Small Molecule Inhibitors of Dual Specificity Protein Phosphatases

Katharine E Pestell1, Alexander P Ducruet1, Peter Wipf2 and John S Lazo*,1

1Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 2Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

One hallmark of neoplasia is the deregulation of cell general acid on a surface loop (Cohn, 1989; Zhang et cycle control mechanisms, which is secondary to altered al., 1994). In contrast, there are no similarities protein phosphorylation. Dual speci®city protein phos- between the PPase and PTPase catalytic domains phatases uniquely dephosphorylate both phosphoserines/ (Charbonneau et al., 1989; Cohn, 1989). This is threonines and phosphotyrosines on the same protein manifest in the dierent mechanisms of catalysis: substrate. As a class they regulate intracellular signaling while the PPases require metal ions and eect catalysis through the mitogen activated and stress activated by direct attack of an activated water molecule at the kinases and govern cellular movement through G1/S phosphorous ion of the substrate (Barford, 1996), the and G2/M cell cycle checkpoints by aecting the activity PTPases proceed via a covalent phosphocysteine of cyclin-dependent kinases. In particular, the Cdc25 intermediate (Zhang, 1997). The macromolecular phosphatases, which dephosphorylate cyclin-dependent structural organization of the enzymes also diers: kinases, are overexpressed in many human tumors and while the PPases exist in vivo as holoenzymes with this increased expression is associated with a poor multiple subunits; the PTPases are monomers contain- prognosis. In addition to expression levels, the intracel- ing a catalytic domain of up to 250 residues ¯anked lular activity of Cdc25 phosphatases is determined by by amino or carboxyl terminal extensions with their subcellular distribution and physical proximity to targeting or regulatory functions. substrates. Small molecules that either inhibit the The importance of tyrosine phosphorylation in catalytic activity or alter the subcellular distribution of cellular regulation is revealed by its role in a wide these dual speci®city protein phosphatases could provide range of cellular pathways. For example, tyrosine eective tools to interrogate the role of phosphorylation phosphorylation is a key reaction in the initiation and pathways and may aord new approaches to the propagation of insulin action (Myers and White, 1996; management of cancer. Oncogene (2000) 19, 6607 ± White and Kahn, 1994). Thus, PTP1B (Ahmad et al., 6612. 1995; Kenner et al., 1996; Kole et al., 1996), LAR (Kulas et al., 1995; Zhang et al., 1996) and PTBa Keywords: tyrosine phosphatases; dual speci®city phos- (Lammers et al., 1997; Moller et al., 1995) have been phatases; cdc25; cancer implicated as negative regulators of insulin receptor signaling. Consequently, protein phosphatase inhibitors may have use in the treatment of diabetes and Introduction obesity. Other PTPases have been associated with X- linked recessive myotubular myopathy, a congenital Approximately one-third of all intracellular proteins in muscle disorder (Kioschis et al., 1996) and in Lafora's mammalian cells are phosphorylated; this is a funda- disease, an autosomal recessive form of progressive mental regulatory process catalyzed by protein kinases. myoclonus epilepsy characterized by seizures and Most cellular protein phosphorylation occurs on serine cumulative neurological deterioration (Minassian et and threonine residues with tyrosine phosphorylation al., 1999). accounting for just 0.01 ± 0.05% of total protein The mitogen (MAPK) and stress activated protein phosphorylation (Hunter and Sefton, 1980). Dephos- kinase (SAPK) signaling pathways, which control phorylation of these residues is catalyzed by two major cellular proliferation, dierentiation, development, protein phosphatase families: phosphoserine/phospho- in¯ammatory responses and apoptosis, are regulated threonine speci®c protein phosphatases (PPases; e.g., by both kinases and phosphatases. Examples of such PP1 and PP2A) and protein-tyrosine phosphatases phosphatases are the DSPases VHR, which terminate (PTPases; e.g., PTP1B), which include the dual the catalytic activity of the extracellular regulated speci®city protein phosphatases (DSPases; e.g., VHR kinases (ERKs) (Todd et al., 1999) and MKP-5, which and Cdc25) that not only dephosphorylate phospho- inactivates p38 and SAPK/JNK (Tanoue et al., 1999). tyrosine but also phosphothreonine and phosphoserine Phosphatases may also represent novel targets for on the same protein substrate. the development of antibiotics. The pathogenic bacter- The PTPases have similar conserved structural ium Yersinia encodes a PTPase essential for its elements: a phosphate-binding loop, which contains virulence (Bolin and Wolf-Watz, 1988). Yersinia pestis the signature motif C(X)5R in a catalytic crevice was the pathogen responsible for the Bubonic Plague between a b strand and a helix, and frequently a or Black Death (Butler, 1985), which is predicted to have killed 200 million during