Review General

Targeting the PTPome in human disease Lutz Tautz, Maurizio Pellecchia & Tomas Mustelin†

1. Introduction to the human PTPome 2. PTPs and human disease 3. PTPs as drug targets 4. Expert opinion

†Infectious

and Inflammatory Disease Center and Cancer Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA

Protein tyrosine phosphatases (PTPs) play vital roles in numerous cellular processes and are implicated in a growing number of human diseases, ranging from cancer to cardiovascular, immunological, infectious, neurological and metabolic diseases. There are at least 107 genes in the human genome, collectively referred to as the human ‘PTPome’. Here the authors review the involvement of PTPs in human disease, discuss their potential as drug targets, and current efforts to develop PTP inhibitors for the treatment of human disease. Finally, the authors present their view of the future for PTPs as drug targets. Keywords: Alzheimer’s disease, autoimmune disease, cancer, Charcot-Marie-Tooth syndrome, diabetes, epilepsy, genetic disease, myelodysplastic syndrome, Noonan syndrome, protein tyrosine phosphatase (PTP), phosphatase inhibitor Expert Opin. Ther. Targets (2006) 10(1):157-177

1. Introduction

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to the human PTPome

It has been estimated that at least one third of all cellular proteins contain covalently-bound phosphate. Among the many phospho-acceptor amino acids, phosphorylation on serine is the most prevalent, constituting some 95% of all protein-bound phosphate, followed by phosphate on threonine in ∼ 5% of phosphoproteins. By comparison, phosphorylation of proteins on tyrosine [1] is rare, only 0.01 – 0.1% of phosphoproteins, but nevertheless, plays an extremely important role in many processes that are characteristic of higher eukaryotes, such as cell-to-cell communication and functions that coordinate the behaviour of cell populations within these multicellular organisms [2-4]. However, tyrosine phosphorylation has recently also been found in bacteria and Archea [5-7], the sequenced genomes of which usually contain several genes for protein tyrosine phosphatases (PTPs). Thus, tyrosine phosphorylation may have flourished after the emergence of multicellular organisms, but it already existed from much earlier times. Tyrosine phosphorylation is a rapidly reversible post-translational modification catalysed by protein tyrosine kinases (PTKs) and reversed by PTPs. Thus, the state of phosphorylation of a protein, at a given moment in time, is the net result of the opposing activities of the relevant PTK(s) and PTP(s). A change in phosphorylation state can be the result of a change in the activity (or access) of either enzyme. With very few exceptions (e.g., c-Src Y527), the balance is skewed very far towards the dephosphorylated state: most tyrosine phosphorylated proteins are not phosphorylated at all under ‘resting’ conditions and become phosphorylated to a stoichiometry of only a few percent even under the strongest inducing conditions. Thus, one could argue that PTPs are more important than kinases in setting the levels of tyrosine phosphorylation and that they should be much better drug targets. Indeed, PTPs often play very specific, non-redundant, highly regulated and very active roles in many cellular processes [8-16]. PTPs often participate in cellular processes as ‘positive’ components [17-19] and many PTP knockout mice have unique and complex phenotypes [20-28]. Finally, the completion of the human genome has demonstrated that there are more PTP genes than PTK genes [3,29]; 107 versus 90. The authors 10.1517/14728222.10.1.157 © 2006 Ashley Publications ISSN 1472-8222

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Figure 1. Common catalytic mechanism of class I – III cysteine-based protein tyrosine phosphatases.

refer to the human genome complement of PTPs as the ‘human PTPome’. In the authors’ definition, the human PTPome includes all genes that encode proteins with structural homology to the catalytic domains of the known enzymes with PTP activity [3]. Importantly, this classification is structural and not based on biochemical specificity. The human PTPome consists of four evolutionary distinct families: the class I, II and III Cys-based PTPs, and the Asp-based phosphatases, exemplified by the Eya (eyes absent) tyrosine phosphatases [13]. These Asp-based PTPs are part of the haloacid dehalogenase family, which is now emerging as a very large protein family with representatives in plants, prokaryotes and mammals and includes numerous enzymes with other than Tyr-specificity. This family is not discussed further in this review. All Cys-based PTPs share a common catalytic mechanism based on a cysteine with a low pKa that forms a thiophosphate intermediate during catalysis (Figure 1). The class I Cys-based PTPs are structurally related to the first PTP, PTP1B, whose amino acid sequence was determined [30]. There are at least 99 members of this family in the human genome [3] and they can be further subclassified into the classical PTPs (receptor-like and nonreceptor), and the VH1-like phosphatase group, which contains the mitogen-activated protein kinase (MAPK) phosphatases (MKPs), the atypical dual-specificity phosphatases (aDSPs), the slingshots, the PRLs, the CDC14s, the PTEN group, and the myotubularins. The two latter dephosphorylate inositol phospholipids [31,32]. Within all these homologous phosphatases, it appears that the aDSPs represent the evolutionary most ancient members of the family. Genes with a high degree of similarity can 158

be found across all kingdoms of life, including Archea and plants [33]. In contrast, the classical PTPs, particularly the receptor-like group, seem to be more recent groups that have flourished and diversified in multicellular organisms. The class II Cys-based PTPs comprise a small group of cell-cycle regulators known as the CDC25 phosphatases. Although the core of their catalytic machinery is very similar to that of the class I enzymes, they are structurally unrelated and instead bear considerable resemblance to bacterial rhodanese enzymes [34], from which are thought to have evolved relatively late in eukaryote evolution. Interestingly, the MAPK phosphatases, which belong to the class I family, have incorporated a catalytically inactive rhodanese-like domain, which functions as a MAPK docking module [35]. This region of homology between the CDC25s and the noncatalytic portion of the MAPK phosphatases should not be confused with the lack of homology between their catalytic domains. The class III Cys-based PTPs are widely distributed in all kingdoms of life and most bacteria have the genes for one or two such enzymes in their genomes. In Eserichia coli, one such PTP dephosphorylates a transmembrane tyrosine autokinase, which, in turn, controls the synthesis of polysaccharides of the bacterial capsule [36]. In the Gram-negative Bacillus subtilis, the two class III phosphatases YfkJ and YwlE have clearly distinct properties and bacterial knockout strains have distinct phenotypes [37]. The human genome contains a single gene for a class III PTP, the low molecular weight PTP (LMPTP), which undergoes alternative splicing to yield two active and one inactive isoforms. Although a polymorphism in this gene correlates with numerous common human diseases [4], the function of LMPTP has remained obscure.

