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The therapeutic potential of phosphatase inhibitors

Viktor V Vintonyak1, Andrey P Antonchick1, Daniel Rauh3 and Herbert Waldmann1,2
Protein phosphatases (PPs) constitute a large family of enzymes, which are crucial modulators of cellular phosphorylation events. Malfunction in PP activity has been associated with human diseases, including diabetes, obesity, cancer, and neurodegenerative and autoimmune disorders, and makes this class of enzymes attractive targets for chemical biology and medicinal chemistry research. A number of strategies are currently explored for the identication and development of various classes of PP modulators and have resulted in a plethora of chemically distinct inhibitors. Limited selectivity and adverse pharmacological properties of PP inhibitors are still major bottlenecks for further clinical development and resulted in only a few molecular entities currently in clinical trials.
Addresses 1 Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany 2 Technical University Dortmund, Otto-Hahn-Strasse 6, D-44221 Dortmund, Germany 3 Chemical Genomics Centre of the Max Planck Society, Otto-HahnStrasse 15, D-44227 Dortmund, Germany Corresponding author: Rauh, Daniel (daniel.rauh@cgc.mpg.de) and Waldmann, Herbert (herbert.waldmann@mpi-dortmund.mpg.de)

Current Opinion in Chemical Biology 2009, 13:272283 This review comes from a themed issue on Next-generation therapeutics Edited by Karl-Heinz Altmann and Dario Neri Available online 4th May 2009 1367-5931/$ see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2009.03.021

Introduction
Phosphorylation and dephosphorylation are among the most important modications by which nature modulates protein function in nearly all biological systems to transduce information between distinct cellular sites. The specic transfer of the g-phosphate from ATP to substrate proteins is catalyzed by protein kinases (PKs) and can lead to conformational changes, promote proteinprotein interactions, or turn on/off enzymatic activity to allow cells to translate a wide variety of environmental signals into functional changes. Protein phosphatases (PPs) are dened by their ability to catalyze the hydrolysis of phosphates to restore the protein substrate to its dephosphorylated state and can be considered as physiological
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counterparts of PKs. The balanced and highly dynamic interplay between these two enzyme classes is crucial for the control of cell signaling cascades. To achieve the essential temporal and spatial specicity in modulating enzyme activity in signaling events, nature often uses the compartmentalization of PK and PP activities. In consequence, aberrantly regulated PKs and PPs play causative roles in diseases such as cancer, diabetes, and neurological and autoimmune disorders, making these enzymes an important set of therapeutic targets across almost all disease areas [1,24,5,6]. PPs are classied by their substrate specicities into serine/threonine-phosphatase (STP), protein histidine-phosphatase (PHP), protein tyrosine-phosphatase (PTP) and dual-specic phosphatases (DSPs) which catalyze the dephosphorylation of both serine/threonine or tyrosine residues. The detailed understanding of the different catalytic mechanisms, substrate specicities, and associated conformational changes in phosphatase structure has proven to be crucial for the design and development of potent and selective inhibitors. STPs have metal ions in their catalytic center (zinc, manganese, or magnesium) which are coordinated by side chains of His and Asp amino acids. A highly polarized water molecule bridges the two metal centers and can act as a nucleophile attacking the phosphate group to initiate its hydrolysis. PTPs follow a different mechanism and do not require metal ions. A structural characteristic crucial for the design of most PTP inhibitors is a deep pocket to accommodate the pTyr of the substrate. A WPD-loop (Trp-Pro-Asp) closes on this site, regulates catalysis by its Asp residue, and allows proper substrate recognition. In the closed substrate bound state, the Asp forms a hydrogen bond to the phenolic oxygen of the pTyr and polarizes this site for the nucleophilic attack of the adjacent catalytic cysteine. This Cys is positioned at the N-terminus of a long helix in the middle of the catalytic domain. The helix induces a dipole and thereby alters the properties of the Cys thiolate side chain. The phosphate gets transferred onto the Cys to form a thiophosphate, which is subsequently hydrolyzed by a water molecule. DSPs follow the same mechanism but possess a shallower substrate pocket to accommodate pThr and pSer. The human genome encodes for a total of 518 kinases, including 90 tyrosine kinases. Similarly, more than 130 PPs are encoded in the human genome, including 107 tyrosine phosphatases [7]. This represents a fairly balanced complement of PTKs and PTPs and suggests an equal partnership of these enzymes in regulating
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Therapeutic potential of phosphatase inhibitors Vintonyak et al. 273

