o-(chloroacetylcarbamoyl)fumagillol and fumagillin

Angiogenesis inhibition, long studied in the treatment of malignancies, has begun to emerge as a potential therapeutic approach in managing inflammatory arthritis, particularly rheumatoid arthritis. The growth of new vessels is required for the development of the rheumatoid pannus, which then leads to extensive synovial inflammation and joint destruction. Vascular endothelial growth factor is the best studied mediator of angiogenesis, and several therapies have been developed that specifically target this molecule. Several other angiogenesis mediators, such as the angiopoietin-TIE system, hypoxia inducible factor and integrin alpha(V)beta(3), as well as naturally occurring inhibitors of angiogenesis, are also being investigated as potential therapeutic targets. Additionally, there are a number of drugs, including paclitaxel, 2-methoxyestradiol and fumagillin analogs, that might have a role in inhibiting angiogenesis and, thus, in treating proliferative synovitis.

Topics: Angiopoietins; Cyclohexanes; Epoxy Compounds; Fatty Acids, Unsaturated; Humans; Hypoxia-Inducible Factor 1; Integrin alphaVbeta3; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Receptor, TIE-2; Sesquiterpenes; Synovitis; Tubulin Modulators; Valine; Vascular Endothelial Growth Factor A

Fumagillin is an active amebicide and anti-infective isolated from the fungus Aspergillus fumigatus. Since its characterization in 1951, fumagillin has been studied extensively for its anti-infective properties. Although fumagillin is not approved for systemic use in the USA, this compound has one of the highest efficacies for the treatment of microsporidial infections in HIV-positive patients. Fumagillin does exhibit some side effects that have deterred its acceptance as a viable treatment, but the current body of research on the synthesis of novel analogs of this molecule shows an exciting and promising revival of this drug as both an anti-infective and antiangiogenic agent.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Animals; Anti-Infective Agents; Cyclohexanes; Fatty Acids, Unsaturated; Glycoproteins; Humans; Methionyl Aminopeptidases; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes

Angiogenesis is the outgrowth of new vessels from pre-existing ones. Tumour growth and metastasis is dependent on angiogenesis and many stimulatory and inhibitory factors have been described which play an active role in this process. Inhibition of tumour neovasculature may be one strategy to inhibit tumour growth. Naturally occurring inhibitors of angiogenesis have been discovered and synthetic agents have been designed. Many of these inhibitors are currently being evaluated in clinical trials for the treatment of cancer. This review discusses the mechanism of action of these anti-angiogenics as well as a description of the clinical trials in which they are being evaluated.

Topics: Angiogenesis Inhibitors; Animals; Antibodies; Clinical Trials as Topic; Collagen; Cyclohexanes; Endostatins; Endothelial Growth Factors; Fatty Acids, Unsaturated; Humans; Interleukin-12; Lymphokines; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Neoplasms; O-(Chloroacetylcarbamoyl)fumagillol; Peptide Fragments; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Receptors, Vitronectin; RNA, Catalytic; Sesquiterpenes; Suramin; Thalidomide; Triazoles; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Vasculogenic mimicry (VM) is the formation of a blood supply system that confers aggressive and metastatic properties to tumors and correlates with a poor prognosis in cancer patients. Thus, the inhibition of VM is considered an effective approach for cancer treatment, although such a mechanism remains poorly described. In the present study, we examined methionine aminopeptidase‑2 (MetAP2), a key factor of angiogenesis, and demonstrated that it is pivotal for VM, using pharmacological and genetic approaches. Fumagillin and TNP‑470, angiogenesis inhibitors that target MetAP2, significantly suppressed VM in various human cancer cell lines. We established MetAP2‑knockout (KO) human fibrosarcoma HT1080 cells using the CRISPR/Cas9 system and found that VM was attenuated in these cells. Furthermore, re‑expression of wild‑type MetAP2 restored VM in the MetAP2‑KO HT1080 cells, but the substitution of D251, a conserved amino acid in MetAP2, failed to rescue the VM. Collectively, our results demonstrate that MetAP2 is critical for VM in human cancer cells and suggest fumagillin and TNP‑470 as potent VM‑suppressing agents.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Cell Line, Tumor; CRISPR-Cas Systems; Cyclohexanes; Fatty Acids, Unsaturated; Fibrosarcoma; Gene Knockdown Techniques; Humans; Metalloendopeptidases; Methionyl Aminopeptidases; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes

Identification of methionine aminopeptidase-2 (MetAP-2) as the molecular target of the antiangiogenic compound TNP-470 has sparked interest in N-terminal Met excision's (NME) role in endothelial cell biology. In this regard, we recently demonstrated that MetAP-2 inhibition suppresses Wnt planar cell polarity (PCP) signaling and that endothelial cells depend on this pathway for normal function. Despite this advance, the substrate(s) whose activity is altered upon MetAP-2 inhibition, resulting in loss of Wnt PCP signaling, is not known. Here we identify the small G protein Rab37 as a MetAP-2-specific substrate that accumulates in the presence of TNP-470. A functional role for aberrant Rab37 accumulation in TNP-470's mode of action is demonstrated using a Rab37 point mutant that is resistant to NME, because expression of this mutant phenocopies the effects of MetAP-2 inhibition on Wnt PCP signaling-dependent processes.

Topics: Adaptor Proteins, Signal Transducing; Aminopeptidases; Angiogenesis Inhibitors; Animals; Base Sequence; Cell Polarity; Cell Proliferation; Cyclohexanes; Dishevelled Proteins; Embryo, Nonmammalian; Fatty Acids, Unsaturated; Gene Knockdown Techniques; Growth Inhibitors; Human Umbilical Vein Endothelial Cells; Humans; Metalloendopeptidases; Molecular Sequence Data; Mutation; O-(Chloroacetylcarbamoyl)fumagillol; Phosphoproteins; rab GTP-Binding Proteins; Sesquiterpenes; Wnt Signaling Pathway; Zebrafish; Zebrafish Proteins

The fumagillin family of natural products is known to inhibit angiogenesis through irreversible inhibition of human type 2 methionine aminopeptidase (MetAP2). Recently, fumagillin and TNP-470 were reported to possess antimalarial activity in vitro, and it was hypothesized that this inhibition was mediated by interaction with the putative malarial ortholog of human MetAP2. In this report, we have overexpressed and purified to near-homogeneity PfMetAP2 from bacteria, yeast, and insect cells. Although none of the recombinant forms of PfMetAP2 exhibited enzymatic activity in existing assays, PfMetAP2 proteins expressed in both yeast and insect cells were able to bind to fumagillin in a pull-down assay. The interaction between fumagillin and analogs with PfMetAP2 was further demonstrated using a newly established mammalian three-hybrid assay incorporating a conjugate between dexamethasone and fumagillin. Unlike human (Hs)MetAP2, it was found that PfMetAP2 is bound to fumagillin noncovalently. Importantly, a new analog of fumagillin, fumarranol, was demonstrated to interact with PfMetAP2 and inhibit the growth of both chloroquine-sensitive and drug-resistant Plasmodium falciparum strains in vitro. Antiparasite activity of fumagillin and fumarranol was also demonstrated in vivo using a mouse malaria model. These findings suggest that PfMetAP2 is a viable target, and fumarranol is a promising lead compound for the development of novel antimalarial agents.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Antimalarials; Cell Proliferation; Cyclohexanes; Escherichia coli; Fatty Acids, Unsaturated; Female; Humans; Malaria; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Plasmodium falciparum; Protein Binding; Protozoan Proteins; Recombinant Proteins; Saccharomyces cerevisiae; Sequence Alignment; Sesquiterpenes

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.