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Why are there so many PTPs in the human genome? A likely answer is that PTPs have a high degree of specificity, important and non-redundant functions, and, consequently, relatively few substrates each. However, these premises remain largely untested and there is some evidence also for the opposite: although many of the > 30 reported PTP knockout mice support the notion of important and unique functions (e.g., the embryonically lethal deletions of PTP-PEST [20] and SHP2 [21]), some of these mice have much milder phenotypes than expected, for example, MKP1 [38] or PEP [39]. Thus, it is best to assume that closely related PTPs have at least partly overlapping sets of substrates. This has very important consequences for the consideration of PTPs as drug targets: strictly monospecific PTP inhibitors may have a much smaller impact than inhibitors of a small group of closely related PTPs. Another possible explanation for the abundance of PTP genes in the human genome would be that many genes have a restricted expression profile. This does not appear to be the case: most PTPs are quite broadly expressed (albeit at varying levels) and individual cell types express a large portion of the PTPome. The authors have found that white blood cells express between 65 and 75 different PTPs with each cell lineage having its own profile of PTPs at certain relative expression levels. Interestingly, this profile undergoes both qualitative and quantitative changes in response to external stimuli, cell activation, differentiation and so on. Remarkably, each response is unique, and identical stimuli elicit different responses in different cells types. Finally, there is individual variations in PTP expression profile between healthy blood donors, suggesting that polymorphisms and genetic heterogeneity affect PTP expression patterns. All these levels of complexity will need to be considered when contemplating the question of redundancy between PTPs and the use of PTPs as drug targets. Clearly, a more systematic analysis of tissue expression profiles, relative expression levels and differential regulation of PTP expression during embryogenesis and development will be needed. 2. PTPs

and human disease

In support of the notion that PTPs have important and nonredundant functions in cell physiology, nearly half of all PTP genes in the human genome have already been implicated in human disease [3,4,40]. Disease-related perturbations range from loss of expression to point-mutations and single amino acid substitutions. There are also examples of amplification, overexpression and ectopic expression of PTPs in human disease (e.g., in cancer). Perhaps the most striking finding is that very subtle alterations in PTP function can underlie lifelong suffering and even lethal ailments. It also seems that genetic polymorphisms in PTPs play a significant role in disease predisposition and in the genetic heterogeneity that underlies individual variation in immunity and disease susceptibility, severity and recovery.

Given the importance of tyrosine phosphorylation and the very limited studies performed so far, the authors predict that a very large number of human health concerns will be found to involve a central role for PTPs. They also predict that the pharmaceutical industry will become increasingly interested in PTPs as drug targets. 2.1 PTPs

in cancer As might be expected from the known role of tyrosine phosphorylation in cell growth and the existence of PTK oncogenes, loss of PTPs has been reported in numerous experimental and clinical cancers. At least 30 different PTPs have been reported to be affected and loss can occur by genetic (e.g., chromosomal abnormalities, frameshift mutations or point-mutations) or epigenetic (e.g., promoter methylation or changes in transcription) mechanisms. One of the earliest cases of PTP dysregulation in cancer was provided by Zanke and co-workers, who cloned the haematopoietic tyrosine phosphatase HePTP [41] and found that its gene was located on chromosome 1q32.1, a site of frequent abnormalities in the preleukemic myelodysplastic syndrome, as well as in haematopoietic malignancies [42]. Indeed, a patient with acute myeloblastic leukaemia and grossly overexpressed HePTP was found by these authors [42]. Similarly, the SH2-containing PTP SHP1, which acts as a negative regulator of many survival and growth signalling pathways in haematopoietic cells [10], is frequently lost in myelodysplastic syndrome [43] and lymphomas [44]. Reduced expression of SHP1 appears to be an early step in carcinogenesis and is indicative of more aggressive disease with rapid progression [43]. The most commonly lost PTP in human cancer is PTEN, a class I cysteine-based phosphatase specific for phosphate at the D3-position of inositol phospholipids rather than phosphotyrosine. Loss of PTEN is seen in over half of all glioblastomata and in a high portion of breast and prostate cancer, in lymphomas and many other common malignancies [45-54]. Because of its substrate-specificity, PTEN directly counteracts the many growth, survival and motility promoting effects of phosphatidylinositol-3-kinase, which involve the Ser/Thr-protein kinase Akt and other pleckstrin homology domain-containing proteins, and signalling pathways downstream of them. Several transmembrane PTPs have also been found to be lost in malignant cells, for example DEP1 (PTPRJ) in colon cancer [55], and GLEPP1 (PTPRO) in hepatocellular carcinomas [56] and colon cancer [57]. A more comprehensive analysis of the PTPome was reported by Wang and co-workers [58], who found that RPTPρ (PTPRT), RPTPγ (PTPRG), LAR (PTPRF), PTPH1 (PTPN3), PTP-BAS (PTPN13), and PTPD2 (PTPN14) are frequently mutated in colon cancer. They tested several mutants and found many to exhibit reduced catalytic activity. The finding that PTPs sometimes are overexpressed, rather than reduced, in cancer cells illustrates the growing notion that PTPs not only act as tumour suppressors, but also as ‘positive’ components of signalling pathways and cell functions. It

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appears that many cellular processes, such as cell motility, require a dynamic balance between kinases and phosphatases. This may explain the high level overexpression of the small farnesylated phosphatase PRL3 in metastatic colon cancer [59], but not in the main tumour. Overexpression of MKP1 in prostate cancer [60], again may be to prevent excessive activation of MAPKs, which can result in cell-cycle arrest and cell senescence. Finally, the increased levels of PTPα in breast cancer [61] is most likely related to the activation of Src family PTKs by dephosphorylation of their negative regulatory site by PTPα. Finally, there are many PTPs that regulate cell-cycle progression, such as the Cdc25A, Cdc25B, Cdc25C, Cdc14A and Cdc14B phosphatases, which dephosphorylate and activate or inactivate cyclin-dependent kinases at key transition points in the cell cycle. These phosphatases have long been considered as drug targets for cancer therapy. 2.2 PTPs

