ovalicin and fumagillin

The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Binding Sites; Cyclohexanes; Drug Design; Edetic Acid; Escherichia coli; Fatty Acids, Unsaturated; Humans; Leucine; Metals; Methionyl Aminopeptidases; Models, Molecular; Molecular Structure; Protease Inhibitors; Protein Folding; Sesquiterpenes; Substrate Specificity

This review focuses on the synthetic strategies used for the construction of fumagillin, ovalicin, and other natural products of this family that are known angiogenesis inhibitors. These compounds are comprised of a cyclohexane framework, two epoxides, and five or six contiguous stereogenic centers. The first total syntheses of fumagillin and ovalicin were reported by Corey in 1972 and 1985, respectively. There were numerous studies directed at these natural products in the decades that followed with many reports appearing in the year 2000 or later. Despite the relatively small size of these molecules, their syntheses highlight the efficient construction of stereogenic centers in organic synthesis.

Topics: Angiogenesis Inhibitors; Cyclohexanes; Epoxy Compounds; Fatty Acids, Unsaturated; Molecular Structure; Sesquiterpenes; Stereoisomerism; Structure-Activity Relationship

Therapies for microsporidiosis in humans are limited, and fumagillin, which appears to be the most broadly effective antimicrosporidial drug, is considered to be moderately toxic. The purpose of this study was to apply an in vitro drug screening assay for Encephalitozoon intestinalis and Vittaforma corneae and an in vivo athymic mouse model of V. corneae infection to assess the efficacy of TNP-470 (a semisynthetic analogue of fumagillin), ovalicin, and eight ovalicin derivatives. TNP-470, ovalicin, and three of the ovalicin derivatives inhibited both E. intestinalis and V. corneae replication by more than 70% in vitro. Another three of the ovalicin derivatives inhibited one of the two microsporidian species by more than 70%. None of the treated athymic mice survived the V. corneae infection, but they did survive statistically significantly longer than the untreated controls after daily treatment with fumagillin administered at 5, 10, and 20 mg/kg of body weight subcutaneously (s.c.), TNP-470 administered at 20 mg/kg intraperitoneally (i.p.), or ovalicin administered at 5 mg/kg s.c. Of two ovalicin derivatives that were assessed in vivo, NSC 9665 given at 10 mg/kg i.p. daily also statistically significantly prolonged survival of the mice. No lesions associated with drug toxicity were observed in the kidneys or livers of uninfected mice treated with these drugs at the highest dose of 20 mg/kg daily. These results thus support continued studies to identify more effective fumagillin-related drugs for treating microsporidiosis.

Topics: Animals; Cyclohexanes; Drug Evaluation, Preclinical; Encephalitozoon; Fatty Acids, Unsaturated; In Vitro Techniques; Male; Mice; Mice, Nude; Microsporidia; Microsporidiosis; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Time Factors; Vittaforma

A promising approach among the numerous efforts to cure cancer is the interruption of the tumour-induced formation of new blood vessels (angiogenesis). By suppressing angiogenesis with drugs, the tumour can neither grow to a life threatening size, nor metastasize. The natural product fumagillin 1 and the structurally related ovalicin 2 are two of the most potent anti-angiogenic compounds. Here, we report the design and synthesis of novel fumagillin and ovalicin analogues lacking reactive epoxy functionalities, which were thought to be responsible for the severe toxic side-effects observed. We also report a new synthetic approach and the determination of the anti-angiogenic properties of these compounds in endothelial cells.

Topics: Angiogenesis Inhibitors; Cyclohexanes; Fatty Acids, Unsaturated; Sesquiterpenes

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.

Topics: Amino Acid Sequence; Aminopeptidases; Angiogenesis Inhibitors; Animals; Calcium; Calmodulin; Cyclohexanes; Endothelium, Vascular; Fatty Acids, Unsaturated; Fibroblast Growth Factors; Humans; Metalloendopeptidases; Methionine; Mice; Molecular Sequence Data; Neoplasm Metastasis; S100 Calcium-Binding Protein A4; S100 Proteins; Sesquiterpenes; Spodoptera; Tumor Cells, Cultured

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer and other human diseases. Fumagillin and ovalicin compose a class of structurally related natural products that potently inhibit angiogenesis by blocking endothelial cell proliferation. A synthetic analog of fumagillin, TNP-470, is currently undergoing clinical trials for treatment of a variety of cancers. A common target for fumagillin and ovalicin recently was identified as the type 2 methionine aminopeptidase (MetAP2). These natural products bind MetAP2 covalently, inhibiting its enzymatic activity. The specificity of this binding is underscored by the lack of inhibition of the closely related type 1 enzyme, MetAP1. The molecular basis of the high affinity and specificity of these inhibitors for MetAP2 has remained undiscovered. To determine the structural elements of these inhibitors and MetAP2 that are involved in this interaction, we synthesized fumagillin analogs in which each of the potentially reactive epoxide groups was removed either individually or in combination. We found that the ring epoxide in fumagillin is involved in the covalent modification of MetAP2, whereas the side chain epoxide group is dispensable. By using a fumagillin analog tagged with fluorescein, His-231 in MetAP2 was identified as the residue that is covalently modified by fumagillin. Site-directed mutagenesis of His-231 demonstrated its importance for the catalytic activity of MetAP2 and confirmed that the same residue is covalently modified by fumagillin. These results, in agreement with a recent structural study, suggest that fumagillin and ovalicin inhibit MetAP2 by irreversible blockage of the active site.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Anti-Bacterial Agents; Catalytic Domain; Cell Division; Cell Line; Cyclohexanes; Cysteine; Endothelium, Vascular; Fatty Acids, Unsaturated; Histidine; Humans; Metalloendopeptidases; Mutagenesis, Site-Directed; Neovascularization, Pathologic; Recombinant Proteins; Sesquiterpenes; Spodoptera; Transfection

Angiogenesis, the formation of new blood vessels, is essential for tumor growth. The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer. Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers. The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown.. Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2). This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation. Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation. There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs. A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined. A significant correlation was found between the two activities.. The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.

Topics: Affinity Labels; Amino Acid Sequence; Aminopeptidases; Animals; Antibiotics, Antineoplastic; Biotin; Cattle; Cells, Cultured; Cyclohexanes; Endothelium, Vascular; Eukaryotic Initiation Factor-2; Fatty Acids, Unsaturated; Metalloendopeptidases; Mice; Molecular Sequence Data; Molecular Structure; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Phosphorylation; Recombinant Proteins; Sesquiterpenes; Yeasts