proliferation, differentiation, adhesion, apoptosis, and metabolic homeostasis. Among the major regulatory mechanisms controlling signal transduction is reversible phosphorylation, which employs multiple kinases and phosphatases. Contrary to kinases, which are now among classic therapeutic targets of carcinogenesis regulation, the field of phosphatase research is still young and the roles of individual phosphatases are only recently recognized in detail. Here we focus on novel aspects of signalling, detection, and targeting of both protein serine/threonine (PPs) and tyrosine (PTPs) phosphatases. The research on PPs is somewhat more advanced since the first discoveries of PPs in mid-60th years [1], whereas identification of the first genuine PTP was reported only as late as in 1988 by Nicholas Tonks and his colleagues [2]. The structures and functions of many PPs and PTPs and their mechanisms of regulation have already been defined. Some are specific for a particular substrate, while others are capable to dephosphorylate vast array of cellular proteins [3]. But only recently, the PPs and PTPs are recognized as not less important components of the same pathways that involve tyrosine or serine/threonine kinases. Even though we are still plagued by the problems of specificity and selectivity, the field significantly advanced forward and the PPs and PTPs holds much promise as future targets of anti-cancer agents. Here we focus on various areas of phosphatase research, which already uncovered findings with potential to improve patient care in terms of improved cancer diagnosis, prognosis, and treatment. Firstly, we focus on microcystins, potent PP inhibitors of growing interest (Fig. 1A). These toxins are produced naturally by blue-green algae (cyanobacteria), a diverse group of photosynthetic bacteria inhabiting wide range of aquatic and terrestrial environments, including drinking water reservoirs, recreational lakes, or coastal waters. H. Fujiki & M. Suganuma focus on the state-of-art knowledge about microcystins and their influence on carcinogenesis in humans, starting with their seminal discovery of tumor promoter function of microcystins in 1992 [4], and discussing the advances in the field until recent days. Despite the acute microcystin intoxication has only rarely fatal consequences, the reverse is true for the chronic exposures, and thus removal of microcystins is particularly important in water treatment plants utilizing water from surface water bodies. Thus, microcystin degradation and removal is subject of complementing paper by D. D. Dionysiou and his colleagues. The techniques described in their study have a potential to completely remove the contaminating microcystins from water even in turbid conditions. These techniques include biodegradation, photochemical degradation including titanium dioxide photocatalysis, sulphate-radical-based oxidation, sonolysis, ozonisation, and chlorination. Current WHO guidelines and local laws in most industrialized countries take in account the most widespread microcystin (microcystin-LR), however the other > 80 microcystin species are left aside without any limits even though they have comparable toxicity to microcystin-LR. Focus on other microcystins, verification of currently applied maximal acceptable limits, and improvement of both detection and elimination of microcystins in water treatment plants is highly desirable. Closely related to the above mentioned topics is review written by P. Kalev & A. A. Sablina, who explore the family of heterotrimeric serine-threonine phosphatases PP2A. This phosphatase family is responsible for the majority of serine/threonine phosphatase activity in mammalian cells, and is among major targets of the above mentioned microcystins. In their paper, they describe current approaches to modulate PP2A signalling in cancer, which includes both activation of PP2A activity, which downregulates major cancer-associated signalling pathways, and inhibition of PP2A activity, which was shown to support the effects of currently used chemotherapeutics due to abrogation cell cycle checkpoints and promotion of premature entry into mitosis. Next we focus on advances in detection of PPs and PTPs. Improved detection methods are crucial to determine the spatiotemporal localization and activity of individual molecules of interest. In first of the two papers, A. Ishida & I. Kameshita explore the current advances in the field of in-gel phosphatase assays. These methods are of great potential in basic and preclinical research as they allow detecting and discriminating between activity of several PPs or PTPs within each sample by incorporation of various substrates to the conventional SDSPAGE gel. Employment of fluorogenic instead of radioactive substrates makes them now more user-friendly and facilitates their wider use. However, compared to in-tube methods, the in-gel assays are still limited due to the necessary ability of the enzyme to renaturate after SDSPAGE, and are efficient only when used to detect the changes caused by covalent modifications (such as phosphorylation or oxidation). In the second paper on PTP detection, S. J. Chung and his colleagues focus on recent advances in development of chemical probes for PTPs, with focus on general and selective fluorescent activity probes. Some of these probes have high potential to be used in either screens for PTP inhibitors or activators, and as diagnostic and prognostic tools. Examples of their current use in basic and preclinical science are discussed. In the next review, K. V. S. Rao and his colleagues deal with interesting emerging phenomenon, regulatory cascades of PPs and PTPs. Broadly defined, these cascades comprise of a series of successive dephosphorylation reactions occurring within a cell and are catalyzed by sequentially activated phosphatases. Despite they closely resemble the very well-known cascades, such as MAPK/Erk pathway, their existence was recognized only recently. As there is frequent crosstalk between the already known phosphatase and kinase cascades, their targeting should definitely be among the major interests of future pharmaceutical research. Several papers in this issue focus in mechanisms of action of emerging cancer-associated PTPs, and evaluate their potential to serve as potential targets for anti-cancer therapy. Our knowledge about the role of PTPs in cancer shifted significantly forward during recent years [5]. Apart of classical target of PTP pharmacological research, PTP1B [6, 7], numerous inhibitors of various PTPs were recently described [8], allowing specific and selective modulation of PTP signaling in basic and preclinical research. However, since the termination of phase II clinical trial of ertiprotafib (Wyeth Research) due to unsatisfactory efficacy, dose-limiting site effects and inconsistency of data from in vivo treatments with the expected outcomes, the market is still waiting for its first clinically approved specific PTP inhibitor, despite several currently used drugs show some degree of PTP inhibitory activity. Among the recently emerging cancer-associated PTPs are PTPN13 / PTPBAS (Fig. 1B; reviewed here by G. Freiss & D. Chalbos), PTPN6 / SHP-1 (reviewed here by B. Colas and her colleagues), emerging marker of metastasis PRL-3 / PTP4A3 (Fig. 1C; reviewed here by K. Guzinska-Ustymowicz), and various MAPK phosphatases (Fig. 1D; reviewed here by R. Pulido and his colleagues). Some PTPs and their fragments may serve as important cancer diagnostic tools. In this regard, S. E. L. Craig and S. M. Brady-Kalnay elucidate here the very interesting phenomenon of shedding of extracellular domains of some receptor PTPs. These shed extracellular domains have a great potential to be used in non-invasive molecular imaging strategies to detect the main tumor. They may be used to mark the tumor margins, and possibly for molecular targeting of tumor cells to deliver therapeutics. Extracellular domain cleavage observed for receptor PTPs resembles those of Notch, where three proteases sequentially digest Notch to release biologically active protein fragments. S. M. Brady-Kalnay pioneered this area of research [9, 10] and offers here exciting state-of-art view of this important emerging subject of cancer research..........