in monogenic inherited syndromes Several inherited genetic diseases have been mapped to genes encoding PTPs [3]. The identified defects include frameshift mutations causing premature termination of translation and loss of the protein product, or single base mutations that result in amino acid substitutions. Examples of the former mechanism include Lafora’s epilepsy, which is caused by loss of a small PTP with a glycogen-binding domain [62], termed laforin [63], and an X-linked muscle dystrophy caused by loss of a phosphoinositide-specific PTP called myotubularin [64]. Another PTP with similar substrate-specificity, myotubularin-related protein 2 (MTMR2), was found to be mutated in the inherited nerve myelination disease Charcot-Marie-Tooth syndrome type 4B [65]. Surprisingly, a subset of patients were found to have an intact MTMR2 gene and instead to have mutations in the catalytically inactive phosphatase MTMR13 [66]. The disease is the same in both cases, raising the question of how the loss of an inactive phosphatase can lead to the same pathology as the loss of an active phosphatase. The answer to this puzzle was provided by the discovery that the two proteins form a heterodimer, in which the catalytically inactive MTMR13 provides critical aid to the function of the active MTMR2. Germline mutations in PTEN are also present in three different monogenic diseases, Cowden disease, Bannayan-Zonana syndrome and Lhermitte-Duclos disease [67-69], which are characterised by a propensity to develop benign hamartomas and a greatly increased risk of malignant tumours. Another monogenic disease with increased risk of leukaemia is Noonan syndrome, which is caused by gain-of-function mutations in the SH2 domain-containing phosphatase SHP2 [70]. This disease is characterised by heart abnormalities, facial dysmorphisms, short stature, cryptorchism, mental retardation and other developmental problems. 2.3 PTP

drug targets in metabolic diseases A major impetus for considering PTPs as drug targets was the phenotype of the PTP1B knockout mouse [71], which 160

included increased responses to insulin and resistance to fat diet-induced obesity. This phenotype leads to the assumption that a small-molecule inhibitor of PTP1B would increase insulin signalling and improve glucose uptake and fatty acid metabolism; both highly desirable effects in patients with Type 2 diabetes. The biology behind PTP1B function is also very interesting; it turns out that PTP1B is mostly located on internal membranes of the endoplasmic reticulum and endosomes and primarily meet the phosphorylated active insulin receptor after it has been internalised. Thus, PTP1B plays a major role only in regulation of the later phases of insulin signalling and in the duration of signalling. Nevertheless, a polymorphism in the gene for PTP1B (PTPN1) correlates with insulin resistance [72]. It also turns out that PTP1B regulates other receptor PTKs in a similar manner and that at least some of the functions of PTP1B are shared with the closely related TCPTP. In addition to PTP1B, it appears that multiple other PTPs participate in the dephosphorylation of the insulin receptor before and early after insulin binding [73]. Another PTP with a major impact on overall metabolism is the SH2 domain-containing phosphatase SHP2, the brain-specific deletion of which leads to a striking weight gain due to decreased catabolism and body temperature [74]. This role of SHP2 in central control of metabolism is probably related to its function in leptin and STAT3 signalling. 2.4 PTPs

in cardiovascular and neurological diseases Endothelial cells express many transmembrane PTPs, which participate in the regulation of intercellular contacts of vascular wall cells [12], and the cadherin-catenin-desmoplakin complexes of tight junctions [75], such as PTPκ (PTPRK), PTPµ (PTPRM), GLEPP1 (PTPRO) and DEP1 (PTPRJ). At least the first two of these are regulated through homophilic interactions during cell–cell contact [76-78] and regulate intercellular signalling in endothelial cells that line blood vessels. Knockout mice have verified an important role for these PTPs in vascular biology: PTPµ-/- mice had defective dilation responses in their mesenteric arteries [25], whereas GLEPP1-/- mice had hypertension due to abnormal podocyte development in their kidneys [25]. It also appears that these and other PTPs that regulate cell–cell junctions between endothelial cells play important roles in the interaction with immune cells, particularly in the process of transmigration of leukocytes from the blood into the surrounding tissues. LMPTP [4] and MKP-1 [79] have been implicated in cardiac hypertrophy, which involves signalling from growth factor receptors and MAPKs. The allelic polymorphism in LMPTP (ACP1) also correlates with the development of Alzheimer’s disease [4], perhaps through an (indirect?) influence on tau phosphorylation. Finally, a case report of a child with severe autism detected a chromosomal abnormality that included loss of the gene for PTP-MEG2 (PTPN9) in addition to one other gene [80]. The aithors’ recent observations concerning the role of PTP-MEG2 in secretory vesicle biology [81] and embryonic development (unpublished data),

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suggest that a causative role of PTP-MEG2 in autism is fully plausible. Considering the critical role of many receptor PTPs in axonal guidance, synaptic morphology and flexibility, glutamate receptor regulation, MAPK control, learning, memory, motor control and many other functions of the CNS, it seems very likely that many of these PTPs are instrumental in pathophysiological processes and may become drug targets in the near future. 2.5 PTPs

in immunodeficiency and autoimmunity An important discovery for our appreciation of PTPs in the immune system was the finding that the motheaten mouse mutant was deficient in the SH2 domain-containing tyrosine phosphatase SHP1 [82]. These mice have activated macrophages and their lymphoid cells show exaggerated responses to antigen stimulation. As a consequence, the mice develop inflammatory pathology of multiple tissues [83]. A few genetic polymorphisms have also been described in the human SHP1 gene (PTPN6) [84], but no associations have yet been reported with human autoimmunity. Instead, deletion of SHP1 has been observed in haematological malignancies, as discussed in Section 2.1. Instead, another PTP, the lymphoid tyrosine phosphatase LYP, has recently been found to cause numerous major human autoimmune diseases. The authors’ laboratory discovered that the gene encoding LYP, termed PTPN22, has a single-nucleotide polymorphism C1858T, which leads to a substitution of arginine for tryptophan at position 622 in the C-terminus of LYP [85]. This change results in loss of binding of LYP to the SH3 domain of the inhibitory Csk kinase and correlates with the development of Type 1 diabetes [85], an autoimmune disease characterised by destruction of the insulin-producing β-cells in the pancreas. This association has now been confirmed by numerous studies [86-90], and extended to rheumatoid arthritis [91-93], juvenile arthritis [94], systemic lupus erythematosus [93,95], Graves’ disease [96,97] and other autoimmune diseases [98]. Interestingly, multiple sclerosis, celiac disease and Sjögren’s syndrome do not seem to associate with the polymorphism in PTPN22 [88,99,100]. The exact molecular mechanisms by which LYP contributes to autoimmune disease are not yet known, but presumably include its negative regulation of the PTKs that mediate lymphocyte activation [10] and the elimination of autoreactive T cells in the thymus. Curiously, a key antigen for autoreactive T cells in the β-islets is also a PTP, namely a receptor-like enzyme termed phogrin (PTPRN) located on the insulin-containing secretory vesicles in β-cells [101]. Abnormalities in another leukocyte-restricted enzyme, the transmembrane PTP CD45, have also been reported in patients with systemic lupus erythematosus [102], and a polymorphism that impairs alternative splicing of CD45 was reported to be associated with multiple sclerosis [103]. The latter association was not replicated in other populations [104], as might be expected in polygenic diseases with linkage disequilibrium and complex interactions with other genetic and environmental factors [105]. Autoimmune disease also develops in