human history (Bruba- ker, 1991). The bacterial pathogen Salmonella typhi- murium causes typhoid fever and food poisoning; it *Correspondence: JS Lazo, Department of Pharmacology, Biomedical Sciences Tower E1340, University of Pittsburgh, 3550 secretes PTPases that are required for its full display of Terrace and DeSoto Streets, Pittsburgh, PA 15261, USA virulence (Kaniga et al., 1996). PTPase activity has also Protein phosphatase inhibitors KE Pestell et al 6608 been detected in parasites such as Leishmania donovani Regulation and structure of Cdc25 (Cool and Blum, 1993), Trypanosoma brucei and Trypanosoma cruzi (Bakalara et al., 1995a,b), although Amino acid sequence alignment of the three mammalian it is less clear if these activities are important for their Cdc25 proteins reveals two general domains: the C- pathogenesis. terminus, which comprises approximately one-third of the protein, is highly conserved and contains the catalytic activity; and the N-terminus, which has a Cyclin-dependent protein phosphatases and cancer regulatory role and is quite variable. The expression and One hallmark of oncogenic transformation is the activity of the Cdc25s are is regulated at multiple levels. deregulation of cell cycle control. Phosphorylation is For example, the expression of Cdc25B protein peaks in an important regulatory mechanism for the cyclin late S and G2 phase, when it is thought to be active dependent kinases (Cdk)/cyclin complexes that drive (Garner-Hamrick and Fisher, 1998). In addition, the cell cycle. Inhibitory phosphorylation on Thr and hyperphosphorylation of the N-terminus of Cdc25B Tyr located in the glycine-rich ATP anchor motif of regulates its ability to dephosphorylate cyclin B1/cdk1 cdks results in inactivation (Figure 1). The DSPase (Gabrielli et al., 1997). Excellent reviews on Cdc25 and Cdc25 re-activates cdks via dephosphorylation of the regulation of its expression appear elsewhere (Draetta inhibitory Thr and Tyr residues (Gautier et al., 1991). and Eckstein, 1997; Nilsson and Homann, 2000). Both There are three mammalian Cdc25 proteins, which the expression and subcellular location of Cdc25 are dephosphorylate cdks at distinct phases of the cell aected by DNA integrity. Thus, DNA damage induces cycle: Cdc25A acts on cdk2 and cdk4 in G1/S (Jinno et a ubiquitin- and proteasome-dependent degradation of al., 1994), and Cdc25B and C on cdk1 in G2/M Cdc25A (Bernardi et al., 2000; Mailand et al., 2000), (Lammer et al., 1998; Strausfeld et al., 1991). In resulting in arrest at the G1/S cell cycle checkpoint. addition to their essential role in controlling these cell DNA damage also actives the chk1 pathway, leading to cycle checkpoints, Cdc25s are attractive targets for new phosphorylation of Cdc25C on Ser-216, binding to a 14- therapeutics because: (a) they have a structure that is 3-3 isoform, sequestration in the cytoplasm and physical distinct from other PTPases; (b) they cooperate with separation from its substrate (Figure 1) (Lopez-Girona other oncogenes in oncogenic transformation (Galak- et al., 1999; Peng et al., 1997). This unique form of tionov et al., 1995b); (c) their expression is potentially enzyme-substrate subcellular separation has also been controlled by at least two oncogenes, namely raf1 and proposed as a regulatory mechanism for Cdc25B c-myc (Galaktionov et al., 1995a, 1996; Xia et al., (Davezac et al., 2000). 1999) and, (d) they are overexpressed in many human The crystal structures of the catalytic domains of tumors. Thus, Cdc25A and B are found overexpressed Cdc25A and B but not Cdc25C have been resolved. in non-Hodkin's (Hernandez et al., 1998, 2000), head The active-site loop of Cdc25A showed similarity to and neck (Gasparotto et al., 1997), colon (Dixon et al., the PTPases and this was shallow compared to other 1998); (Galaktionov et al., 1995b), gastric (Kudo et al., PTPases (Fauman et al., 1998). The catalytic site 1997) and non-small cell lung (Wu et al., 1998) cancers. comprised a small a/b domain with a central ®ve- Elevated expression is associated with poor prognosis stranded parallel b sheet with three helices below and in some cancers (Galaktionov et al., 1995b; Hernandez two above (Fauman et al., 1998). The overall structure et al., 1998, 2000; Kudo et al., 1997). and folding of the Cdc25B catalytic domain was similar to that of Cdc25A, although the Cdc25B domain was able to bind sulfate or other oxyanions in the catalytic site whereas the Cdc25A domain was unable to bind oxyanions (Reynolds et al., 1999). In addition, there is a major dierence between the Cdc25A and Cdc25B crystal structures at their C- termini. In Cdc25B the ®nal C-terminal residues lie along the primary protein molecule producing a cleft extending from the catalytic site. In contrast, the analogous C-terminal region in Cdc25A extends away from the main body of the protein and is disordered (Reynolds et al., 1999). A second cysteine-containing cleft has been identi®ed in the Cdc25B structure that could be important for catalysis and, thus, as a region for small molecule disruption.