Microcystin-LR and nodularin, along with okadaic acid, are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The mechanisms of action of microcystin-LR and nodularin in the liver and that of okadaic acid, a potent tumor promoter on mouse skin, have attracted the attention of the scientists. This paper reviews several topics: new inhibitors of PP1 and PP2A with new chemical structures, structure-function relationships for both receptor binding and inhibition of protein phosphatases, the crystal structure of PP1 or PP2A - toxin complex, induction of gene expression and apoptosis. These subjects were studied by using in vitro and in vivo experimental systems. Two-stage carcinogenesis experiments with microcystin-LR and nodularin for the first time demonstrated that microcystin- LR is a new tumor promoter in rat liver initiated with diethylnitrosamine (DEN), and that nodularin is a potent tumor promoter associated with weak initiating activity in rat liver initiated with DEN. A working group of WHO (IARC) concluded that microcystin-LR is “possibly carcinogenic to humans” and that nodularin is “not classifiable as to carcinogenicity”. Our studies revealed that chemical tumor promoters are inducers of TNF-α in the cells of target tissues and that TNF-α is an endogenous tumor promoter. This advance in carcinogenesis made it possible to look for the link between chemical tumor promoters and endogenous tumor promoters, such as TNF-α and IL-1. The carcinogenic features of TNF-α are described in this review, and the TNF-α inducing protein (Tipα) of elicobacter pylori genome is presented as an example of a tumor promoter of human stomach cancer development.