mice transgenic for a gain-of-function E613R mutant CD45 [106], whereas loss of CD45 was found in a few patients with severe combined immune deficiency [107,108]. Together, these findings are in agreement with the important role that CD45 plays in the regulation of Src family PTKs [18] and lymphocyte activation [10]. Accordingly, CD45 has been considered a drug target for treatment of autoimmune diseases and transplant rejection. Allelic polymorphism in a third PTP, the class III Cys-based LMPTP, ACP1, was found to correlate with increased IgE levels and atopy [4,109]. In this case, the polymorphism affects the alternative splicing of the biochemically distinct isoforms A and B, as well as the total levels of LMPTP expression. As in the case of LYP and CD45, significant human disease seems to arise from relatively small alterations in PTP function, attesting to the delicate balances that govern the accuracy of our immune system. 2.6 PTPs

as virulence factors for lethal pathogens The strong influence that many PTPs have on the immune response [10] has been exploited by several pathogenic bacteria and viruses. Perhaps the best understood example is the highly virulent bacterium that causes bubonic plague, Yersinia pestis, which injects a highly active PTP called YopH directly into the cytoplasm of immune cells, including macrophages, T and B cells, during the early stages of infection [110,111]. Once in the cells, YopH efficiently dephosphorylates key signalling proteins and thereby inhibits activation of these cells and therefore initiation of an immune response, allowing the bacterium the opportunity to multiply unopposed and to cause a rapidly lethal disease. In T cells, YopH efficiently dephosphorylates Lck at Tyr-394 in the activation loop, resulting in a complete paralysis of T cell receptor signalling [112]. Virulent species of Salmonella use the same strategy and inject host cells with the phosphatase SptP, which interferes with activation of MAPKs [113]. Mycobacteria also secrete a tyrosine-specific PTP involved in virulence [114]. All these PTPs are obvious drug targets for pathogen-specific treatments, as well as tools for mechanistic studies of PTP targets in signalling pathways in human immune cells. Theoretically, some of these PTP virulence factors could be harnessed for therapeutic purposes, for example, in cancer treatment or to prevent transplant rejection. 3. PTPs

as drug targets

The rapidly increasing number of human diseases associated with PTP abnormalities (discussed in Section 2) have begun to elicit a growing interest in PTPs as drug targets [115-118]. Much work has already been done both in academic and pharmaceutical settings to develop inhibitors against a small number of PTPs, notably PTP1B for Type II diabetes and obesity [118,119], MKP1 and CDC25 for cancer [120], and YopH [121,122] to combat the lethality of plague in the hands of bioterrorists. Other already considered drug targets include PTPα, CD45, SHP2,

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Figure 2. Structures of orthovanadate (A), thio-vanadyl ester (B), phenylarsine oxide (C), gallium nitrate (D) and disodium aurothiomalate (E).

PRL3 and LMPTP, and the authors predict that the list will grow considerably in the next few years. As drug targets, the PTPs are some 5 – 7 years behind the PTKs, many inhibitors of which are already in clinical trials or even on the market. The reasons for this are largely historical. The first PTP was identified molecularly [30] 10 years after the first PTK. In addition, the enthusiasm was initially greatly dampened by the notion that PTPs were less specific than PTKs and that the structure of the active site of PTPs did not allow for the generation of selective inhibitors. However, it has now become increasingly clear that PTPs indeed have unique, non-redundant and important functions and a great deal of specificity in vivo. The question of how selective small-molecule inhibitors can be is perhaps not quite resolved, although many promising examples have been published. The major concern that phosphomimetics would always remain too hydrophilic for cell penetrance appears to have been solved and the crystallisation of many PTPs has revealed that the surface topology surrounding the catalytic pocket of each PTP has numerous unique features that can be utilised for rational structure-based design of highly selective compounds. The spark that truly ignited the quest for PTP inhibitors with great market potential was the paper reporting the PTP1B knockout mouse [23]. This animal indicated that PTP1B acts as a negative regulator of insulin signalling and that, by extension, inhibition of PTP1B would alleviate insulin resistance in Type II diabetes and would improve the effects of insulin on both glucose balance and fatty acid metabolism. With the trend of increasing weight gain in the industrialised world, the market for Type II diabetes and obesity drugs is one of the largest known to the industry. 162

In the following sections, the authors review the literature on PTP inhibitors from the nonselective phosphate mimics, through phosphotyrosine-like molecules, peptidomimetics, to the newer small-molecule inhibitors that have been rationally designed or found through high-throughput screening. The authors realise, of course, that many newer and more selective inhibitors have been developed but remain unpublished. Finally, new trends and techniques are discussed [123,124] and PTP inhibitors are put into perspective with some thoughts about the future. and other metal containing inhibitors Due to its insulin-mimetic properties in vivo, the general PTP inhibitor orthovanadate (VO43-) (Figure 2A) has been used to treat diabetes mellitus in both experimental animals and in several clinical trials [125-127]. The stimulatory effect of vanadate on the insulin receptor PTK appears to be due to inhibition of several PTPs, including PTP1B [23,128], that dephosphorylate the receptor [128-130]. Vanadate inhibits PTP1B competitively in vitro with a Ki of 0.38 µM, using 3,6-fluorescein diphosphate as substrate [131]. Vanadate resembles inorganic phosphate and binds to the PTP catalytic side as a transition state analogue, generating a thiol-vanadyl ester linkage (Figure 2B) that resembles the five-coordinate thiolphosphate trigonal bipyramidal structure that forms during catalysis [132]. Because the catalytic site is highly conserved among all PTPs (Figure 1), vanadate acts as a general PTP inhibitor and, therefore, has a high potential for side effects. However, the metabolic effect of vanadium compounds in insulin signalling may outweigh such side effects. Other metal-containing PTP inhibitors that act similar to vanadate include phenylarsine oxide (C6H5AsO; Figure 2C), gallium nitrate (Ga(NO3)3; Figure 2D) and disodium aurothiomalate (NaO2CCH2CH(SAu)CO2Na; Figure 2E). Phenylarsine oxide was shown to be a potent inhibitor of CD45 [133,134], as well as other PTPs in intact cells [135,136]. The anticancer drug gallium nitrate [137] was reported to be a potent inhibitor of T cell membrane PTP activity, albeit not a potent inhibitor of CD45, whereas disodium aurothiomalate was shown to inhibit PTP1B, CD45 and other PTPs [138]. 3.2 Suramin