Small molecule inhibitors of Cdc25 Because of the apparent role of Cdc25 in neoplasia and the dierences between the catalytic domains of Cdc25 and other PTP/DSPases, there has been considerable interest in identifying speci®c Cdc25 inhibitors. The Figure 1 Post-translational and spatial regulation of Cdc25C. most common in vitro assays used to probe for The kinases, wee1 and myt1, inactive cdk1 by phosphorylating potential small molecule inhibitors of Cdc25 enzyme Thr-14 and Tyr-15. Dephosphorylation of cdk1 by nuclear Cdc25C activates the kinase. The chk1 pathway is activated by activity involve recombinant, epitope-tagged Cdc25 DNA damage leading to Cdc25C phosphorylation on Ser-216 and that can be readily puri®ed and synthetic substrates, cytoplasmic sequestration due to 14-3-3 protein binding such as o-methyl ¯uorescein phosphate (OMFP)

Oncogene Protein phosphatase inhibitors KE Pestell et al 6609 (Ducruet et al., 2000); ¯uorescein diphosphate (FDP) isoforms in vitro or within cells. Thus, the ortho- (Rice et al., 1997) or; p-nitrophenyl phosphate (pNPP) quinone nocardione A inhibited Cdc25B phosphatase (Koufaki et al., 1996). The choice of substrate, activity in vitro with an IC50 of 17 mM (Otani et al., however, can aect the enzyme kinetics, as can the 2000). In addition to its anti-Cdc25 activity, nocar- domains of the enzyme used. Thus, truncation of either dione A has antifungal activity and is cytotoxic to the amino or carboxyl terminus can markedly alter the HeLa cervical and SBC-5 non-small cell lung carcino- kinetic properties of the enzyme. The phosphorylation mas (Otani et al., 2000). Dnacins, which are benzoqui- status of Cdc25 is also important (Draetta and noid antibiotics with antitumor activity, inhibit Cdc25B Eckstein, 1997). Small molecular weight surrogate non-competitively (Horiguchi et al., 1994), although substrates are popular as substitutes for the more they also cause direct DNA damage through the physiological cdk substrates, because cdks need to be generation of superoxide radicals (Horiguchi et al., isolated, phosphorylated and bound to cyclins. Un- 1994). Naphthoquinones based on menadione or fortunately, small phosphopeptides modeled around vitamin K3 also inhibit Cdc25 phosphatases. Mena- the known cdk dephosphorylation region are poor dione was originally investigated, because it was known substrates for Cdc25 and, thus, are seldom used (Cans to have a broad range of antitumor activity, it aected et al., 1999). Consequently, it is highly desirable to the phosphorylation status and activity of tyrosine validate any inhibition seen with a nonprotein phosphatases, and it resembled mechanism-based substrate in vitro with a cellular or in vivo observation phosphatase inhibitors (Ham et al., 1997). Ham et al. especially because the requisite catalytic acid may demonstrated that menadione could irreversibly inhibit actually be on the protein substrate (Chen et al., 2000). Cdc25A activity and compete with a known active site Several metal anions, most notably vanadate, are Cdc25 inhibitor, suggesting that its inhibition was due well-established, nonspeci®c inhibitors of the PTPase to covalent modi®cation of the active site (Ham et al., family, including Cdc25. Additional and sometimes 1997). Cellular studies with a vitamin K analog, more potent or selective Cdc25 inhibitors have Compound 11, indicate blockage of cell cycle progres- emerged from studies of natural products (Eckstein, sion at the G1/S transition, an increase in cdk2 2000) and several of these are illustrated in Figure 2. phosphorylation and a concomitant decrease in cdk2 A deep-water sponge from the Ircinia genus produces activity, consistent with inhibition of Cdc25A (Ham et sul®rcin, which is a non-speci®c phosphatase inhibitor al., 1998). Another synthetic vitamin K analog, originally identi®ed as an antifungal