The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Systematic gene silencing identified Cryptosporidium nucleoside diphosphate kinase and other molecules as targets for suppression of parasite proliferation in human intestinal cells.

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium. In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium. However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15-30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.

Evidence for a novel function of Awd in maintenance of genomic stability.

The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. Here, we analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. We show that awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, our data demonstrate a novel function for awd in maintenance of genomic stability. Our results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd - induced genomic instability on tumour development and metastasis formation.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization.

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = ß = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites.

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites.

Involvement of nucleotide diphosphate kinase 2 in the reopening of the sensitive period of filial imprinting of domestic chicks (Gallus gallus domesticus).

Filial imprinting is a behavior characterized by the sensitive or critical period restricted to the first few days after hatching. Once the sensitive period is closed, it is widely believed that chicks can never be imprinted under natural conditions. Previously, we showed that the exogenous injection of T3 reopened the sensitive period which was already closed. That study suggested that T3 functioned by way of a rapid non-genomic action; however, the molecular mechanism of how T3 reopens the sensitive period remains unknown. Here, we show that the phosphorylation level of nucleotide diphosphate kinase 2 (NDPK2) was upregulated following T3 injection. Pharmacological deprivation of the kinase activity of NDPK hampered the molecular process prerequisite for the reopening of the sensitive period of filial imprinting. Moreover, it is shown that the kinase activity of NDPK2 participates in the priming process by T3 signaling which endows the potential for learning. Our data indicate that NDPK2 plays a crucial role downstream of T3 action and that its phosphorylation is involved in the non-genomic signaling during imprinting.

Nucleoside mono- and diphosphate prodrugs of 2',3'-dideoxyuridine and 2',3'-dideoxy-2',3'-didehydrouridine.

Despite their close structural similarity to nucleoside analogues such as the anti-HIV drugs AZT and d4T, 2',3'-dideoxyuridine (ddU) and 2',3'-dideoxy-2',3'-didehydrouridine (d4U) are entirely inactive against HIV in their nucleoside form. However, it has been shown that the corresponding triphosphates of these two nucleosides can effectively block HIV reverse transcriptase. Herein we report on two types of nucleotide prodrugs (cycloSal and DiPPro nucleotides) of ddU and d4U to investigate their ability to overcome insufficient intracellular phosphorylation, which may be the reason behind their low anti-HIV activity. The release of the corresponding mono- and diphosphates from these compounds was demonstrated by hydrolysis studies in phosphate buffer (pH 7.3) and human CD4 (+) T-lymphocyte CEM cell extracts. Surprisingly, however, these compounds showed low or no anti-HIV activity in tests with human CD4 (+) T-lymphocyte CEM cells. Studies of the conversion of ddUDP and d4UDP into their triphosphate metabolites by nucleoside diphosphate kinase (NDPK) showed nearly no conversion of either diphosphate, which may be the reason for low intracellular triphosphate levels that result in low antiviral activity.

Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

Diva/BclB regulates differentiation by inhibiting NDPKB/Nm23H2-mediated neuronal differentiation in PC-12 cells.

BACKGROUND: Diva (death inducer binding to vBcl-2 and Apaf-1)/BclB is a Bcl-2 family member, which is known for its function in apoptosis. Diva/BclB has been shown to interact with NDPKB/Nm23H2, which is involved in cellular differentiation. Thus far, there has been no direct evidence of Diva/BclB having a role in differentiation. In the present study, we investigated the expression of Diva/BclB and NDPKB/Nm23H2 during differentiation in PC-12 cell line. RESULTS: Our results show that after differentiation, Diva/BclB expression was decreased and reciprocally, NDPKB/Nm23H2 expression was increased and it translocated into the nucleus. Overexpression of NDPKB/Nm23H2 promoted PC-12 neuronal differentiation by increasing neurite outgrowth and arresting cell cycle progression. There was a concurrent downregulation of Diva/Boo when NDPKB/Nm23H2 was overexpressed, which mirrors the effect of NGF on PC-12 cell differentiation. Overexpression of Diva/BclB did not change the expression level of NDPKB/Nm23H2, but inhibited its nuclear localization. Cells that overexpressed Diva/BclB presented a decreased percentage of differentiated cells and average neurite length was shortened. This was due to an increase in the formation of Diva/BclB and NDPKB/Nm23H2 complexes as well as Diva/BclB and ß-tubulin complexes. Concomitantly, there was a decrease in formation of NDPKB/Nm23H2 and ß-tubulin complexes. Overexpression of Diva/BclB also resulted in a higher percentage of S-phase cells. CONCLUSION: Our results showed a novel role for Diva/BclB in neuronal differentiation. Its downregulation during neuronal differentiation may be necessary to allow NDPKB/Nm23H2 and ß-tubulin interaction that promotes NDPKB/Nm23H2 mediated differentiation.

Activity of nucleoside diphosphate kinase α (NDPK α) capable of binding to outer mitochondrial membrane accounts for less than 10% of total NDPK activity present in cytoplasm of liver cells.