Microcystins are cyclic heptapeptide toxins produced by a number of genera of cyanobacteria. They are ubiquitous in bodies of water worldwide and pose significant hazard to human, plant, and animal health. Microcystins are primarily hepatotoxins known to inhibit serine-threonine phosphatases leading to the disruption of cascade of events important in the regulation and control of cellular processes. Covalent binding of microcystins with phosphatases is thought to be responsible for the cytotoxic and genotoxic effects of microcystins. In addition, microcystins can trigger oxidative stress in cells resulting in necrosis or apoptosis. Their cyclic structure and novel amino acids enhance their stability and persistence in the environment. Humans are primarily exposed to microcystins via drinking water consumption and accidental ingestion of recreational water. Recreational exposure by skin contact or inhalation to microcystins is now recognized to cause a wide range of acute illnesses which can be life-threatening. Microcystins are primarily degraded by microorganisms in the environment, while sunlight can cause the isomerization of the double bonds and hydroxylation in the presence of pigments. Attempts to utilize these organisms in sand and membrane filters to treat water contaminated with microcystins showed complete removal and detoxification. Conventional water treatment processes may not fully eliminate microcystins when there are high levels of organic compounds especially during harmful bloom events. Combination of conventional and advanced oxidation technologies can potentially remove 100% of microcystins in water even in turbid conditions. This review covers selected treatment technologies to degrade microcystins in water.

The kinase oncogenes are well-characterized drivers of cancer development, and several targeted therapies focused on both specific and selectively nonselective kinase inhibitors have now been approved for clinical use. In contrast, much less is known about the role of protein phosphatases, although modulation of their activities might form the foundation for an effective anti-cancer approach. The serine-threonine protein phosphatase 2A (PP2A) is implicated in the regulation of numerous signaling pathways and may function as a tumor suppressor. Recently pharmacological modulation of PP2A activity has been showed to have a potent anti-tumor activity in both in vitro and in vivo cancer models. These studies implicate PP2A as a promising therapeutic target for the treatment of cancer.

Intracellular signaling is governed by protein phosphorylation and dephosphorylation catalyzed by protein kinases and protein phosphatases, respectively. Since there is growing evidence that a variety of protein phosphatases are involved in the pathogenesis of various diseases, protein phosphatases have recently been the focus of intense research interest, not only in basic biology but also in clinical medicine. In the process of these studies, analytical methods for protein phosphatases will be of increasing importance. A major bottleneck in protein phosphatase assays is the selection and preparation of an efficient substrate for the phosphatase to be assayed. To circumvent this difficulty, a variety of protein phosphatase substrates have been devised during the development of novel assay techniques by which protein phosphatase activities can be readily detected. In this review, we focus on the methodology for detecting protein phosphatase activities, with special emphasis on in-gel protein phosphatase assays and related techniques. The utility and limitations of these methods are also discussed.

Protein tyrosine phosphatases (PTPs) are key regulatory enzymes in signal transduction pathways and their aberrancy has been implicated in various diseases such as cancers, metabolic syndromes, and autoimmune disorders. In spite of its great importance, determination of the functional significance of PTPs remains a major challenge, and efficient methodologies are needed to specifically delineate PTP functions. Besides the strategy to use potent and selective PTP inhibitors to study the physiological roles of the enzymes, measurement of PTP activities using specific PTP substrates or activity-based probes (ABPs) has been reported. This review focused on the recent development of small molecular probes to detect PTP activities, consisting of five sub-categories: 1. Conventional fluorescent substrates; 2. Ratiometric fluorescent PTP substrates; 3. Fluorescence substrates with selectivity to a single PTP or a class of PTPs; 4. ABPs specific for PTPs; and 5. A real-time imaging of PTP-substrate complex using a small molecule PTP probe which, offers a measurement of a real-time activity of a certain PTP in cells.