Topics: Administration, Oral; Aminopeptidases; Animals; Antimalarials; Cyclohexanes; Fatty Acids, Unsaturated; Metalloendopeptidases; Mice; Parasitic Sensitivity Tests; Sesquiterpenes

Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity invitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Baculoviridae; Cloning, Molecular; Crystallography, X-Ray; Cyclohexanes; Encephalitozoon cuniculi; Fatty Acids, Unsaturated; Gene Expression; Methionyl Aminopeptidases; Models, Molecular; Molecular Sequence Data; O-(Chloroacetylcarbamoyl)fumagillol; Protein Structure, Tertiary; Sequence Alignment; Sesquiterpenes

The fumagillin family of natural products inhibits angiogenesis through the irreversible inhibition of the type 2 methionine aminopeptidase (MetAP2). Herein is reported a novel fumagillin analogue named fumarranol. It is shown that, like fumagillin, fumarranol selectively inhibits MetAP2 and endothelial cell proliferation. It is also active in a mouse model of angiogenesis in vivo. Unlike TNP-470, fumarranol does not covalently bind to MetAP2. Fumarranol may serve as a lead for a new class of angiogenesis inhibitors.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Animals; Cattle; Cell Proliferation; Cells, Cultured; Collagen; Crystallography, X-Ray; Cyclohexanes; Drug Combinations; Endothelial Cells; Endothelium, Vascular; Fatty Acids, Unsaturated; Glycoproteins; Laminin; Methionyl Aminopeptidases; Mice; Molecular Structure; Proteoglycans; Sesquiterpenes; 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

Improved methods for discovering small-molecule mechanisms of action are needed. In this issue of Chemistry & Biology, Zhang et al. make clever use of the zebrafish to study the mechanism of the angiogenesis inhibitor fumagillin and reveal that it targets the noncanonical Wnt pathway.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cyclohexanes; Embryonic Development; Fatty Acids, Unsaturated; Metalloendopeptidases; Mice; O-(Chloroacetylcarbamoyl)fumagillol; Phenotype; RNA, Antisense; Sesquiterpenes; Signal Transduction; Wnt Proteins; Wnt-5a Protein; Zebrafish; Zebrafish Proteins

Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the antiangiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos phenocopies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/beta-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.

Topics: Adaptor Proteins, Signal Transducing; Aminopeptidases; Angiogenesis Inhibitors; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Line, Tumor; Cyclohexanes; Dishevelled Proteins; Embryonic Development; Endothelial Cells; Eye Abnormalities; Fatty Acids, Unsaturated; Frizzled Receptors; Humans; JNK Mitogen-Activated Protein Kinases; Metalloendopeptidases; Mice; Molecular Sequence Data; O-(Chloroacetylcarbamoyl)fumagillol; Oligonucleotides, Antisense; Phenotype; Phosphoproteins; Receptors, G-Protein-Coupled; rhoA GTP-Binding Protein; RNA, Small Interfering; Sesquiterpenes; Signal Transduction; Tail; Wnt Proteins; Wnt-5a Protein; Zebrafish; Zebrafish Proteins

Inhibition of angiogenesis is emerging as a promising strategy for the treatment of cancer. In our study reported here, the effects of 4 highly potent methionine aminopeptidase 2 (MetAP2) inhibitors, IDR-803, IDR-804, IDR-805 and CKD-732 (designed by structure-based molecular modeling), on angiogenesis and tumor growth were assessed. Concentrations of these inhibitors as low as 2.5 nM were able to inhibit the growth of human umbilical vein endothelial cells (HUVEC) by as much as 50%, arresting growth in the G1 stage of mitosis. An intracellular accumulation of p21(WAF1/Cip1) protein was also observed. Furthermore, at higher concentrations (25 nM) of these 4 MetAP2 inhibitors, a significant induction of apoptosis was apparent in the same HUVEC cultures. As a result of these findings, the possible anticancer effects of these inhibitors were examined, utilizing the SNU-398 hepatoma cell line. Interestingly, pretreatment with these inhibitors led to an increased number of apoptotic cells of up to 60% or more, compared to untreated controls. Moreover, utilizing an in vivo xenografted murine model, these inhibitors suppressed the growth of engrafted tumor. In conclusion, these 4 inhibitory compounds potently exert an antiangiogenic effect to inhibit the growth of cancers in vivo and could potentially be useful for the treatment of a variety of cancers.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cinnamates; Cyclohexanes; Disease Models, Animal; Endothelial Cells; Epoxy Compounds; Fatty Acids, Unsaturated; Humans; Liver Neoplasms; Metalloendopeptidases; Mice; Mice, Nude; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Transplantation, Heterologous; Umbilical Veins

