Selective inhibition of endothelial cell proliferation by fumagillin is not due to differential expression of methionine aminopeptidases

The angiogenesis inhibitors fumagillin and TNP-470 selectively inhibit the proliferation of endothelial cells, as compared with most other cell types. The mechanism of this selective inhibition remains uncertain, although methionine aminopeptidase-2 (MetAP2) has recently been found to be a target for fumagillin or TNP-470, which inactivates MetAP2 enzyme activity through covalent modification. Primary cultures of human endothelial cells and six other non-endothelial cell types were treated with fumagillin to determine its effect on cell proliferation. Only the growth of endothelial cells was completely inhibited at low concentrations of fumagillin. MetAP1 and MetAP2 levels in these cells were investigated to determine whether differential enzyme expression plays a role in the selective action of fumagillin. Western blot analysis and RT-PCR data showed that MetAP1 and MetAP2 were both expressed in these different types of cells, thus, ruling out differential expression of MetAP1 and MetAP2 as an explanation for the cell specificity of fumagillin. Expression of MetAP2, but not of MetAP1, is regulated. Treatment of human microvascular endothelial cells (HMVEC) with fumagillin resulted in threefold increases of MetAP2 protein in the cells, while MetAP1 remained constant. Similar upregulation of MetAP2 by exposure to fumagillin was also observed in non-endothelial cells, eliminating this response as an explanation for cell specificity. Taken together, these results indicate that while MetAP2 plays a critical role in the effect of fumagillin on endothelial cell proliferation, differential endogenous expression or fumagillin-induced upregulation of methionine aminopeptidases is not responsible for this observed selective inhibition.     

Methionine aminopeptidases type I and type II are essential to control cell proliferation

The dependence of cell growth on methionine aminopeptidase (MetAP) function in bacteria and yeast is firmly established. Here we report experimental evidence that the control of cell proliferation in mammalian cells is directly linked and strictly dependent on the activity of both MetAP-1 and MetAP-2. The targeted downregulation of either methionine aminopeptidase MetAP-1 or MetAP-2 protein expression by small interfering RNA (siRNA) significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) (70%-80%), while A549 human lung carcinoma cell proliferation was less inhibited (20%-30%). The cellular levels of MetAP-2 enzyme were measured after MetAP-2 siRNA treatment and found to decrease over time from 4 to 96 h, while rapid and complete depletion of MetAP-2 enzyme activity was observed after 4 h treatment with two pharmacological inhibitors of MetAP-2, PPI-2458 and fumagillin. When HUVEC and A549 cells were treated simultaneously with MetAP-2 siRNA and PPI-2458, or fumagillin, which irreversibly inhibit MetAP-2 enzyme activity, no additive effect on maximum growth inhibition was observed. This strongly suggests that MetAP-2 is the single critical cellular enzyme affected by either MetAP-2 targeting approach. Most strikingly, despite their significantly different sensitivity to growth inhibition after targeting of either MetAP-1 or MetAP-2, HUVEC, and A549 cells, which were made functionally deficient in both MetAP-1 and MetAP-2 were completely or almost completely inhibited in their growth, respectively. This closely resembled the observed growth inhibition in genetically double-deficient map1map2 yeast strains. These results suggest that MetAP-1 and MetAP-2 have essential functions in the control of mammalian cell proliferation and that MetAP-dependent growth control is evolutionarily highly conserved.     

Structure of the angiogenesis inhibitor ovalicin bound to its noncognate target, human Type 1 methionine aminopeptidase

Methionine aminopeptidases (MetAPs) remove the initiator methionine during protein biosynthesis. They exist in two isoforms, MetAP1 and MetAP2. The anti-angiogenic compound fumagillin binds tightly to the Type 2 MetAPs but only weakly to Type 1. High-affinity complexes of fumagillin and its relative ovalicin with Type 2 human MetAP have been reported. Here we describe the crystallographic structure of the low-affinity complex between ovalicin and Type 1 human MetAP at 1.1 A resolution. This provides the first opportunity to compare the structures of ovalicin or fumagillin bound to a Type 1 and a Type 2 MetAP. For both Type 1 and Type 2 human MetAPs the inhibitor makes a covalent adduct with a corresponding histidine. At the same time there are significant differences in the alignment of the inhibitors within the respective active sites. It has been argued that the lower affinity of ovalicin and fumagillin for the Type 1 MetAPs is due to the smaller size of their active sites relative to the Type 2 enzymes. Comparison with the uncomplexed structure of human Type 1 MetAP indicates that there is some truth to this. Several active site residues have to move "outward" by 0.5 Angstroms or so to accommodate the inhibitor. Other residues move "inward." There are, however, other factors that come into play. In particular, the side chain of His310 rotates by 134 degrees into a different position where (together with Glu128 and Tyr195) it coordinates a metal ion not seen at this site in the native enzyme.     