protein function through tyrosine phosphorylation. The majority of protein phosphorylation, however, occurs on serines and threonines and is catalyzed by the remaining 428 protein serine/threonine kinases. The fewer protein STPs imply that these enzymes must display much broader substrate specicities than the complementary kinases. The catalytic domains of PPs are highly conserved and differences in substrate specicity are largely determined by regulatory domains or subunits. Some catalytic domains, in particular those of the protein STPs, can interact with a large variety of regulatory subunits and are controlled by post-translational modications, proteinprotein interactions, or heteromeric compositions. The incorporation of catalytic subunits in multidomain complexes explains why eukaryotes contain fewer genes that encode for PPs than genes that encode for PKs. When the holoenzymes are considered, the numbers of PKs and PPs are more or less balanced [3,810]. PPs are often kinase substrates themselves, underlining their essential regulatory role in signaling cascades. As a consequence, many diseases have been shown to be associated with regulatory malfunction of PPs and the acceptance of PPs as equal in importance to PKs as potential drug targets has been supposed over the last decade. In this review we will focus on recent progress in the development of inhibitors directed against clinically relevant PPs (Table 1).

Clinically relevant phosphatases

PTP1B

Acquired Type 2 diabetes and obesity often can be considered as lifestyle-related diseases of civilization and are linked to insulin resistance and loss of proper glucose homeostasis. Type 2 diabetes and obesity are often coupled in human and can lead to the metabolic syndrome, which dramatically increases the risk of cardiovascular incidences and decreases lifespan. Some predictions for the year 2015 estimate 70% of the population of the western hemisphere to be overweight and 40% of these to be obese. The social impact and costs associated with this health risk will be substantial and drugs regulating metabolic disorders are in high demand.
Table 1 Selected phosphatases as target proteins Family Serine/threonine phosphatases Tyrosine phosphatases Phosphatases PP1, PP2A PP2B, PP2C PTP1B MptpA/B CD45 SHP-1/2 Dual-specic phosphatases VHR Cdc25 PRL-1/2/3

The discovery of the receptor tyrosine phosphatase PTP1B as a negative regulator of the insulin and leptin receptor pathways [11,12,13] and animal studies demonstrating that PTP1B-decient mice show an enhanced insulin sensitivity, improved glycemic control, and resistance to high fat diet induced obesity [14,15] made this enzyme an attractive drug target. Although the molecular causality of insulin resistance and obesity is not fully understood, the hormone insulin induces autophosphorylation of its receptor and thereby triggers a kinase cascade that nally induces synthesis of the short-term energy storage glycogen and synthesis of fatty acids and proteins. Dephosphorylation of the receptor by PTP1B leads to its inactivation and shut-down additional downstream processes. The pharmacological blockage of PTP1B should therefore counteract insulin resistance. At the latest when Abbott demonstrated that antisense oligonucleotides (ASOs) designed to downregulate expression of PTP1B normalized blood glucose and improved insulin sensitivity without changing the regular diet of mice [16], nearly every major pharmaceutical company launched a program for the identication of potent PTP1B inhibitors. Besides its central role in the insulin cascade PTP1B is also involved in a variety of clinically relevant pathways [17,18] and is overexpressed or upregulated in human breast, colon, and ovarian cancers [1921]. However, the discovery of clinically useful PTP1B inhibitors has proven to be very difcult because of limited inhibitor selectivity and low bioavailability [4,5,22].
Structure-based design and active-site inhibitors

Structure-based design is widely applied to phosphatase inhibitor research and best exemplied by more than 80 complex crystal structures of PTP1B deposited in the Protein Data Base (PDB) (Figure 1a). One of the rst crystal structures of PTP1B (pdb code: 1pty) featured an enzymatically dead mutant variant (catalytic Cys mutated to Ser) in complex with phosphotyrosine. The substrate is coordinated in the active site of the phosphatase by a network of hydrogen bonds. Interestingly, a second pTyr

Disease, therapeutic approach Tumor suppression, malignancy Cystic brosis, immune suppression, asthma, cardiovascular Diabetes, obesity Tuberculosis Alzheimers disease, autoimmune disease, inammation, organ transplantation Neuron protection, obesity, Noonan syndrome, leukemia, regulation of RAS/MAP-kinases Regulation of MAP-kinases Cell cycle progression, tumor therapy (various human cancers) Promoting tumor cells, leukemia, Hodgkins disease, prostate cancer