Modification of fumagillin was conducted to develop MetAP-2 inhibitors with desirable pharmacological properties. Replacement of the C4 side chain by benzyloxime preserves the inhibitory activity against MetAP-2 enzyme. Fumagillin analogues containing the C4 benzyloxime moiety were found to be very sensitive to the nature of the C6 substituent on the inhibition activity of HUVEC proliferation. This lack of correlation between MetAP-2 and HUVEC activities might be due to the cellular metabolism of the compounds by epoxide hydrolase, which is present in the cell. Compound (E)-3d, containing ethylpiperazinyl carbamate at C6 position, exhibited antiangiogenic effects similar to TNP-470 on matrigel plug assay and rat corneal micropocket assay.

Topics: Angiogenesis Inhibitors; Animals; Cell Line; Cyclohexanes; Fatty Acids, Unsaturated; Humans; Mice; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Sesquiterpenes

The pharmacokinetics of CKD-732 (6-O-4-[dimethyl-aminoethoxy)cinnamoyl]-fumagillol x hemioxalate) was investigated in male SD rats and beagle dogs after bolus intravenous administration. The parent compound and metabolites obtained from in vitro and in vivo samples were determined by LC/MS. The main metabolite was isolated and identified as an N-oxide form of CKD-732 by NMR and LC/MS/MS. CKD-732 was metabolized into either M11 or others by rapid hydroxylation, demethylation, and hydrolysis. The blood level following the intravenous route declined in first-order kinetics with T(1/2)beta values of 0.72 to approximately 0.78 h for CKD-732 and 0.92 to approximately 1.09 h for M11 in rats at a dose of 7.5 to approximately 30 mg/kg. In dogs, T(1/2)beta values of CKD-732 and M11 were 1.54 and 1.79 h, respectively. Moreover, AUC values increased dose dependently for CKD-732 and M11 in rats and dogs. The CLtot and Vdss did not change significantly with increasing dose, indicating linear pharmacokinetic patterns. The excretion patterns through the urine, bile, and feces were also examined in the animals. The total amount excreted in urine, bile, and feces was 2.13% for CKD-732 and 1.29% for M11 in rats, and 1.58% for CKD-732 and 2.28% for M11 in dogs.

Topics: Absorption; Angiogenesis Inhibitors; Animals; Area Under Curve; Bile; Biotransformation; Chromatography, High Pressure Liquid; Cinnamates; Cyclohexanes; Dogs; Epoxy Compounds; Fatty Acids, Unsaturated; Feces; In Vitro Techniques; Infusions, Intravenous; Male; Mice; Mice, Inbred ICR; Microsomes, Liver; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Species Specificity; Tissue Distribution