agent (Cebula et Compound 5, was independently identi®ed as a al., 1997). Sul®rcin has been used as a basic partial-competitive inhibitor of Cdc25 activity (Tamura pharmacophore for the development of a small analog et al., 2000). Interestingly, Compound 5 causes a time- library with reduced structural and stereochemical dependent, irreversible inhibition of Cdc25 (Tamura et complexity. In this series Cdc25A inhibition seemed to al., 2000). Consistent with inhibition of cellular depend on the presence of a long aliphatic side chain Cdc25A, B and C, Compound 5 increases the tyrosine and not on the sulfate moiety, which could be readily phosphorylation of cdk2 and cdk4, decreases Rb replaced with malonate without any appreciable loss phosphorylation, and produces profound G1 and G2/ in activity (Cebula et al., 1997). Another marine M phase cell cycle arrests (Tamura et al., 2000). sponge product, dysidiolide, was initially found to Both quinolines and isoquinolines have been re- inhibit Cdc25A activity in vitro (Gunsakera et al., ported to inhibit Cdc25 (El-Subbagh et al., 1999; 1996) although subsequent de novo synthesis and re- Fritzen, et al., 2000). We have re-synthesized both the analysis indicated that pure dysidiolide either has quinoline-3-carboxylate and the naphthyridine-3-car- much less (Takahashi et al., 2000) or no Cdc25 boxylate, which were reported to have anti-Cdc25 inhibitory activity (Blanchard et al., 1999). Presumably activity (El-Subbagh et al., 1999) but were unable to the anti-Cdc25 activity found in the original dysidio- demonstrate inhibition with our assay conditions (Rice lide preparation was an unidenti®ed contaminant in et al., 1997; unpublished results). A broad screening the extract. Nonetheless, using dysidiolide as a project allowed Fritzen et al. (2000) to identify pharmacophore, Takahashi et al. (2000) and Peng et isoquinolines with weak in vitro inhibitory activity al. (1998) synthesized compounds that mimicked against Cdc25B. They used a solid phase, combinator- substructures of dysidiolide. Pyrolysis of a cholesteryl ial, synthetic approach to generate several inhibitors of acetate derivative produced Compound 7, which Cdc25B including Compound 12 (Figure 2), which had inhibited Cdc25A activity in the low mM range (Peng an IC50 of 15 mM (Fritzen et al., 2000). Although little et al., 1998). Several derivatives of Compound 7 were has been reported about their speci®city, these developed in an eort to produce a simpler inhibitor tetrahydroisoquiolines might be a useful platform for structure. Compound 15, a dithiocarboniccarboxylic future inhibitor design. acid derivative of cholestanol, was slightly more Natural products have also been used in the design potent that the parental compound. Compound 15, of pharmacophore platforms for combinatorial syn- like other compounds in this series, exhibited growth thetic approaches (Rice et al., 1997). Thus, SC-aad9is inhibitory activity against human cancer cell lines a competitive inhibitor of all 3 human Cdc25 isoforms (Peng et al., 2000). Analogs of vitamin D3, namely in vitro with low mMKi values (Rice et al., 1997). SC- Compound 3b, possess Cdc25A-inhibitory activity in aad9 also inhibits cell cycle progression at both the G1 vitro and cause G1 arrest in HL60 cells, as expected of and G2/M phases in synchronized murine mammary Cdc25A inhibitors (Dodo et al., 2000). carcinoma cells, causes enhanced tyrosine phosphoryla- Quinones also have been found to have activity tion of cdk1, cdk2 and cdk4 and, decreases cdk4 kinase against Cdc25 although, like almost all of the described activity (Tamura et al., 1999). A limited structure- compounds, little is known about their selectivity activity study indicated the critical importance of a against other PTPases or even against other Cdc25 lipophilic nonyl-residue on SC-aad9 presumably due to