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Figure 1

Inhibitors in complex with the catalytic domain of PTP1B. (a) 88 PTP1B-inhibitor complexes found in the PDB and aligned to the first reported PTP1B crystal structure (PDB code: 1pty). Most inhibitors target the phosphate binding sites and mimic substrate binding. (b) Close-up view of the active site shows binding of the pTyr of the substrate peptide (gray sticks) to the N-terminal end of the central helix (blue) to position the phosphate in close proximity to the catalytic Cys215 (ball and sticks). The WPD-loop (yellow) controls access to the active site. Binding of a second pTyr molecule (blue ball and sticks) adjacent to the active site stimulated the design of molecules that bind to both pTyr sites and resulted in the development of the potent bidentate inhibitor 8 (green) (PDB code: 2qbp). (c) In inactive PTP1B conformations the C-terminal helix (orange) adopts an extended orientation and exposes a new cavity (green). Allosteric modulators such as 3 inhibit PTP1B activity by binding to this allosteric site and preventing the WPD-loop from adopting a catalytically competent conformation. The inhibitors bind around the side chain of Phe280 and form favorable pp interactions. Figures were prepared with PyMol (http://www.pymol.org).

molecule was identied in an adjacent, noncatalytic site and stimulated ideas to fuse the two moieties by a suitable linker which resulted in bidentate inhibitors with dramatically increased afnities (Figure 1b). This second site has since then been highly important in lead structure optimization for PTP1B. Fragment-based approaches both by protein X-ray crystallography and NMR techniques were employed to identify, for example, small organic acids as binders to this second site and resulted in an impressive array of potent nM inhibitors when fused to phosphomimics (Figure 2). Most active-site-directed PTP inhibitors reported to date are nonhydrolyzable pTyr mimics that take advantage of the positively charged active site. However, this conserved mode of action reduces selectivity and often results in the potent inhibition of multiple phosphatases. Most pTyr mimics possess a high charge density, which in turn is often detrimental to cell penetration. The most useful and potent pTyr surrogate devised for PTP1B thus far contains the nonhydrolyzable diuorophosphonomethylphenylalanine (F2Pmp) group [23]. The uorine
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atoms have been proposed to interact via hydrogen bonds with NH-groups of the PTP and to increase the binding afnity by several orders of magnitude. Compound 1a identied from a combinatorial chemistry approach displays a Ki value of 2.4 nM for PTP1B and exhibits notable selectivity in favor of PTP1B against a panel of PTPs including Cdc25A, SHP-2, and VHR [24]. Because of its high polarity compound 1a is not cell permeable and shows no cellular activity. To enhance penetration across membranes, derivatives of 1a were prepared either by coupling to a cell permeable (D)-Arg8-peptide tag (1b) [25] or to a highly lipophilic fatty acid (1c) [26]. Compound 1b showed a signicant improvement in leptindependent suppression of food intake in leptin-resistant rats [27]. Furthermore, the concept of a prodrug was utilized in 2 [28]. Several strategies have been applied to identify pTyr mimics with more favorable pharmacological properties. One such approach, referred to Breakaway Tethering, introduces a Cys residue on the surface of the enzyme, which acts as a tethering point. The modied enzyme is
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Therapeutic potential of phosphatase inhibitors Vintonyak et al. 275

Figure 2

Structures of selected PTP1B and Cdc25 inhibitors, IC50 or Ki values are given. www.sciencedirect.com Current Opinion in Chemical Biology 2009, 13:272283