During incubation of a constant volume of rat liver cytosol with an increasing quantity of mitochondrial protein in the presence of 3.3 mM MgCl(2), the binding of nucleoside diphosphate kinase (NDPK) from the cytosol to mitochondrial membranes is described by a saturation curve. The highest bound NDPK activity accounts for less than 9% of the added activity. Analysis of the results suggests that only one NDPK isozyme is bound to the membranes. Western blotting showed it to be NDPK α, a homolog of human NDPK-B. Substrates of NDPK, hexokinase, and glycerol kinase, as well as N,N'-dicyclohexylcarbodiimide and palmitate, did not influence the association of NDPK with mitochondrial membranes. We conclude that the sites of NDPK binding to the outer mitochondrial membrane are not identical to those of hexokinase and glycerol kinase.

AMPK directly inhibits NDPK through a phosphoserine switch to maintain cellular homeostasis.

AMP-activated protein kinase (AMPK) is a key energy sensor that regulates metabolism to maintain cellular energy balance. AMPK activation has also been proposed to mimic benefits of caloric restriction and exercise. Therefore, identifying downstream AMPK targets could elucidate new mechanisms for maintaining cellular energy homeostasis. We identified the phosphotransferase nucleoside diphosphate kinase (NDPK), which maintains pools of nucleotides, as a direct AMPK target through the use of two-dimensional differential in-gel electrophoresis. Furthermore, we mapped the AMPK/NDPK phosphorylation site (serine 120) as a functionally potent enzymatic "off switch" both in vivo and in vitro. Because ATP is usually the most abundant cellular nucleotide, NDPK would normally consume ATP, whereas AMPK would inhibit NDPK to conserve energy. It is intriguing that serine 120 is mutated in advanced neuroblastoma, which suggests a mechanism by which NDPK in neuroblastoma can no longer be inhibited by AMPK-mediated phosphorylation. This novel placement of AMPK upstream and directly regulating NDPK activity has widespread implications for cellular energy/nucleotide balance, and we demonstrate in vivo that increased NDPK activity leads to susceptibility to energy deprivation-induced death.

The advanced glycation end product-lowering agent ALT-711 is a low-affinity inhibitor of thiamine diphosphokinase.

Advanced glycation end products (AGEs) are involved in age-related diseases, including the complications of diabetes and chronic renal impairment with arterial stiffening. Alagebrium chloride (ALT-711) is an AGE-lowering agent with beneficial effects in renal structural and functional parameters in diabetes, decreased diabetes-accelerated atherosclerosis, and age-related myocardial stiffening. ALT-711 exhibits a structural homology to thiamine, and it was suggested to interfere with thiamine metabolism. Thiamine is converted to thiamine diphosphate (TDP) by thiamine diphosphokinase (TDPK). TDP is a cofactor for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase. A decreased activity of these enzymes due to TDP deficiency results in disorders such as beriberi and Wernicke-Korsakoff syndrome. Therefore, we investigated whether ALT-711 is an inhibitor of TDPK. Molecular modeling studies showed that ALT-711 fits into the thiamine-binding pocket of TDPK, and there are three interactions between the thiazolium ring and the enzyme, as well as parallel stacking between the phenyl ring and the indole ring of Trp222B. Enzyme kinetic experiments also showed that ALT-711 dose-dependently decreased TDPK activity with K(i)s, calculated by different experiments and fitting models ranging from 0.88 to 1.09 mM. Fitting of the kinetic data favored mixed-mode inhibition with a major role for competitive inhibition. In summary, our results suggest that ALT-711 is a low-affinity inhibitor of TDPK, but is unlikely to interfere with thiamine metabolism at therapeutic concentrations. However, when new AGE-crosslink breakers based on thiamine are designed, care should be taken that they do not act as more potent competitive inhibitors than ALT-711.

Measurement of human breast tumor cell-secreted shNDPK-B in a murine breast cancer model suggests its role in metastatic progression.

Human breast cancers metastasize early in tumorigenesis and distant lesions, though dormant are very likely extant at the time of diagnosis and treatment in the majority of cases. Removal of primary tumors by surgeons as an imperative of the current treatment approach, also removes inhibitory factors secreted by the primary tumor that had maintained the dormancy of the metastases. We have identified a factor secreted by human breast cancer cells that supports the formation of blood vessels and may be a principal early factor supporting the growth and development of metastases in human disease. Here we demonstrate for the first time that this factor, secreted (s) human (h) nucleoside diphosphate kinase type B (shNDPK-B), product of the nm23-h2 gene, can be detected specifically with high sensitivity (50 pg/ml; 2.5 pM) in an ELISA assay of our own design. We further demonstrate that shNDPK-B is released into the circulation in immunocompromized mice carrying the human breast carcinoma cell MDA-MB-231. These data support the hypothesis that shNDPK-B may be responsible for the early events in angiogenesis supporting both primary and metastatic tumor growth and development.