The protein processing enzyme, methionine aminopeptidase-2 (MetAP-2), has been identified as a molecular target of fumagillin and its derivative, TNP-470, compounds known to inhibit endothelial cell proliferation and angiogenesis. A high-throughput screening program was undertaken to identify selective, reversible inhibitors of MetAP-2 in an attempt to discover structurally novel anti-angiogenic agents for potential therapeutic use in oncology. Approximately 90 small-molecule, reversible, selective inhibitors of rhMetAP-2 were identified. The most potent of these compounds contained a singly-substituted triazole moiety which exhibited an IC50 of 8 nM (95% confidence limits 5 to 13 nM) and was highly selective for MetAP-2 over MetAP-1 (approximately 60-fold difference in IC50 values). Unlike fumagillin, these MetAP-2 inhibitors failed to significantly inhibit growth factor-stimulated endothelial cell (HUVEC) proliferation or to suppress angiogenesis in the in vitro aortic ring explant model of microvessel outgrowth. The MetAP-2-inhibitory activity of these compounds was dependent on the divalent cation used as the metalloenzyme activating cofactor for MetAP-2. These inhibitors were identified using cobalt(II)-activated recombinant human MetAP-2 for screening compound libraries. When manganese (Mn2+) was substituted for cobalt following EDTA treatment and extensive dialysis of the MetAP-2 protein, these inhibitors were significantly less potent (40-fold increase in IC50) as inhibitors of MetAP-2. These results support the recent hypothesis that cobalt may not be the relevant divalent metal ion cofactor for MetAP-2 in cells and may explain the observed absence of cell-based activity using potent triazole inhibitors of cobalt-activated MetAP-2.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Aorta; Cell Proliferation; Cyclohexanes; Drug Evaluation, Preclinical; Endothelium, Vascular; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glycoproteins; Humans; Metalloendopeptidases; Methionyl Aminopeptidases; Neovascularization, Physiologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Structure-Activity Relationship; Umbilical Veins

A concise, diastereoselective synthesis of (+/-)-fumagillol (3) and formal, enantioselective syntheses of the potent angiogenesis inhibitors fumagillin (1) and TNP-470 (2) are reported. The origin of asymmetry is a highly diastereoselective Diels-Alder reaction using a diene with a chiral oxazolidinone auxiliary. The stereochemical course of a key conjugate addition reaction is controlled by the cup-shaped architecture of a cis-fused bicyclic enal. Other key steps include a facile hetero-Claisen rearrangement and a site-selective Sharpless epoxidation.

Topics: Bridged Bicyclo Compounds; Cyclohexanes; Fatty Acids, Unsaturated; Molecular Conformation; Molecular Structure; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Stereoisomerism

Fumagillin is a potent anti-angiogenic drug used in cancer treatments. It is also one of the few molecules active against the Enterocytozoon and Encephalitozoon parasites responsible for various clinical syndromes in HIV-infected or immunosuppressive treated patients. Its toxicity, however, makes desirable the design of more specific molecules. The fumagillin target, as anti-angiogenic agent, is the methionine aminopeptidase, an ubiquitous metallo-enzyme responsible for the removing of the N-terminal methionine in nascent proteins. By analogy, it has been proposed that this enzyme could also be the target in the parasites. As a first approach to verify this and to determine if it would be possible to design a more specific derivative, we have built a homology model of the E. cuniculi aminopeptidase. The charges of the two cobalt ions present in the active site and of the side-chains involved in their binding were computed using ab-initio methods. A preliminary comparison of the interactions of the fumagillin and of a related compound, the TNP-470, with both the human and the parasitic enzymes strongly support the hypothesis that the parasitic aminopeptidase is indeed the target of the fumagillin. It also suggests that the TNP-470 interact identically with both enzymes while there could be small differences in case of the fumagillin.

Topics: Amino Acid Sequence; Aminopeptidases; Angiogenesis Inhibitors; Antiprotozoal Agents; Binding Sites; Cobalt; Cyclohexanes; Drug Design; Fatty Acids, Unsaturated; Humans; Methionyl Aminopeptidases; Microsporidia, Unclassified; Models, Molecular; Molecular Sequence Data; O-(Chloroacetylcarbamoyl)fumagillol; Protein Binding; Sequence Alignment; Sesquiterpenes; Static Electricity

Inhibition of angiogenesis has emerged as a key focus for the treatment of cancer, necessitating a better understanding of the downstream molecular targets of angiogenesis inhibitors. Endostatin, thrombospondin-1, fumagillin, and its synthetic derivative, TNP-470, are potent inhibitors of endothelial cell proliferation and migration in culture and of angiogenesis in vivo. To identify targets that mediate the effects of these inhibitors, we compared two-dimensional gel electrophoresis patterns from lysates of treated and untreated human endothelial cells. Among the proteins identified were cofilin and hsp27, two proteins involved in actin dynamics. Western blotting and immunofluorescence experiments confirmed that the phosphorylation states and subcellular localization of these two proteins were affected by all of the inhibitors tested and that treated cells had a more extensive network of actin stress fibers and more numerous focal adhesion plaques compared with untreated cells. Endothelial monocyte activating polypeptide II, another angiogenesis inhibitor, elicited the same response in the actin cytoskeleton and focal adhesions of endothelial cells. This more adherent phenotype may explain the shared ability of these inhibitors to block endothelial migratory signals. Starting with a proteomics approach, we have identified common effector molecules used by a panel of angiogenesis inhibitors that perturb the cytoskeleton to prevent endothelial migration.

Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Angiogenesis Inhibitors; Cell Adhesion; Cell Movement; Cells, Cultured; Cyclohexanes; Cytokines; Cytoskeletal Proteins; Cytoskeleton; Endostatins; Endothelium, Vascular; Fatty Acids, Unsaturated; Heat-Shock Proteins; Humans; Microfilament Proteins; Neoplasm Proteins; O-(Chloroacetylcarbamoyl)fumagillol; Phosphorylation; RNA-Binding Proteins; Sesquiterpenes; Subcellular Fractions; Thrombospondin 1

Novel bicyclic tetrahydropyrano[3,2-d]oxazolones derivatives, analogues of Fumagillin, were synthesised via a stereocontrolled oxidative-rearrangement of furylcarbinols and subsequent treatment with the appropriate isocyanate. These compounds demonstrated potent antiangiogenic activity.

Topics: Allantois; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Chick Embryo; Chorion; Cyclohexanes; Fatty Acids, Unsaturated; Half-Life; Humans; Mice; Molecular Conformation; Neovascularization, Physiologic; O-(Chloroacetylcarbamoyl)fumagillol; Oxazolone; Sesquiterpenes; Structure-Activity Relationship; Tumor Cells, Cultured

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Antiprotozoal Agents; Cyclohexanes; Encephalitozoon; Fatty Acids, Unsaturated; Methionyl Aminopeptidases; Molecular Sequence Data; O-(Chloroacetylcarbamoyl)fumagillol; Polymerase Chain Reaction; Sequence Alignment; Sesquiterpenes; Spores

Topics: Aminopeptidases; Animals; Anti-Bacterial Agents; Cyclohexanes; Disease Models, Animal; Encephalitozoon cuniculi; Encephalitozoonosis; Enzyme Inhibitors; Fatty Acids, Unsaturated; Female; Leucine; Mice; Mice, Nude; O-(Chloroacetylcarbamoyl)fumagillol; Parasitic Sensitivity Tests; Peptides; Sesquiterpenes

New fumagillin analogues were designed through structure-based molecular modeling with a human methionine aminopeptidase-2. Among the fumagillin analogues, cinnamic acid ester derivative CKD-731 showed 1000-fold more potent proliferation inhibitory activity on endothelial cell than TNP-470.

Topics: Amino Acid Sequence; Aminopeptidases; Angiogenesis Inhibitors; Animals; Cattle; Cell Line; Cinnamates; Cyclohexanes; Drug Design; Epoxy Compounds; Fatty Acids, Unsaturated; Growth Inhibitors; Humans; Leukemia P388; Metalloendopeptidases; Mice; Models, Molecular; Molecular Sequence Data; Sequence Homology, Amino Acid; Sesquiterpenes; Structure-Activity Relationship