276 Next-generation therapeutics

incubated with a library of disulde-containing smallmolecule fragments under reducing conditions, which promote thiol exchange and formation of an SS bond between the ligand and the surface Cys residue [29]. This tethering approach in combination with mass spectrometry allows for capturing compounds that display even weak afnity for the target protein. Using this approach Erlanson et al. identied a novel PTP1B inhibitor with enhanced bioavailability. In a more recent analysis 1,2,5-thiadiazolidin-3-one-1,1dioxide was identied as a novel phosphomimic and the isothiazolidinone group was utilized in the synthesis of the peptide-based inhibitor 4, which shows an IC50 of 40 nM [30]. In addition, using the same mimic, nonpeptide-based compound 5 was identied, which displays high potency in biochemical assays (IC50 of 35 nM). The same compound also shows cellular activity and increases the level of phosphorylation on the insulin receptor (2.8-fold) at 80 mM [31]. More recently, Zhang et al. identied aryl diketoacids 6 as novel pTyr surrogates and showed that neutral amide-linked aryl diketoacid dimers 7 also exhibit PTP inhibitory activity. Detailed studies by enzyme kinetics and protein X-ray crystallography revealed that these derivatives stabilize PTP1B in its inactive, WPDloop open conformation and act as noncompetitive inhibitors [32]. Using a high-throughput screening approach a series of monocyclic thiophenes were identied as PTP1B inhibitors [33]. Further optimization resulted in the development of the potent inhibitor 8. Introduction of a tetrazole ring or 1,2,5-thiadiazolidine-3-one-1,1-dioxide as a carboxylate mimic led to the discovery of two unique starting points that improved cell permeability and increased potency up to 300 nM [34]. Additional examples for active-site-directed PTP1B inhibitors include arylbenzonaphthothiophenes and arylbenzonaphthofurans. Both scaffolds were shown to improve insulin sensitivity in rodents. One compound from these efforts, ertiprotab (9), progressed to clinical trials for the treatment of Type 2 diabetes. Development was discontinued in phase II because of insufcient efcacy and strong unwanted side effects [35]. Although great progress has been made to increase potency of PTP1B inhibitors, initial successes were compromised by unwanted cross reactivity with Tcell protein tyrosine phosphatase (TCPTP) (77% sequence identity with PTP1B) and the discovery that TCPTP knockout mice are born healthy but die after about four weeks. Even more disturbing was the nding that parallel knockout of TCPTP and PTP1B turned out to be lethal. The design and synthesis of selective PTP1B inhibitors that are less potent against TCPTP can be considered a particular challenge in current PTP1B medicinal chemistry research.
Allosteric inhibitors

directed phosphatase inhibitors was presented in 2004 when researchers at Sunesis reported an allosteric site located at the back of the phosphatase about 20 A distant to the catalytic site. The crystal structure of PTP1B in complex with the allosteric inhibitor 3 (pdb code: 1t4j) proved that the ligand stabilizes the inactive phosphatase conformation by preventing the WPD-loop from adopting a catalytically competent conformation [36] and revealed a novel but general mechanism to inhibit tyrosine phosphatases (Figure 1c). In the allosteric site, the inhibitor binds close to a central Phe residue and forms favorable pp interactions. Interestingly, the structural homolog TCPTP holds a Cys residue at this position and 3 binds only with reduced afnity. Future work will show if the selectivity problem can be tackled adequately by this new mode of action. Another allosteric inhibitor of PTP1B, trodusquemine (MSI-1436) (10), is being developed by Genaera Corp. for the potential treatment of Type 2 diabetes and obesity and proceeded to phase Ib [37]. Two previous phase I studies showed that single doses of 10 administered to more than 60 subjects were well-tolerated with an acceptable adverse event prole, produced dose-dependent weight loss and improved insulin sensitivity. Genaera Corp. expects to verify the clinical potential and positive efcacy results of this drug in ongoing clinical trials [38]. Meanwhile a new clinical focus is on ASOs which are directed against PTP1B. The main advantage of oligonucleotides compared to small-molecular-weight inhibitors is their genetic selectivity for PTP1B without perturbing TCPTP function. Several PTP1B ASOs have been developed by ISIS Pharmaceuticals Inc. and a second generation ASO ISIS-113715 is currently in phase II clinical trials for the treatment of Type 2 diabetes.
Cancer-related PTPs

A number of PTPs have been identied as critical oncogenes in human malignancy and are considered potential drug targets for the development of novel anticancer therapeutics [5,6,39,40].
Cdc25

An alternative approach to overcome the current limitations in selectivity and bioavailability of active-siteCurrent Opinion in Chemical Biology 2009, 13:272283