p67/MetAP2 suppresses K-RasV12-mediated transformation of NIH3T3 mouse fibroblasts in culture and in athymic mice

In many tumor cells, the activation and activity of extracellular signal-regulated kinases (ERK1/2) are very high because of the constitutive activation of the Ras-mediated signaling pathway. Here, we ectopically expressed the human homologue of rat eukaryotic initiation factor 2-associated glycoprotein, p67/MetAP2, in EGF-treated mouse embryonic NIH3T3 fibroblasts and C2C12 myoblasts and NIH3T3 cell lines expressing the constitutively active form of MAP kinase kinase (MEK) to inhibit the activation and activity of ERK1/2 MAP kinases. In addition, we also ectopically expressed rat p67/MetAP2 in oncogenic Ras-induced transformed NIH3T3 fibroblasts and inhibited their transformed phenotype both in culture and in athymic nude mice possibly by inhibiting angiogenesis. This inhibition of ERK1/2 MAP kinases is due to the direct binding with rat p67/MetAP2, and this leads to the inhibition of activity of ERK1/2 MAP kinases both in vitro and in vivo. Furthermore, expression of p67/MetAP2 siRNA in both NIH3T3 fibroblasts and C2C12 myoblasts causes activation and activity of ERK1/2 MAP kinases. Our results thus suggest that ectopic expression of rat p67/MetAP2 in transformed cells can inhibit the tumorigenic phenotype by inhibiting the activation and activity of ERK1/2 MAP kinases and, thus, that p67/MetAP2 has tumor suppression activity.     

Fumagillin suppresses HIV-1 infection of macrophages through the inhibition of Vpr activity

HIV-1 viral protein R (Vpr) is one of the human immunodeficiency virus type 1 encoded proteins that have important roles in viral pathogenesis. However, no clinical drug for AIDS therapy that targets Vpr has been developed. Here, we have established a screening system to isolate Vpr inhibitors using budding yeast cells. We purified a Vpr inhibitory compound from fungal metabolites and identified it as fumagillin, a chemical already known to be a potent inhibitor of angiogenesis. Fumagillin not only reversed the growth inhibitory activity of Vpr in yeast and human cells, but also inhibited Vpr-dependent viral gene expression upon the infection of human macrophages.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

Angiogenesis inhibitors specific for methionine aminopeptidase 2 as drugs for Malaria and Leishmaniasis

Methionine aminopeptidase 2 (MetAP2) is responsible for the hydrolysis of the initiator methionine molecule from the majority of newly synthesized proteins. We have cloned the MetAP2 gene from the malaria parasite Plasmodium falciparum (PfMetAP2; GenBank accession number AF348320). The cloned PfMetAP2 has no intron, consists of 1,544 bp and encodes a protein of 354 amino acids with a molecular mass of 40,537 D and an overall base composition of 72.54% A + T. PfMetAP2 has 40% sequence identity with human MetAP2 and 45% identity with yeast MetAP2, and is located in chromosome 14 of P. falciparum. The three-dimensional structure of Pf MetAP2 has been modeled based on the crystal structure of human MetAP2, and several amino acid side chains protruding into the binding pocket that differ between the plasmodial and human enzyme have been identified. The specific MetAP2 inhibitors, fumagillin and TNP-470, potently blocked in vitro growth of P. falciparum and Leishmania donavani, with IC(50) values similar to the prototype drugs. Furthermore, in the case of P. falciparum, the chloroquine-resistant strains are equally susceptible to these two compounds.     

Antiparasitic activities of novel,orally available fumagillin analogs

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin based inhibitors against parasitic targets by inhibition of MetAP2.     