Oncogene Protein phosphatase inhibitors KE Pestell et al 6610

Figure 2 Chemical structures of representative published inhibitors of Cdc25. In vitro IC50 values and the Cdc25 isoform are indicated

interactions with a conserved hydrophobic region To explore the active site of Cdc25, Bergnes et al. adjacent to the catalytic site. Anity for this (1999) designed a group of mechanism-based inhibitors hydrophobic region may account for the observed using a four component Ugi reaction. One diamide, inhibition of Cdc25A in vitro with Compound 11, an Compound 13, is the ®rst submicromolar inhibitor of N-methylmorpholino derivative of alkylphosphoether Cdc25 reported to date and has a seven- and 120-fold lipids (Figure 2; Koufaki et al., 1996). These selectivity for Cdc25A compared with VHR and phosphoether lipids have antitumor activity against PTP1B, respectively (Bergnes et al., 1999). Surprisingly, human xenografts (Koufaki et al., 1996). Rigidifying in the context of the Ugi products, the phosphate the SC-aad9 pharmacophore yielded FY21-aa09, which moiety is not essential for inhibition. This ®nding is is slightly more potent as an inhibitor of Cdc25 and reconciled by the inhibitory kinetics: these compounds has improved selectivity for the Cdc25 class of are noncompetitive inhibitors suggesting that they do phosphatases (Ducruet et al., 2000). Consistent with not directly interact with the active site of the enzyme its antiphosphatase activity, FY21-aa09 inhibits MCF- (Bergnes et al., 1999). 7 and MDA-MB-231 breast cancer cell proliferation in While DNA damage causes the redistribution and a vitro and causes cell cycle arrest at the G2/M transition presumptive inactivation of Cdc25C, little is known (Ducruet et al., 2000). about small molecules that might alter the subcellular

Oncogene Protein phosphatase inhibitors KE Pestell et al 6611 distribution of Cdc25A, B or C. The DNA topoisome- inhibitors have been identi®ed, their potency and rase II inhibitor and DNA damaging agent, etoposide, speci®city are quite limited. The intracellular spatial inhibited nuclear localization of Cdc25B in S phase, modulation of Cdc25 provides an additional approach possibly by invoking a sequestration cascade (Woo et to modify the activity of these phosphatases. al., 1999). These studies suggest it may be possible to identify reagents that selectively aect the subcellular distribution of dierent Cdc25 phosphatases in combi- nation with agents causing DNA damage. These would Abbreviations be valuable tools to interrogate the functional role of The abbreviations used are: cdk, cyclin dependent kinase; Cdc25 sequestration. MAPK, mitogen activated protein kinase; SAPK, stress activated protein kinase; DSPases, dual speci®city phosphatases; ERK, extracellular regulated kinase; IC50,med- Conclusions ian inhibitory concentration; PTPases, protein tyrosine phosphatases; PPases, phosphoserine/phosphothreonine phosphatases Cdc25 phosphatases have a vital role in cell proliferation and checkpoint control. There is increasing evidence that aberrant Cdc25 functionality is important Acknowledgments in oncogenesis. Recent crystal structures of the The authors are supported in part by NIH Grant CA catalytic region of Cdc25 indicate their unique 43917, Army Breast Grant DAMD17-97-1-7229, NIH topology and suggest selective active site inhibitors of Training Grant T32 GM08424 (AP Ducruet) and the Fiske these enzymes may be approachable. Although several Drug Discovery Fund.

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Oncogene