Cdc25s belong to the family of DSPs and regulate cyclindependent kinases (Cdks), which are the key participants in cell division induced in response to extracellular signals including growth factors. Cdc25 phosphatases are also the key components of the checkpoint pathway and are inactivated or degraded to induce cell cycle arrest in response to DNA damage, leading to DNA repair or apoptosis [41]. Dysregulation of these processes can contribute to genomic instability and lead to cancer. In humans, the three isoforms Cdc25A, Cdc25B, and Cdc25C seem to have overlapping substrate specicities for the different Cdkcyclin complexes [42]. Cdc25A and Cdc25B overexpression has been reported in various human cancers, including breast, ovarian, prostate, lung,
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Therapeutic potential of phosphatase inhibitors Vintonyak et al. 277

colorectal, pancreatic, gastric, thyroid, hepatocellular, and neuroblastoma and often put in context with more aggressive tumors and poorer clinical outcome [41]. The inhibition of Cdc25 phosphatases may represent a novel approach for the development of anticancer therapeutics. Two recent reviews by Garuti et al. [43] and ContourGalcera et al. [44] provide a comprehensive overview of current Cdc25 inhibitor development. The most potent Cdc25 inhibitor reported to date is the naphthoquinone NSC95397 (11) with IC50 values of 22, 125, and 56.9 nM for Cdc25A, Cdc25B, and Cdc25C, respectively [45]. In addition, it displayed signicant inhibition when tested against human and murine carcinoma cells and blocked the G2/M phase transition during the cell cycle. Another potent compound is NSC663284 (12), which inhibits Cdc25s in vitro in an irreversible, time-dependent manner with Ki values in the mid nM range for all Cdc25 isoforms. It arrests cells in the G1 and G2/M phases of the cell cycle and induces signicant growth inhibition of human breast cancer cell lines [46]. Contour-Galcera et al. recently reported the identication of a new thiazoloquinone, BN82685 (13), which inhibits Cdc25 in biochemical assays (IC50 = 250, 250, and 170 nM for Cdc25A, Cdc25B, and Cdc25C, respectively) and is active against human xenografts [47]. In addition, low concentrations of BN82685 in combination with the mitotic inhibitor paclitaxel (Taxol) inhibit the proliferation of colon cancer cells [48]. Most quinone-based Cdc25 inhibitors reported to date are irreversible binders and act via arylation of the nucleophilic catalytic Cys. The redox properties of quinones can also generate reactive oxygen species (ROS), which may cause toxicity to normal tissues and thus reduce therapeutic potential. In order to overcome this problem, Carr et al. recently synthesized a series of nonquinone sulfone analogs of vitamin K3, H32 (14) [49]. Such compounds preferentially inhibit Cdc25 by reversibly binding to the catalytic cysteine and lead to G1 phase arrest during the cell cycle in Hep3B cells. An alternative strategy in the design of Cdc25 inhibitors is the utilization of phosphate surrogates, which anchor the ligand in the active site. Dysidiolide (15a), a sesterterpenoid isolated from the Caribbean sponge Dysidea etheria was the rst natural product reported to be active on Cdc25 (inhibition of Cdc25A with an IC50 of 9.4 mM). The g-hydroxybutenolide moiety is thought to mimic the substrate phosphate. Facilitating 15a as a biologically validated starting point, we used solid-phase synthesis for the generation of a small collection of dysidiolide analogs [50,51] and were able to identify the inhibitors of Cdc25C that are more potent than the parent natural product 15a. Analogs 15b and 15c displayed IC50 values of 5.1 and 0.8 mM, respectively. Moreover, these analogs proved considerable biological activity in cytotoxicity assays employing different cancer cell lines. Using the structures of dysidiolide and vitamin D3 as starting points Shimazawa et al. designed several potent Cdc25 inhibitors. Compound 16 inhibits Cdc25A and Cdc25B with
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IC50 values of 0.44 and 1.9 mM, respectively [52]. Recently 2-methoxyestradiol (2-ME) was reported as a potent, selective, and relatively nontoxic inhibitor of hepatoma growth both in vitro and in vivo. It was suggested that 2ME binds to the catalytic site Cys [53]. Another class of Cdc25 inhibitors is maleic anhydride derivatives bearing a fatty acid chain at the C-4 position [54]. Compound 17 inhibits Cdc25s with IC50 values in the low mM range and induces G0/G1 phase accumulation with subsequent inhibition of Cdk2 activity. Moreover, apoptosis was triggered in the presence of 17 within a 48-hour treatment without oxidative burst. N-Arylmaleimide derivatives are potent electrophiles and reagents for thiol-selective modications and were introduced as a novel class of Cdc25 inhibitors [55]. PM-20 (18) is selective for Cdc25A with an IC50 value of 1 mM. Furthermore, PM-20 inhibits the growth of several human tumor cells, especially Hep3B cells with an IC50 of 0.7 mM. More recently several Cdc25 phosphatase inhibitors with micromolar activities were discovered from structure-based virtual screening [56]. The most active of them was compound 19, which inhibits Cdc25A and Cdc25B with IC50 of 0.8 and 2.0 mM, respectively.
SHP-2