We evaluated the effects of angiogenesis inhibitor, TNP-470, on hepatocellular carcinoma (HCCs) induced by a choline-deficient L-amino acid defined (CDAA) diet in rats. Male Fisher 344 rats were fed CDAA for 68 weeks. Rats were treated by subcutaneous injection of TNP-470 (15 mg/kg) or saline (control) three times per week from 53 to 68 weeks. At the end of the experiment, we determined the frequency and size of HCCs and glutathione S-transferase placental form (GSTP)-positive lesions, histology of liver cirrhosis, liver function, and liver weight per body weight. We also determined, using histologic and immunohistochemical semiquantification analyses, the degree of vascularity, apoptosis and proliferation in HCC and adjacent tissues. Treatment with TNP-470 resulted in a reduction of the size and frequency of HCC compared to untreated rats. However, TNP-470 did not influence the histology of liver cirrhosis and liver function. The liver weight per body weight of TNP-470-treated rats was slightly heavier in comparison with that of the controls. Treatment with TNP-470 significantly reduced tumor vascularity relative to the controls. There were no significant differences in the Ki-67 labeling index of HCCs between TNP-470 treated and control rats. The frequency of apoptotic hepatoma cells in TNP-470-treated rats was higher than in control rats. Our results indicate that TNP-470 suppresses the progression of CDAA-induced HCCs in rats through inhibition of angiogenesis, and suggest that TNP-470 might be useful clinically for HCCs.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Carcinoma, Hepatocellular; Choline; Cyclohexanes; Diet; Fatty Acids, Unsaturated; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Rats, Inbred F344; Sesquiterpenes

The antitumor and antimetastatic effects of TNP-470, an angiogenesis inhibitor, on human gastrointestinal tumors xenotransplanted into nude mice were investigated. When two gastric cancer (MT-2 and MT-5) and two colon cancer (TK-4 and TK-13) xenografts are transplanted orthotopically into nude mice, liver metastasis develops 6 weeks after transplantation. TNP-470 30 mg/kg had a significant inhibitory effect on primary tumor growth of gastric cancers when given on alternate days from 7 days after transplantation. However, when given from 10 days or 14 days after transplantation, no inhibitory effect on the growth of any tumor xenograft was observed. In contrast, liver metastasis of each xenograft type was inhibited significantly by TNP-470. The effect of TNP-470 on prognosis was investigated using a hepatic metastatic model of rat hepatoma. Although all untreated rats that received AH-130 cell implants died within one month of massive hepatic metastasis, >50% of rats treated with TNP-470 survived for 4 months. The number of apoptotic cells in hepatic metastatic foci was significantly increased by TNP-470 administration. These results suggest that TNP-470 may provide a new approach to the treatment of digestive organ malignancies.

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Body Weight; Cyclohexanes; Digestive System Neoplasms; Fatty Acids, Unsaturated; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Organ Size; Rats; Sesquiterpenes; Transplantation, Heterologous

Relatively few effective compounds are available for treating microsporidiosis in humans. In this study, several compounds were assayed for activity against Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) and Vittaforma corneae Shadduck, Meccoli, Davis et Font, 1990 in vitro. Of the benzimidazoles tested, albendazole was most effective and the MIC50 values were 8.0 ng/ml and 55.0 ng/ml for E. intestinalis and V. corneae, respectively. Fumagillin and its analogue, TNP-470 were nearly equally effective against both E. intestinalis and V. corneae. The MIC50 values of fumagillin were 0.52 ng/ml and 0.81 ng/ml, and the MIC50 values of TNP-470 were 0.35 ng/ml and 0.38 ng/ml for E. intestinalis and V. corneae, respectively. In addition, 12 of 44 purines and pteridines with putative tubulin binding activity that were synthesized at Southern Research Institute (SRI), inhibited microsporidial replication by more than 50% at concentrations that were not toxic to the host cells. Several chitin synthesis/assembly inhibitors inhibited growth of the microsporidia in vitro but were toxic for the host cells making it difficult to interpret the results. One exception was lufenuron, which caused no significant toxicity to the host cells and expressed approximate MIC50 values of 2.95 micrograms/ml and 6.3 micrograms/ml against E. intestinalis and V. corneae, respectively. These results warrant further studies on albendazole, fumagillin, TNP-470, lufenuron, and the selected SRI purines and pteridines for developing therapeutic strategies for microsporidiosis.