– a polyanionic molecule Suramin is a large, polysulfonated, symmetrical urea compound (Figure 3), which interacts with a variety of cellular proteins, leading to biological activities that include immunosuppressive [139] and anticancer effects [140]. Originally synthesised by Bayer AG in 1916, it has been widely used for the treatment of sleeping sickness and onchoceriasis over the last 80 years [141]. Recently, this compound was identified to cause dramatic increase of tyrosine phosphorylation of several cellular proteins in epidermal carcinoma and several other cancer cell lines [142,143]. Suramin was also found to inhibit plasma membrane CD45 in an irreversible and noncompetitive manner [144]. In 1998, Zhang and co-workers reported that suramin acts as an active site-directed, reversible and

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tight-binding inhibitor of PTP1B, VHR and YopH [145]. More recently, the same group reported suramin derivatives that inhibit Cdc25A, a potential human oncogene overexpressed in a number of tumours [146,147], with IC50 values in the submicromolar range [148]. However, suramin is not very selective and it is unclear which of its many cellular activities are due to inhibition of phosphatases. On the other hand, because suramin is already in clinical use, this molecule is a promising starting point. 3.3 Peptide-based

inhibitors As substrates for PTPs, phosphopeptides and phosphoproteins have as much as 10,000 times lower Km values than free phosphotyrosine [149], suggesting that interactions of the substrate with structures of the PTP outside of the phosphotyrosine-binding pocket have a major impact on substrate affinity and specificity. Moreover, Km values for the same phosphopeptide can vary by several orders of magnitude between different PTPs, suggesting that these interactions indeed confer substrate selectivity. The structural basis for this can be found in the striking differences in surface topology and surface

charge distribution around the catalytic pocket among the PTPs [3]. Some of these features interact with amino acid residues or phosphate groups at some distance from the target phosphotyrosine in substrate phosphopeptides or proteins. High-affinity peptide substrates can be converted into competitive PTP inhibitors by changing the phosphotyrosine moiety to a nonhydrolyzable phosphotyrosyl mimetic. Phosphonates are the most successfully applied mimetics to date, in particular the phosphonodifluoromethyl phenylalanine (F2Pmp)-containing peptides [150] (Figure 4). The two fluorine atoms probably restore or enhance hydrogen bonding to side chains in the active site of the PTP, which normally would interact with the phenolic oxygen of phosphotyrosine. As a result, the binding affinity increases by several orders of magnitude [151]. The crystal structure of a catalytically inactive mutant of PTP1B in complex with either phosphotyrosine or bis(para-phosphophenyl) methane recently revealed a second aryl phosphate binding site in the vicinity of the catalytic site [152]. Because this second site is not conserved among PTPs, this finding constitutes a new paradigm for the design of ligand with high affinity and selectivity. In an effort to develop bidentate inhibitors for PTP1B, which would bind both the active site and this adjacent second phosphotyrosine binding pocket, a peptide containing two F2Pmp moieties (Figure 5) was reported to be the most potent PTP1B inhibitor to date (Ki = 2.4 nM) [153]. This compound showed excellent selectivity of up to three orders of magnitude for a range of PTPs, and even tenfold better inhibition of PTP1B than of TCPTP, its closest relative [154]. However, because this compound was not able to penetrate cell membranes, a derivative containing a lipophilic fatty acid moiety (Figure 5B) was synthesised. This compound (Ki = 25 nM) entered cells and was active against PTP1B [155]. Peptidyl carboxylic acid inhibitors were also heavily investigated, resulting in a series of potent PTP1B inhibitors with the best being a tripeptide featuring a carboxymethyl salicylic acid moiety as a phosphotyrosine mimetic (Figure 6A) (Ki = 0.22 µM) [156].

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Figure 5. Structures of phosphonodifluoromethyl phenylalanine (F2Pmp)-containing inhibitors for PTP1B.

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O N H

HO O O

O

OH

OH O

O

O

O

F

OH O

O

O

OH

OH

OH

OH

NH

O

O

O

H N

O OH

HO

OH

NH2

O

NH2

N H

N H

O

O

NH2 O A

B

C

OH

D

Figure 6. Structures of a peptidyl carboxylic acid inhibitor (A), O-malonyl-tyrosine (OMT; B), fluoro-O-malonyl-tyrosine (FOMT; C) and the tripeptide substituted cinnamic acid CinnGEE (D).

Furthermore, O-malonyl-tyrosine (Figure 6B) and fluoroO-malonyl-tyrosine (Figure 6C) containing peptides have been found to bind Src homology 2 domains and to inhibit PTP1B [157,158]. Parasubstituted cinnamic acid tripeptides were reported as PTP1B inhibitors with the most potent CinnGEE (Figure 6D) having a Ki of 79 nM [159], whereas peptides containing cinnamic aldehyde show slow binding and covalent inhibition for PTP1B, SHP-1, and VHR [160,161]. Other phosphotyrosyl mimetics have been reported including sulphotyrosyl [162], thiophosphoryltyrosyl [163], O-boranophosphoryltyrosyl [164], O-dithiophosphoryltyrosyl [164] and isothiazolidinone compounds [165]. In summary, peptide-based inhibitors are useful to study substrate–PTP interactions in vitro and with certain modifications they may even be useful for in vivo studies. However, their use in cellular assays and animal treatment is limited by poor membrane permeability and degradation. 164

3.4 Natural

products and their derivatives Natural products have been a rich source of therapeutic agents, including several phosphatase inhibitors. Although these natural products may provide interesting lead molecules, they likely still need to be optimised to increase potency and selectivity before they can be therapeutically useful. Alendronate (ALN, Figure 7A), an amino-bisphosphonate used in the treatment and prevention of osteoporosis, was found to inhibit the enzymatic activity of RPTPε, which is highly expressed in osteoclastic cells, with an IC50 of 2 µM. ALN suppressed in vitro osteoclast differentiation as well as bone resorption by freshly isolated osteoclast [166]. The aporphine alkaloids nornuciferine (Figure 7B) and anonaine (Figure 7C) were isolated and identified as CD45 inhibitor metabolites of Rollinia ulei with IC50 values of 5.3 µM and 17 µM, respectively. Treatment of T cells with these compounds resulted in inhibition of IL-2 production upon T cell receptor stimulation [167]. The same alkaloids

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OH O

O

O-Na+

O

P HO OH

NH

O

NH2

NH

O

P O

OH

A

C

B

Figure 7. Structures of alendronate (A), nornuciferine (B) and anonaine (C).