SHP-2 is a nonreceptor PTP that mediates cell signaling by growth factors and cytokines acting via the RAS/MAPkinase pathway [40]. Consistent with its overall role in cell signaling, mutations of the SHP-2 gene (PTPN11) can cause hyperactivation of its catalytic activity and have been identied in the Noonan syndrome, a developmental disorder that is frequently associated with short stature [57], and in various childhood leukemia [58]. The incidence of the Noonan syndrome is relatively frequent and affects 1 in 1002500 children, while leukemia accounts for 2% of adult cancers and 1/3 of childhood cancers. Because mutation associated SHP-2 overactivation seems to be the cause of a great portion of these incidents, pharmacological modulation of SHP-2 activity represents an attractive way to prevent disease development in patients bearing these mutations. The exploration of selective SHP-2 inhibitors not only is thought to be of use for the treatment of cancer but may also become the basis for future treatments of infectious diseases. The pentavalent antimony derivative sodium stibogluconate, a known agent against leishmaniasis, has recently been found to inhibit SHP-2 activity [59]. Moreover, sodium stibogluconate is the rst SHP-1/2 inhibitor that reached the clinic. In September 2006 VioQuest Pharmaceuticals initiated a phase I/IIa clinical trial for sodium stibogluconate as chemotherapeutic in patients with advanced malignancies. Although SHP-2 represents an attractive target for the treatment of cancer, only a few SHP-2 inhibitors are known from the literature. Recently the design and synthesis of a compound collection containing SHP-2 inhibitors inspired by furanodictines and the concept of biology-oriented synthesis was reported [60].
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Figure 3

Structures of selected inhibitors of SHP-2, PRLs, MptpA, MptpB, and CD45. IC50 or Ki values are given.

Inhibitor 20 (Figure 3) exhibits an IC50 of 2.5 mM against SHP-2. A screening initiative at the National Cancer Institute resulted in the identication of NSC-87877 (21) [61] and NSC-117199 (22) [62] which inhibit SHPCurrent Opinion in Chemical Biology 2009, 13:272283

2 with IC50 values of 0.32 and 47 mM, respectively. On the basis of the oxindole 22 a focused library was designed and 23 was identied as a selective inhibitor of SHP-2 over SHP-1 and PTP1B with an IC50 value of 0.8 mM for
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Therapeutic potential of phosphatase inhibitors Vintonyak et al. 279

SHP-2 [62]. Recently, Birchmeier et al. performed an in silico screen of low-molecular-weight compounds that may bind to the catalytic site of SHP-2. This analysis resulted in the discovery of PHPS1 (24) as a potent and cell-permeable inhibitor, which is selective for SHP-2 over SHP-1 and PTP1B [63]. PHPS1 efciently inhibits the activation of Erk1/2 by a leukemia-associated mutant variant of SHP-2 and blocks growth of a variety of human tumor cell lines. In addition, Geronikaki et al. reported the synthesis and biological evaluation of thiazolidin-4-one derivatives as a novel class of SHP-2 inhibitors [64]. Compound 25 exhibited the best inhibitory activity with a Ki of 11.7 mM.
PRL

low inhibitory potency with IC50 values ranging from 10 to 50 mM.