Coordinated coupling of biochemical reactions involving protein phosphorylation and dephosphorylation represents the hallmark of the intracellular signal transduction machinery. Distinct classes of enzymes known as kinases and phosphatases respectively drive these reactions. Alterations in activity of such signaling intermediates, either due to mutations in the corresponding genes or epigenetic modulation of their expression levels, is often the cause of many cancers. The role of kinases during signal transduction has been extensively investigated over the past several decades and the consensus view is that subsets of kinases form distinct cascades of signaling pathways. Further, the extensive crosstalk that exists between these cascades leads to a complex network configuration for the signaling machinery. Inhibitors of many of these kinases are now being exploited in cancer therapy. In contrast to this, regulation by cellular phosphatases has generally been considered to occur through isolated interactions between a given phosphatase and its target substrate. Emerging evidence, however, is beginning to suggest that phosphatases also inter-regulate each other and that such interactions can lead to the formation of discrete phosphatase-specific cascades. A phosphatase cascade may be defined broadly as a series of successive dephosphorylation reactions that occur within a cell and are catalyzed by phosphatases which are activated sequentially. In general, the term ‘phosphatase cascade’ refers to cascades that include two or more phosphatase members [1-4]. The crosstalk between such regulatory axes of phosphatase and kinase cascades provides for complex modes of regulation, with non-linear signal input/output relationships. This review discusses the implications of such phosphatase-constituted regulatory elements for both signal processing and transmission. Further, we also explore the potential that insights on the functioning of phosphatase cascades offers, for the development of new and selective strategies for cancer therapy.

Protein tyrosine phosphorylation plays a major role in many cellular functions implicated in cancer development and progression, but only a few of the known protein tyrosine phosphatases have yet been clearly classified as oncogenes or tumor suppressors. PTPL1 interacts with tumor-associated proteins, suggesting a link between PTPL1, the PTPN13 gene product, and tumorigenesis or cancer progression. However, the impact of PTPL1 on cancer is divided between its capacity to counteract the activity of oncogenic tyrosine kinases and its inhibitory interaction with the death receptor, Fas. In this manuscript, we review the PTPL1-interacting proteins implicated in cancer. In addition, we examine the phenotypic arguments concerning both the PTPL1/Fas interaction and the ability of PTPL1 to inhibit signaling from growth factor receptors or oncogenes with tyrosine kinase activity. Finally, we compare the alterations in expression and the genetic and epigenetic arguments supporting an oncogenic or an anti-oncogenic impact of PTPL1.

The reversible phosphorylation of tyrosine residues in proteins, which is governed by the balanced action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is a key element of the signaling pathways that are involved in the control of cell proliferation. Deregulation of either of these key regulators leads to abnormal cell signaling, which is largely associated with human pathologies including cancer. This review focuses on recent studies on the role of the protein tyrosine phosphatase SHP-1 on cell-cycle regulation and its possible roles in tumour onset and progression. SHP-1 is a PTP with two SH2 domains that is expressed in haematopoietic cells and, moderately, in many other cell types, especially malignant epithelial cells. SHP-1 regulates cell proliferation, whether it is by controlling mitogenic pathways activated by receptors with tyrosine kinase activity, or by regulating components of the cell-cycle machinery such as CDK2, p27 and cyclin D1. Since several inhibitors targeting SHP-1 have demonstrated their value in cancer treatment, this phosphatase has been proposed as a therapeutic target for this pathology.

PRL-3 protein belongs to the family of protein tyrosine phosphatases with unique COOH-terminal prenylation motif, which determines the functions of this protein and its location in the cell. Numerous research studies revealed that apart from performing the poorly investigated physiological role, PRL-3 takes part in the process of carcinogenesis. Specifically, it is involved in reconstructing of the cytoskeleton, regulating adhesion and cell cycle of the cancer cells, and in epithelial-mesenchymal transition. Through these mechanisms PRL-3 protein participates in invasion, migration, metastasis and angiogenesis. Numerous studies indicate that PRL-3 expression is particularly important in colorectal, as well as in gastric, ovarian and breast carcinomas. Recently, several studies on PRL-3 protein in other types of cancer have been published. They reveal a significant role of this protein in the process of angiogenesis and metastasis. It has been proven that a higher expression of PRL-3 correlates with tumor progression and its severity. While the degree of overexpression of PRL-3 varies in different types of tumors, most research shows that in the metastases of these tumors, whether to the lymph nodes or to other organs, the level of expression is extremely high. Overexpression of PRL-3 protein was repeatedly confirmed in metastases, but not with primary tumors. PRL-3 seems to be an adequate marker in diagnosing the stage of tumor advancement for various types of carcinomas, especially for colorectal carcinoma investigated thoroughly in this study. PRL-3 overexpression predicts poor prognosis in patients with various carcinomas and is a promising target in the cancer treatment.