HO

OH O

O O

HO

O O

OH

O

O

9

O 13

OH

O

O

O A

13 O

O B

Figure 8. Structures of RK-682 (A) and its dimeric derivative (B).

were also isolated from the fruit of Annona muricata as active components of its tranquillising and sedative properties [168]. Extracted from Guatteria amplifolia, nornuciferine also demonstrated significant activity against Leishmania mexicana and Leishmania panamensis [169]. The tetronic acid derivative RK-682 (3-hexadecanoyl-5hydroxymethyltetronic acid, Figure 8A) was isolated from a Streptomyces strain as a PTP inhibitor in a microbial metabolites screening [170]. In vitro, RK-682 inhibited CD45 and VHR with IC50 values of 54 and 11.6 µM, respectively. In intact cells, RK-682 enhanced the phosphotyrosine level of B cell leukaemia Ball-1 cells, but not the level of phosphoserine and phosphothreonine. Furthermore, RK-682 inhibited cell cycle progression of Ball-1 cells, arresting them at the G1/S cell cycle phase transition, unlike orthovanadate, which caused cycle arrest at the G2/M boundary. RK-682 did not inhibit Cdc25B and Cdc25C, which are required for G2/M transition, but orthovanadate did. To further explore the inhibitory action of RK-682, the same group synthesised a tetronic acid library of 36 derivatives. Structure–activity relationship studies revealed that the acidic 3-acyltetronic acid group functions as a

phosphate mimic and that a long acyl chain at the C3-position is important for inhibitory activity [171]. Kinetic studies also revealed that two molecules of tetronic acid derivative bind to each VHR molecule. Based on this observation, a VHR-(RK-682)2 binding model was constructed, in which the second tetronic acid moiety makes favourable interactions with the guanidino group of Arg158 which lies at the bottom of a pocket close to the active site. Interestingly, the corresponding Arg residue, although conserved in many PTPs, has a different environment for each enzyme and is not exposed to the surface, for example, in the Yersinia PTP YopH or in PTP1B. In an approach to target both the active site and the basic Arg158 pocket, a dimeric compound (Figure 8B) was synthesised. It showed improved inhibition of VHR with an IC50 value of 1.83 µM [172]. Another potent VHR inhibitor, 4-isovenaciolide (Figure 9A), was isolated from a fungal strain and found to bind covalently to VHR with an IC50 of 1.2 µM. Mass spectrometry and mechanistic studies suggested that it acts as a Michael acceptor and binds to the thiol group of the catalytic cysteine residue [173]. Dysidiolide (Figure 9B), a sesterterpene with a γ-hydroxybutenolide group was isolated from the Caribbean sponge Dysidea etheria and identified as an inhibitor of Cdc25A (IC50 = 9.4 µM). No inhibition of calcineurin, CD45, or LAR was observed at a concentration of 12.4 µM. Moreover, dysidiolide was found to inhibit growth of the A-549 human lung carcinoma and P388 murine leukaemia cell lines with IC50 values of 4.7 and 1.5 µM, respectively [174]. The synthesis and evaluation of a small collection of dysidiolide analogues was reported, showing that the C4 keton analogue was a potent inhibitor of Cdc25C in vitro (IC50 = 0.8 µM) [175]. Similar to the γ-hydroxybutenolide group in the dysidiolide structures, the 3-pyrrol-2-one core moiety of pulchellalactam (Figure 9C) serves likely as a surrogate phosphate. The compound was isolated from the marine fungus Corollospora pulchella and was found to inhibit CD45 with an IC50 value of 124 µg/ml [176]. Sulfircin (Figure 9D) was isolated from the marine sponge Ircinia sp. [177] and was found to inhibit Cdc25A and VHR with an IC50 values of 7.8 µM and 4.7 µM, respectively. Investigations of synthesised analogues showed that the sulfate group can be replaced by malonate without loss of potency [178].

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O O

O

H

H N

OSO3-

H O

O

O

6

C

H

A

HO

D H

4

B OH

O

O

Figure 9. Structures of 4-isovenaciolide (A), dysidiolide (B), pulchellalactam (C) and sulfircin (D).

OH

NO

O

ON N

N

N

OH OH

OH

OH OH

A

B

C

Figure 10. Structures of dephostatin (A), ethyl-3,4-dephostatin (B) and hexyl-methoxime-3,4-dephostatin (C).

Dephostatin

(2,5-dihydroxy-N-methyl-N-nitrosoaniline,

Figure 10A), isolated from Streptomyces, was found to be a

competitive inhibitor of CD45 with an IC50 of 10.7 µM [179,180]. Because dephostatin is labile, alkyl-3,4-dephostatins were synthesised as stable analogues, which were highly potent against PTP1B. Ethyl-3,4-dephostatin (Figure 10B) inhibited PTP1B with an IC50 of 3.2 µM in vitro and enhanced insulin-related signal transduction in vivo [181]. In an attempt to develop dephostatin analogues that lack the potentially carcinogenic nitrosamine moiety, hexyl-methoxime-3,4-dephostatin (Figure 10C) was designed by molecular modelling, using the concept of CH/π interactions by means of the alkyl side chain making favourable interactions with π-electrons of Phe182 within the active site of PTP1B. It inhibited PTP1B with an IC50 of 3.7 µM in vitro and enhanced the accumulation of glucose in 3T3-L1 adipocytes in an dose-dependent manner [116]. 3.5 Structure-based design of small-molecule PTP inhibitors

Apart from using an active natural product as a lead structure for PTP inhibitor development, more rational approaches 166

have been successfully deployed recently. Although a high-throughput library screening is still indispensable as a first step in many inhibitor development projects, the impact of structure-based information on generating high quality leads is increasing. Based on the many reported three-dimensional structures of all major subfamilies of PTPs during the last ten years, molecular modelling can now be used to design ligands that can bind both competitively and selectively to a particular PTP. Moreover, because catalytic domains within the subfamilies are highly conserved, protein structure homology modelling can result in precise 3D-models of PTP structures that still remain to be resolved experimentally [3]. Thus, any of the 103 Cys-based PTP in the human genome could be subjected to in silico docking and virtual screening for inhibitors. A major drawback of small-molecule PTP inhibitors that contain peptidomimetic scaffolds based on phosphonate or fluorophosphonate groups is their multi-charged nature and poor drug-likeness according to Lipinski’s rules [182,183], resulting in lack of cell-membrane permeability. Mono carboxylic acids carry only one negative charge and have been reported as potent PTP inhibitors in vitro and in vivo. From a series of compounds synthesised at Wyeth Research, the novel carboxylic acid ertiprotafib (Figure 11A) has been developed as a PTP1B inhibitor (IC50 = 384 nM) and was shown to improve insulin sensitivity in rodents [184]. This compound progressed to Phase II clinical trials for treatment of Type 2 diabetes. However, because of unsatisfactory efficacy and dose-limiting side effects, the trial was terminated in 2002. Because the data from the in vivo treatments were inconsistent with PTP1B inhibition as the sole mechanism for improved insulin sensitivity, further investigations were conducted and revealed that ertiprotafib was also an agonist of peroxisome proliferator-activated receptors PPARα and PPARγ with EC50 values of about 1 µM. Thus, ertiprotafib improved glycaemic control via multiple mechanisms [185]. A series of benzofuran/benzothiophene biphenyl oxoacetic acids and sulfonyl-salicylic acids were also developed as

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Br

O

OH O O O

S

HO

A

O

B Br

OH O O

O

S

S O

OH

Br C

Figure 11. Structures of benzofuran- and benzothiophene-derived PTP1B inhibitors.