Synthesis and antitumor activity of 1,3,4-oxadiazole possessing 1,4-benzodioxan moiety as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,3,4-oxadiazole derivatives containing 1,4-benzodioxan moiety (7a–7q) have been designed, synthesized and evaluated for their antitumor activity. Most of the synthesized compounds were proved to have potent antitumor activity and low toxicity. Among them, compound 7a showed the most potent biological activity against Human Umbilical Vein Endothelial cells, which was comparable to the positive control. The results of apoptosis and flow cytometry (FCM) demonstrated that compound 7a induce cell apoptosis by the inhibition of MetAP2 pathway. Molecular docking was performed to position compound 7a into MetAP2 binding site in order to explore the potential target.     

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC₅₀: 1 μM) led to the most potent compound 27 (IC₅₀: 0.038 μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.     

Eukaryotic initiation factor 2-associated glycoprotein, p67, shows differential effects on the activity of certain kinases during serum-starved conditions

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. P67 has five conserved amino acid residues at the D251, D262, H331, E364, and E459 positions. To determine the roles of these conserved amino acid residues in eIF2alpha phosphorylation during serum-starved conditions, we constitutively expressed D251A, D262A, H331A, E364A, and E459A mutants in rat tumor hepatoma cells. We find that the point mutants D251A, H331A, and E364A lower the levels of eIF2alpha phosphorylation. These low levels of phosphorylation decrease when serum-starved cells are grown in medium containing serum. To understand the mechanism of action of the p67 mutants in eIF2alpha phosphorylation during serum-starvation, we performed detailed biochemical analyses with the D251A mutant. We find that neither the O-GlcNAc modification on the D251A mutant nor the binding of D251A mutant with eIF2gamma has significant effects on eIF2alpha phosphorylation during serum-starved conditions. However, the D251A mutant inhibits p67's activity to suppress the activity of ERK1/2. Our data suggest that both p67 and the D251A mutant bind to ERK1, thus strengthening the idea that p67 regulates the activity of ERK1. During serum-starvation conditions, both PKR and PERK are phosphorylated and the D251A mutant shows increased stability of PERK as well as a slight decrease in its activity. Altogether, our data provide evidence to suggest that p67 modulates the expression and activity of certain eIF2alpha-specific kinases.     

Two methionine aminopeptidases from Acinetobacter baumannii are functional enzymes

Drug resistance in Gram-negative bacteria, such as Acinetobacter baumannii, is emerging as a significant healthcare problem. New antibiotics with a novel mechanism of action are urgently needed to overcome the drug resistance. Methionine aminopeptidase (MetAP) carries out an essential cotranslational methionine excision in many bacteria and is a potential target to develop such novel antibiotics. Two putative MetAP genes were identified in A. baumannii genome, but whether they actually function as MetAP enzymes was not known. Therefore, we established an efficient E. coli expression system for their production as soluble and metal-free proteins for biochemical characterization. We demonstrated that both could carry out the metal-dependent catalysis and could be activated by divalent metal ions with the order Fe(II) ≈ Ni(II) > Co(II) > Mn(II) for both. By using a set of metalloform-selective inhibitors discovered on other MetAP enzymes, potency and metalloform selectivity on the A. baumannii MetAP proteins were observed. The similarity of their catalysis and inhibition to other MetAP enzymes confirmed that both may function as competent MetAP enzymes in A. baumannii and either or both may serve as the potential drug target.     

Nosema ceranae Escapes Fumagillin Control in Honey Bees

Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.     

Methionine aminopeptidase 2 is a new target for the metastasis-associated protein, S100A4

S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.     

Stabilization of P/CAF,as a ubiquitin ligase toward MDM2, suppresses mitotic cell death through p53-p21 activation in HCT116 cells with SIRT2 suppression