The protein tyrosine phosphatase family (PTP) contains a group of dual-specificity phosphatases (DUSPs) that regulate the activivity of MAP kinases (MAPKs), which are key effectors in the control of cell growth and survival in physiological and pathological processes, including cancer. These phosphatases, named as MKP-DUSPs, include the MAPK phosphatases (MKPs) as well as a group of small-size atypical DUSPs structurally and functionally related to the MKPs. MKP-DUSPs, in most of the cases, are direct inactivators of MAPKs by dephosphorylation of both the Thr and the Tyr regulatory residues at the MAPKs catalytic loop. In some other cases, MKPDUSPs regulate the activity of MAPKs indirectly, acting through upstream MAPK pathways components. The active involvement of MKP-DUSPs in oncogenesis or resistance to cancer therapies is now well documented, making the search and validation of MKP-DUSPs inhibitors a prominent area in clinical cancer research. Here, we review the current knowledge on the role of MKP-DUSPs in human cancer, the status of the preclinical development and validation of specific MKP-DUSP inhibitors, and the potential of MKP-DUSPs as targets for anti-cancer drugs.

Receptor protein tyrosine phosphatases (RPTPs) are involved in many cellular processes, including the regulation of adhesion, migration and cellular signaling. Many RPTPs are putative tumor suppressors because of the transcriptional and translational changes observed in their expression during tumorigenesis. Recently, RPTPs were shown to be post-translationally regulated during tumorigenesis by proteolysis in a manner similar to proteolysis of the Notch receptor. There is accumulating evidence that proteolysis of RPTPs influence their cellular function and that RPTP fragments may function as oncogenes. By exploiting what is known about RPTP ligand binding domains and crystal structures of ligand-RPTP interfaces, we describe novel molecular diagnostics that have been or can be developed to identify tumor margins and target tumor tissues.

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.

Phosphatases are well known drug targets for diseases such as diabetes, obesity and other autoimmune diseases. Their role in cancer is due to unusual expression patterns in different types of cancer. However, there is strong evidence for selective targeting of phosphatases in cancer therapy. Several experimental and in silico techniques have been attempted for design of phosphatase inhibitors, with focus on diseases such as diabetes, inflammation and obesity. Their utility for cancer therapy is limited and needs to be explored vastly. Quantitative Structure Activity relationship (QSAR) is well established in silico ligand based drug design technique, used by medicinal chemists for prediction of ligand binding affinity and lead design. These techniques have shown promise for subsequent optimization of already existing lead compounds, with an aim of increased potency and pharmacological properties for a particular drug target. Furthermore, their utility in virtual screening and scaffold hopping is highlighted in recent years. This review focuses on the recent molecular field analysis (MFA) and QSAR techniques, directed for design and development of phosphatase inhibitors and their potential use in cancer therapy. In addition, this review also addresses issues concerning the binding orientation and binding conformation of ligands for alignment sensitive QSAR approaches.

Protein tyrosine phosphatases (PTPs) are a large family of signaling enzymes playing critical role in signal transduction and regulation of cellular processes. Dysfunction of PTP activity is associated with diabetes, cancer, autoimmune disorders, and neural diseases. PTP inhibitors therefore emerged as promising therapeutic targets. Recent research indicates that besides small organic molecules, metal ions and metal complexes can also strongly inhibit PTPs both in vitro and in vivo, resulting in the increase of phosphorylation of corresponding substrates and the modulation of cellular process. Structure of metal complexes influences the potency and selectivity of PTP inhibition. Detailed studies on this subject are not only expected to yield metal-based drugs targeting individual PTPs, but also to support understanding the function of metals in organisms. This review focuses on recent advancements in this area of research.