PTP1B inhibitors at Wyeth, and showed good oral antihyperglycaemic activity in a diabetic mouse model, with compound B (Figure 11B, IC50 = 320 nM) having the best in vivo effect. This compound normalised plasma glucose levels at 25 mg/kg (per os) and 1 mg/kg (intraperitoneal). The salicylic acid derivative (Figure 11C) inhibited PTP1B in vitro with an IC50 of 30 nM and normalised plasma glucose levels at 100 mg/kg (per os) [186]. Based on X-ray crystallographic data of the co-crystallised complexes, the binding mode for these types of compounds has been defined. As predicted, the glycolic acid moiety and the salicylic acid, respectively, bind to the active site of PTP1B, with the biphenyl ring proximal to the glycolic acid group and the aromatic portion of the salicylic acid moiety, respectively, sandwiched between Tyr46 and Phe182. Interestingly, the salicylic acid is involved in direct hydrogen bonding with residues Lys120 and Tyr46, and interacts with one of the two water molecules that interact with Arg221, whereas the acetic acid only binds to the two water molecules that bridge the guanidino group of Arg221. Moreover, the lipophilic tails of the two types of compounds occupy an almost opposite orientation at the PTP1B surface. In contrast to other compounds, the 2-benzyl-benzothiophene tail-piece of the salicylates forms nonspecific van der Waals interactions with a distinctive hydrophobic region near the active site at the protein surface of PTP1B. In a recently published report from the authors’ own laboratory, high-throughput library screening and structure–activity relationship analysis was used to identify competitive and selective inhibitors for the Yersinia PTP YopH [122]. Several drug-like lead structures were taken into in silico docking studies to examine binding modes and inhibitory properties. This approach identified the novel pharmacophore furanyl salicylate (Figure 12), which acts as a phosphotyrosine mimetic, but is

much less polar than phosphotyrosine. The four most potent inhibitors had IC50 values of 180 nM (Figure 12A), 260 nM (Figure 12B), 390 nM (Figure 12C) and 460 nM (Figure 12D), respectively. Flexible ligand docking studies with the X-ray coordinates of the catalytic domain of YopH showed that the salicyl-furanyl moiety occupied the active site pocket, where it was found to be involved in a complex network of hydrogen bonding interactions [122]. In addition, the oxygen atom of the furanyl ring was also invariably involved in hydrogen bonding interactions with the side chains of Gln357 and/or Arg404 (Figure 13), both of which are unique to YopH among PTPs. Selectivity was further demonstrated in silico by attempts to dock the inhibitors into the crystal structures of PTP1B and VHR clearly demonstrated that none of them fit into their active sites. Indeed, IC50 values for PTP1B and VHR were found to be 30 – 39-fold and 70 – 135-fold higher than for YopH. Although both docking studies and the structure–activity relationship analysis showed that most of the binding energy of the inhibitors came from interactions of the furanyl–salicylic acid moiety with the catalytic pocket and its immediate surroundings, the more variable distal end of the molecules also contributed to specificity and selectivity: the thio-barbituric acid moiety in compound B was involved in an additional hydrogen bonding interaction with the side chains of Gln357 and Gln446, whereas the two methyl groups in compound A made favourable hydrophobic contacts with the YopH surface in very close proximity to an additional groove (Figure 13). Interestingly, the corresponding depression in PTP1B is blocked by a large protrusion, preventing A from binding to PTP1B in the optimal orientation, making this compound the most selective inhibitor for YopH over PTP1B (Figure 14). The structurally less-related VHR does not even have such an additional pocket.

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O O N

S

OH

OH B

OH

OH

N

O

N

O

O

S

O

N H

O A

HN

O

HN

N

OH

O

OH

O

S

N

N

OH

H N

O

N

O OH

O

O D

C

Figure 12. Structures of furanyl salicylate compounds that are potent and selective inhibitors of the Yersinia protein tyrosine phosphatase YopH.

A

B

C

D

Figure 13. Docking of furanyl salicylate compounds into the active site of the Yersinia protein tyrosine phosphatase YopH. Crossed stereo images of hydrogen bond interactions between compound and protein, as well as surface representations are shown for compound A (A, B) and B (C, D), respectively. The colour code of the MOLCAD surfaces in B and D represents the electrostatic potential (red: most positive, purple: most negative).

168

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A. A

B. B

C. C P3 P3

P3 P3

P2 P2

P2 P2

P1 P1

P1 P1

P1 P1

Figure 14. Furanyl salicylate compound A can be docked into the active site of YopH (A), but does not fit into PTP1B (B) or VHR (C). P1 indicates the catalytic pocket, P2 and P3 are additional pockets in the vicinity of the active site in YopH and PTP1B. PTP: Protein tyrosine phosphatase.

The authors believe that these results illustrate how the large differences in structure, surface topology, charge and hydrophobicity can be utilised to achieve a high degree of specificity and selectivity for PTP inhibitors. It should also be mentioned that furanyl–salicylate compounds readily entered live T cells and inhibited the activity of YopH to restore tyrosine phosphorylation, T cell antigen receptor signalling and T cell activation [122]. Finally, it should be emphasised that the search for PTP inhibitors is fraught with numerous pitfalls. Many small molecules can oxidise PTPs and/or bind irreversibly to PTPs, sometimes outside of the catalytic pocket. Some oxidising classes of molecules are notorious for being frequently caught in high-throughput screens. However, some of these molecules can also yield useful leads. For example, although many quinones bind covalently and/or oxidise the catalytic cysteine of PTPs, reversible competitive inhibitors of this class have also been reported (e.g., indolyldihydroxyquinones as inhibitors for Cdc25) [187], and other quinones as inhibitors for Cdc25B [188], PTP1B [189] and CD45 [190]. Others, like quinolines (e.g., quinoline-5,8-dione), show a wide spectrum of biological activities such as antitumour, antifungal and antimalaria effects, as well as inhibition of Cdc25 [191]. 4. Expert

opinion

The PTP field is heading into increasingly exciting times. The entire set of PTP genes in humans and mice is now known and it is finally possible to venture into more global studies of the ‘PTPome’ and to address questions of specificity, redundancy, and tissue expression of all PTPs in healthy and diseased tissues. This task is somewhat complicated by the capacity of many PTP genes to generate alternatively spliced products, the existence of single-nucleotide polymorphisms