Inhibition of Nm23H2 gene product (NDPK-B) by angiostatin, polyphenols and nucleoside analogs.

Human breast cancer cells (MDA-MB-435s) secrete a nucleoside diphosphate kinase (NDPK-B) as a phosphoprotein capable of converting diphosphate nucleosides to triphosphate nucleotides for one round in the absence of a phosphoryl donor. Incubation of the partially purified NDPK-B (Nm23-H2 by Western blot) from [gamma32P]Pi-labeled cells with non-radioactive ADP results in the formation of [gamma32P]ATP (Proc. West. Pharmacol. Soc. 44: 61-63, 2001). The presence of a secreted protein that can maintain ATP levels in the vicinity of capillary and lymph vessels may support cancer metastasis in several ways based on the known actions of ATP at P2Y receptors: facilitate intravasation of breast cancer cells that migrate from a solid tumor, support their extravasation at a distal site, and stimulate angiogenesis. The putative role of angiostatin (AS) as an ATP-synthase inhibitor led us to test the notion that AS blocks NDPK-B activity. Addition of commercial AS (kringles 1-4) did not alter enzyme activity. However, AS produced by us and never lyophilized, blocked NDPK activity in a dose-dependent fashion consistent with the notion that extracellular ATP generation by tumor cells may be important to the development of metastases. The ability of 0.5 mg/ml angiostatin to block NDPK-B activity to approximately 75% of control activity compared poorly with the polyphenol inhibitors of. The catechin gallates, theaflavins and ellagic acid inhibited NDPK-B completely with the rank order of potency: EA > theaflavins > EGCG > ECG > PAPS. Our results suggest that the biological activity of angiostatin as a putative metastasis inhibitor may be in part the result of nm23 inhibition and that the production, lyophilization, packaging or storage of commercial angiostatin leads to the alteration of its biological activity against NDPK-B. Ellagic acid is a potent (IC50 = 10.5 microM) NDPK-B inhibitor that may prove useful in elucidating the role of cancer-cell secreted NDPK-B in tumor development.

Synthesis and biological evaluation of nucleoside dicarboxylates as potential mimics of nucleoside diphosphates.

A series of nucleotide analogues wherein the diphosphate moiety has been replaced by a dicarboxylate were synthesized and tested for inhibitory activity against nucleoside diphosphate (NDP) kinase as well as several pathogenic bacterial strains.

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

Peptidic determinants and structural model of human NDP kinase B (Nm23-H2) bound to single-stranded DNA.

Isoform B of human NDP kinase (NDPK-B) was previously identified as a transcription factor stimulating in vitro and ex vivo the transcription of the c-myc oncogene, which involves this enzyme in carcinogenesis. We have studied the enzymatic properties of NDPK-B in the presence of several single-stranded oligonucleotides. We show that the oligonucleotides are competitive inhibitors of the catalytic activity, indicating that the active site acts as a binding template for the anchorage of the oligonucleotide. Furthermore, the presence of a guanine at the 3'-end of several different aptamers increases its affinity 10-fold. To define the surface of the protein contacting the DNA within the nucleoprotein complex, we used single nanosecond laser pulses as the cross-linking reagent and MALDI-TOF mass spectrometry to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Using 11-mer and 30-mer single-stranded oligonucleotides, the same three different nucleopeptides were identified after irradiation of the complexes, indicating a common binding mode for these two aptamers. Taken together, these results allowed us to propose a structural model of NDPK-B bound to single-stranded DNA.

Increased activity of membrane-associated nucleoside diphosphate kinase and inhibition of cAMP synthesis in failing human myocardium.

OBJECTIVE: Chronic heart failure is associated with a decreased responsiveness of the heart to beta-adrenergic receptor agonists. We recently demonstrated a receptor-independent activation of G proteins and modulation of cardiac adenylyl cyclase activity by sarcolemmal membrane-associated nucleoside diphosphate kinase. We wondered whether changes in the activity of nucleoside diphosphate kinase occur in heart failure and contribute to or compensate for the impairment in myocardial receptor-mediated cAMP generation. METHODS: Sarcolemmal membranes were purified from non-failing and failing human left ventricular myocardium. The protein level and activity of nucleoside diphosphate kinase were quantified. The influence of nucleoside diphosphate kinase on adenylyl cyclase activity was determined by measuring the effect of GDP on adenylyl cyclase activity in the absence and presence of nucleoside diphosphate kinase inhibitors. RESULTS: The amount and activity of nucleoside diphosphate kinase in sarcolemmal membranes from failing hearts (n=13) were increased 3- to 4-fold compared to levels in membranes from non-failing myocardium (n=5). This increase in sarcolemmal nucleoside diphosphate kinase activity resulted in a 50% inhibition of adenylyl cyclase activity over a range of GDP and ATP concentrations. CONCLUSION: The amount and activity of nucleoside diphosphate kinase are increased in sarcolemmal membranes of failing human myocardium, resulting in a substantial receptor-independent inhibition of adenylyl cyclase activity.