MptpA and MptpB

Members of the phosphatase of regenerating liver (PRL) subfamily are DSPs, consisting of PRL-1, PRL-2, and PRL-3, and represent an intriguing group of potential target proteins for the treatment of various cancers [6,65]. PRL-3 overexpression correlates with metastasis in many malignancies and several recent reports suggest that PRLs may play key causal roles in promoting tumor cell motility and invasion. PRL-3 expression is upregulated in the tumors of colorectal cancers metastasized to liver [66], brain, lung, or ovary [67] and is also elevated in pediatric acute myeloid leukemia, Hodgkins lymphoma cells, and prostate cancer. In addition, alteration of PRL1 expression in a number of cancer cell lines leads to changes in cell adherence and invasive properties. The genetic knockdown of PRL-3 with interfering RNA in cancer cells can abrogate cell motility and the ability to metastasize in a mouse model [68]. However, the discovery of PRL phosphatase inhibitors has lagged behind the extensive pharmacological and structural studies on this target protein and only a few inhibitor classes have been reported so far. The bis-benzamidine derivative and antileishmaniasis drug pentamidine (26) were reported to inhibit all three PRL isoforms in vitro and induced tumor shrinkage in a melanoma mouse xenograft model [69]. In addition, recent studies by Lee et al. showed that the combination of pentamidine with the phenothiazine antipsychotic agent chlorpromazine exerts synergistic antiproliferative effects. Pentamidine treatment resulted in chromosomal segregation defects and delayed progression through mitosis, which is consistent with the inhibition of PRL [70]. Another class of PRL-3 inhibitors was identied by screening of chemical libraries by Ahn et al. Benzylidene rhodanine derivatives showed good inhibitory activity against PRL-3. Compound 27 was the most active with an IC50 of 0.9 mM in vitro and showed reduced invasion in cell-based assays [71]. In addition, biavonoids isolated from young branches of Taxus cuspidata inhibit PRL-3 with IC50 values in the low mM range [72]. More recently Park et al. identied 12 novel inhibitors of PRL-3 by means of virtual screening and docking simulations [73]. The discovered inhibitors revealed structural diversity but
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Tuberculosis (TB) continues to be a major cause of morbidity and mortality throughout the world. According to the World Health Organization, one-third of the worlds population is infected with Mycobacterium tuberculosis [74] and about 35 million people are expected to die from TB in the rst 20 years of this century. Because of the increasing occurrence of drug-resistant mycobacteria and the need of the extended use of current drugs, new targets and drugs for therapeutic interventions are in high demand. M. tuberculosis protein tyrosine phosphatase A (MptpA) and MptpB are two enzymes secreted by growing mycobacteria and believed to mediate M. tuberculosis survival in host macrophages by the dephosphorylation of proteins that are involved in interferon signaling, which represents a crucial pathway of the immune system [75]. The genetic knockout of MptpB suppressed growth of M. tuberculosis in activated macrophages and guinea pigs [76] and suggests that MptpA/B could qualify as potential drug targets in the treatment of TB. In addition, inhibitors might also prove useful as probe molecules in chemical biology approaches to dissect the role of MptpA/B phosphatases in hostpathogen invasion. Recently the identication of MptpA inhibitors from screening natural-product inspired compound libraries was reported [77]. Compound 28 is an analog of roseophilin, a natural product found in Streptomyces species and proved to be an inhibitor of MptpA with an IC50 value of 0.9 mM. Utilization of fragment-based library design resulted in the discovery of several novel classes of MptpA inhibitors, among which compound 29 was the most active one, exhibiting a Ki value of 1.6 mM. Additional MptpA inhibitors are based on the indolizine1-carbonitrile scaffold [78]. Using biology-oriented synthesis (BIOS) as an efcient approach to the discovery of new compound classes for medicinal chemistry and chemical biology research, also inhibitors of MptpB were identied [60,79]. The indole derivatives 30, 31, and 32 were at least 100-fold more selective for MptpB and displayed IC50 values in the low mM and high nM range. Alber et al. reported the development of a potent and selective (oxalylamino-methylene)-thiophene sulfonamide inhibitor for MptpB (33) (OMTS) [80]. OMTS (33) has an IC50 value of 440 50 nM and >60-fold specicity for MptpB over six human PTPs. The crystal structure of MptpB in complex with OMTS revealed substantial structural rearrangements of the enzyme, with some residues shifting >27 A relative to the MptpB:PO4 complex. In addition, extensive contacts with the catalytic loop provided a potential basis for inhibitor selectivity. More recently, Ellman et al. developed a substratebased fragment-based approach termed substrate activity screening (SAS) to identify novel PTP inhibitors with submicromolar inhibitory activities [81]. Application of
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this method to MtptB resulted in the discovery of the isoxazole-based inhibitor 34. With a Ki of 220 nM compound 34 is the most potent MptpB inhibitor known from literature to date. Moreover 34 was shown to be selective for mycobacterial (MptpA) against a panel and human PTPs (VHR, TCPTP, CD45, and LAR). Recently, the discovery of a new class of MptpB inhibitors was facilitated by the previously mentioned BIOS approach. A stereoselective solid-phase synthesis of macroline derivatives yielded 120 natural product analogs. Kinetic studies and extensive NMR spectroscopy suggest that inhibitors identied from these macrolines inhibit MptpB not via the substrate-binding site, but rather via an allosteric mechanism yet to be identied [82]. Compounds 35 and 36 selectively inhibit MptpB with IC50 values of 7.0 and 9.6 mM, respectively and did not show inhibition of other phosphatases (MptpA, VE-PTP, PTP1B, TCPTPN2, and Cdc25A) up to a tested concentration of 100 mM. More recently, Terenzi et al. reported the identication of synthetic chalcones as a new class of MptpA inhibitors [83]. Compound 37 turned out to be the most active representative with an IC50 value of 8.4 mM.
CD45