that alter function, and the regulatory influences of many post-translational modifications and protein–protein interactions. However, the rate of progress in this undertaking is likely to be accelerated by many emerging new technologies in high-throughput screening, global proteomics and rapid content-based imaging. The identification of the physiological substrates for PTPs, a critical step in elucidating their function, is likely to be aided by the development of mass spectrometry technologies to study the entire phosphoproteome of cells in a comprehensive, detailed and quantitative manner. Genetic approaches and RNA interference will likely promote discovery of physiological functions and will help address important questions of redundancy. All these issues are important for the full utilisation of PTPs as drug targets and the treatment of many human diseases in which PTPs play regulatory or even causative roles. The authors predict that discoveries in the coming years will reveal that many more PTPs are involved in human disease and, therefore, should be considered for their potential as drug targets. This will stimulate the efforts to design increasingly potent and selective PTP inhibitors with optimal drug-like properties. However, the authors would caution against the notion that PTP inhibitors need to be monospecific. Although the true redundancy of PTPs in vivo remains unclear, the authors interpret the existing information to mean that groups of related PTPs have at least partly overlapping functions and that even more distantly related PTPs can participate in the same biological process. Thus, the greatest biological activities are likely to come from spectrum-selective PTP inhibitors (i.e., inhibitors that inhibit a subgroup of two to five PTPs rather than a single PTP). A major challenge in PTP inhibitor development is posed by the hydrophilic and multiply charged nature of the natural substrate for PTPs, which makes it challenging to design

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O HO

O

O O

OH N

O

HN

OH

O

H N O O

Figure 15. Structure of a bidentate inhibitor of PTP1B-derived from a fragment-based approach. The oxalylaminobenzoic acid moiety mimics the natural phosphotyrosine and occupies the catalytic pocket, whereas the salicylic acid methylester group interacts with an adjacent site. PTP: Protein tyrosine phosphatase.

substrate-like inhibitors (i.e., phosphotyrosine mimetics) that are sufficiently hydrophobic to penetrate the plasma membrane. Fortunately, many cell-permeable lead compounds have recently been reported and the authors believe that these, as well as many new ones, will serve as starting points for the development of efficient and selective PTP inhibitors with optimal pharmacological properties. An important lesson from the progress made in the last few years is the realisation that although all PTPs have very similar catalytic cores, they differ dramatically in surface topology and charge distribution in the terrain that surrounds the catalytic pocket [3]. In a biological context, these striking differences reflect distinct preferences in substrate selection and protein–protein interactions between the PTP and multiple surface features of the substrates other than the phosphotyrosine target residue. However, these same features can be utilised for the development of small-molecule inhibitors with a high degree of specificity. This notion is already supported by several published examples, but the authors believe that the true potential of structure-based specificity and selectivity of PTP inhibitors still remains to be discovered. PTP assays with natural substrates, or at least phosphopeptides, are likely to be helpful in these efforts. The unique features of the substrate-binding surface of PTPs also renders these proteins ideal targets for fragment-based drug discovery approaches [192], as was recently done using nuclear magnetic resonance spectroscopy to identify high-affinity and selective PTP1B inhibitors [193]. In this work, a library of 10,000 compounds was screened using heteronuclear [15N,1H] correlations spectra to assess binding. This technique can identify even small fragments that bind fairly weakly and can yield information on the mode of binding [123]. In the above study [194], the authors identified a compound that mimicked the natural phosphotyrosine substrate (which had Ki ∼ 100 µM), which was then joined by a linker to a fragment identified to occupy an adjacent site. The resulting ‘bidentate’ compound inhibited PTP1B with

170

a Ki of 18 nM (Figure 15). This approach has a lot of promise for the development of efficient and specific PTP inhibitors. Accelerated progress may also come from virtual screening of large libraries. In a recent published example, inhibitors of PTP1B were searched for using both virtual screening (‘in silico’) and high-throughput screening using a 400,000-compound chemical library [194]. Eventually, 365 high-scoring docked compounds were tested experimentally. The hit rate for these 365 molecules was 1700-fold higher than that by high-throughput screening and the docking hits were more drug-like. Intriguingly, there was no overlap among the high-throughput screening and docking hit lists, suggesting that the two techniques are complementary. With the continuing improvement of the algorithms for flexible ligand docking and high-resolution crystal structures of the relevant PTPs in their substrate-bound closed conformation, the authors believe that virtual screening will play an important role in PTP inhibitor development in the coming years. Finally, the authors believe that another bottleneck in PTP inhibitor development is the paucity of well-established cell-based assays to evaluate hits in intact cells. Ideally, such assays should reveal both specific target inhibition as well as off-target effects. For example, a specific PTP inhibitor should cause hyperphosphorylation of the substrates for the target PTP, and downstream pathways, but should not have any effects on overall tyrosine phosphorylation, other pathways, cell viability, or other parameters. In the authors’ opinion, these assays are critical for success and allow for rapid triage of compounds with poor cell permeability, toxicity, or other unwanted properties. Cell-based assays also establish efficacy and dose-response curves that are important for testing in animal models. In conclusion, the authors are convinced that the human PTPome contains numerous potential drug targets and that the next decade will see many attempts to develop specific PTP inhibitors for the treatment of human disease. In the authors’ opinion, it is likely that these efforts eventually will succeed. The authors predict that the first PTP inhibitor on the market will greatly increase the enthusiasm for PTPs and will inspire more research and drug development in this area.

Acknowledgments The authors apologise to all co-workers whose papers we could not cite here due to space limitations. The authors thank Nunzio Bottini, Michael David, Jack Dixon, Rob Edwards, Gen-Sheng Feng, Adam Godzik, Andrei Osterman, Robert Rickert, Zhong-Yin Zhang and many other co-workers and friends for many stimulating discussions about phosphatases. This work was supported by grants AI35603, AI48032, AI53114, AI53585, AI55741, AI55789 and CA96949 from the National Institutes of Health.

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Affiliation

Lutz Tautz1 PhD, Maurizio Pellecchia2 PhD & Tomas Mustelin†3 MD, PhD †Author for correspondence 1Postdoctoral Researcher, Infectious and Inflammatory Disease Center and Cancer Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA 2Associate Professor, Infectious and Inflammatory Disease Center and Cancer Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA 3Professor, Program Director, Infectious and Inflammatory Disease Center and Cancer Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA Tel: +1 858 713 6270; Fax: +1 858 713 6274; E-mail: [email protected]

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Synthesis and PTP1B inhibition of 1,2naphthoquinone derivatives as potent antidiabetic agents. Bioorg. Med. Chem. Lett. (2002) 12:1941-1946.

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