We previously reported that the suppression of SIRT2, an NAD + -dependent protein deacetylases, induces p53 accumulation via degradation of p300 and the subsequent MDM2 degradation, eventually leading to apoptosis in HeLa cells. The present study identified a novel pathway of p53 accumulation by SIRT2 suppression in HCT116(p53+/+) cells in which SIRT2 suppression led to escape from mitotic cell death caused by spindle assembly checkpoint activation induced by microtubule inhibitors such as nocodazole but not apoptosis or G1 or G2 arrest. We found that SIRT2 interacts with P/CAF, a histone acetyltransferase, which also acts as a ubiquitin ligase against MDM2. SIRT2 suppression led to an increase of P/CAF acetylation and its stabilization followed by a decrease in MDM2 and activation of the p53-p21 pathway. Depression of mitotic cell death in HCT116(p53+/+) cells with SIRT2 suppression was released by suppression of P/CAF or p21. Thus, the P/CAF-MDM2-p53-p21 axis enables the escape from mitotic cell death and confers resistance to nocodazole in HCT116(p53+/+) cells with SIRT2 suppression. As SIRT2 has attracted attention as a potential target for cancer therapeutics for p53 regulation, the present study provides a molecular basis for the efficacy of SIRT2 for future cancer therapy based on p53 regulation. These findings also suggest an undesirable function of the SIRT2 suppression associated with activation of the p53-p21 pathway in the suppression of mitotic cell death caused by spindle assembly checkpoint activation.     

A new chronological survival assay in mammalian cell culture

c-Abl is activated by oxidative stress but its precise function in cell response to this stress is elusive. Studies of c-Abl-/- osteoblasts revealed that c-Abl played a negative role in the induction of peroxiredoxin I (Prx I, Prdx I), an anti-oxidant protein with tumor suppression activity, but it provided a protective role against oxidative stress. In contrast, Atm, a signaling molecule that interacts with c-Abl and is required for c-Abl activation, served a totally different function. The significance of these findings is discussed here in the context of aging and tumorigenesis and their links to reactive oxygen species.     

Tyrosine Phosphorylation of the p21 Cyclin-dependent Kinase Inhibitor Facilitates the Development of Proneural Glioma

Phosphorylation of Tyr-88/Tyr-89 in the 3₁₀ helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 3₁₀ helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.     

Negative regulation of the protection of eIF2alpha phosphorylation activity by a unique acidic domain present at the N-terminus of p67

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, has protection of eIF2alpha phosphorylation (POEP) activity, and this activity requires lysine-rich domains I and II of p67. Another unique acidic residue-rich domain is also present at the N-terminus of p67. In this study we analyzed the role of this acidic residue-rich domain in POEP activity. Our data revealed that constitutive expression of a mutant form of p67 (D6/2) in mammalian cells resulted in increased POEP activity, and this activity was partially inhibited when second-site alanine substitutions at the conserved amino acids D251, D262, E364, and E459 were introduced in the D6/2 mutant. In contrast, a similar mutation at the conserved H331 position did not show any effect on POEP activity. Individual alanine substitutions at the above conserved amino acids in wild-type p67 did not show any significant effect on POEP activity except the E459 position where alanine substitution caused approximately 50% increase in POEP activity as compared to the wild type. Although, the levels of endogenous p67 and p67-deglycosylase did not correlate with the POEP activity, we found that the D6/2 mutant of p67 was glycosylated at a higher level in mammalian cells as compared to wild-type p67. The increased POEP activity of the D6/2 mutant also correlated with the higher rate of overall protein synthesis in mammalian cells constitutively expressing this mutant form of p67. Taken together, these data suggest that the acidic residue-rich domain present at the N-terminus of p67 may have a negative role in POEP activity.     

The stability of eukaryotic initiation factor 2-associated glycoprotein, p67, increases during skeletal muscle differentiation and that inhibits the phosphorylation of extracellular signal-regulated kinases 1 and 2

Eukaryotic initiation factor 2-associated glycoprotein, p67, protects eIF2 from phosphorylation by its kinases. To understand the roles of p67 during skeletal muscle differentiation of mouse C2C12 myoblasts, we measured the level of p67 during myotube formation. We noticed that the level of p67 increases during myoblast differentiation and this increased level is controlled at the translational stage. The stability of p67 in the myotubes is due to its low turnover rate. The phosphorylation of the extracellular signal-regulated kinases (ERKs 1 and 2) is high in growth-factor-mediated cycling of C2C12 myoblasts and this phosphorylation decreases at 96 h when these myoblasts are grown in differentiation medium. At this time of differentiation, the level of p67 is higher compared to 0 h of differentiation. p67 binds to ERK2 and inhibits its activity in vitro. Taken together, these results suggest that the stability of p67 increases during myotube formation while inhibiting the phosphorylation of ERKs 1 and 2.