for CD45 over other PTPs including PTP1B. A variety of benzimidazole derivatives have been identied through high-throughput screening as potent CD45 inhibitors and led to the development of TU-572 (39), which inhibits CD45 with an IC50 of 0.28 mM [90]. In addition, 2-amino2-thioxoacetamide derivatives were also reported to inhibit CD45 [91]. However, the most active compound 40 exhibited only moderate activity with an IC50 of 29 mM.

Conclusions
Because of the physiological role of phosphatases as antagonists of kinase activity, there is a high therapeutic potential in targeting these enzymes. Fostered by the enthusiasm and strong indications provided by initial PTP1B inhibitor development, the discovery of phosphatase-modulating agents has progressed steadily in the past years and resulted in the generation of a variety of potent inhibitors. However, because most of the chemical scaffolds that qualify as PP-inhibitors embody phosphate mimics, several challenges remain in current phosphatase-related medicinal chemistry endeavors. These include limited selectivity, limited cell permeability, and insufcient pharmacological properties. As a direct consequence, only a few PP inhibitors have entered clinical trials and new inhibitor classes are in high demand. In addition, relatively few phosphatases are being explored chemically and new targets may be more promising although general limitations such as polarity of activesite-directed inhibitors prevail. Because PPs often function in multienzyme complexes, targeting protein protein interactions either by stabilizers or disruptors might offer a suitable strategy to overcome the current limitations of small molecules directed against the active site of the enzyme. The very recent development of short interfering RNAs (siRNAs) for therapeutic approaches [92,93] may be a viable new alternative in targeting phosphatases. Interfering RNAs lead to the degradation of messenger RNA and facilitate nucleic acids as drugs against otherwise challenging drug targets [94]. Recently, Sirna Therapeutic Inc. claimed to have developed a RNAi directed against PTP1B that potently reduces cellular PTP1B expression levels [95]. However, at this point only time will tell whether any of the discussed therapeutic approaches will succeed. As soon as the rst phosphatase-targeted drug will reach the market a ood of new development programs is to be expected just as for kinases at the end of the 1980s.

CD45 is the rst and the best characterized transmembrane phosphatase. It is expressed in hematopoietic cells and plays a crucial role in T-cell receptor mediated signaling. It regulates the phosphorylation status and thereby activity of Src-family protein tyrosine kinases and their substrates [84]. CD45 has gathered particular attention because its inhibition by antibodies blocks Tcell activation in vitro and graft rejection in mice [85]. These studies highlight the potential value of selective inhibitors of CD45 in the treatment of autoimmune diseases and transplant rejection. Point mutations in the CD45 gene have been associated with autoimmune diseases such as multiple sclerosis [85] and autoimmune hepatitis. In addition the negative regulation of cytokine receptor signaling by CD45 could rationalize the loss of CD45 activity that has been observed in several cancers, such as leukemia. CD45 has been correlated with the proliferation of myeloma cells and could therefore be a potential target for the treatment of multiple myelomas [86]. In addition, recently it was reported that targeted radiotherapy with monoclonal CD45 antibodies induces apoptosis and breaks b-irradiation-resistance and doxorubicin-resistance in leukemia cells [87]. In spite of the potential therapeutic utility of CD45-specic inhibitors, comparatively little has been reported in the literature on their development. The rst comprehensive review on the development of CD45 inhibitors was composed by Lee and Burke [88]. 9,10-Phenanthrenediones (e.g. 38) and 1,2-naphthalenediones have been reported by researchers from AstraZeneca as potent inhibitors of CD45 [89]. Simple structural modications resulted in the identication of compounds that also inhibit T-cell proliferation at low mM concentrations and are selective
Current Opinion in Chemical Biology 2009, 13:272283

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