Topics: Albendazole; Animals; Antiprotozoal Agents; Benzamides; Benzimidazoles; Cell Line; Cyclohexanes; Drug Evaluation, Preclinical; Encephalitozoon; Fatty Acids, Unsaturated; Humans; Kidney; Microsporida; Nosema; O-(Chloroacetylcarbamoyl)fumagillol; Pteridines; Purines; Rabbits; Sesquiterpenes; Time Factors

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Antibiotics, Antineoplastic; Binding Sites; Cattle; Cyclohexanes; Fatty Acids, Unsaturated; Humans; Kinetics; Mammals; Metalloendopeptidases; Methionyl Aminopeptidases; Molecular Sequence Data; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Saccharomyces cerevisiae; Sequence Alignment; Sequence Homology, Amino Acid; Sesquiterpenes

Presently, the two most commonly used drugs for treating microsporidiosis in persons with AIDS are albendazole and fumagillin. Albendazole is effective for treating disseminated infections due to Encephalitozoon spp. but is variably effective against Enterocytozoon bieneusi infections. Fumagillin is highly effective when used topically to treat ocular infections with Encephalitozoon hellem or Encephalitozoon intestinalis but is too toxic for systemic use. In this study, the fumagillin analog TNP-470 was assayed for antimicrosporidial activity in vitro. The MICs of TNP-470 at which 50% of isolates were killed (MIC50s) were 0.35 +/- 0.21 and 0.38 +/- 0.11 ng/ml for E. intestinalis and Vittaforma corneae, respectively, and were similar to the MIC50s of fumagillin for these organisms, which were 0.515 +/- 0.002 and 0.81 +/- 0.014 ng/ml, respectively. The MIC50 of albendazole for E. intestinalis was 8.0 +/- 4.23 ng/ml, significantly less (P < 0.01) than its MIC50 for V. corneae, which was 55.0 +/- 7.07 ng/ml. TNP-470 inhibited replication of E. intestinalis in RK-13 cells if it was given at the same time as infection or if treatment was initiated 7 days later. In addition, treatment of the infected cultures with TNP-470 at a dose of 10 ng/ml for 2 weeks, followed by discontinuation of the drug treatment, resulted in no significant increase in E. intestinalis shedding during the following 3 weeks in culture. Because TNP-470 acts against both E. intestinalis and V. corneae, and because TNP-470 was found by others to be less toxic in vivo, TNP-470 may be a promising new drug for the treatment of microsporidiosis.

Topics: Albendazole; Animals; Antiprotozoal Agents; Cyclohexanes; Drug Evaluation, Preclinical; Encephalitozoon; Fatty Acids, Unsaturated; Microbial Sensitivity Tests; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes

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

The process of angiogenesis occurs in many physiological states, but it is also essential for the growth of solid tumours and metastasis formation. An abnormal arterial vascularization has been shown in prolactin-secreting pituitary adenomas induced by prolonged treatment with oestrogens in Fischer 344 (F344) rats. It is thought that anti-angiogenic agents might be useful in therapy for these tumours. Fumagillin and its analogue TNP-470 are known to inhibit endothelial cell proliferation selectively, but their effect on lactotroph cell secretory function and prolactinoma formation has not yet been described. The aim of the present study was to examine the effects of fumagillin and TNP-470 on prolactin secretion, and morphological and vascular changes within the anterior pituitary in long-term oestrogen-treated male F344 rats in vivo and in vitro. As expected, 7 weeks after s.c. implantation of Silastic tubes containing 10 mg diethyl-stilboestrol (DES), a very high rise in serum prolactin levels was found. Both angiogenesis inhibitors injected s.c. at doses of 10 mg/kg body weight for 24 days attenuated the stimulatory effect of DES on prolactin production and release. They also diminished prolactin cell density and inhibited cell proliferation expressed as the number of anterior pituitary cells labelled with bromodeoxyuridine (BrdU), but the effect of TNP-470 was minor compared with fumagillin. Both angioinhibitors suppressed neo-vascularization within the anterior pituitary with similar potency but, on the other hand, they did not affect DES-induced increases in prolactin secretion from cultured rat pituitary cells and cell proliferation in vitro. In conclusion, our results provide strong evidence for the anti-tumour and anti-prolactin activity of angiogenesis inhibitors in the experimentally oestrogen-induced pituitary adenoma; this might be mediated indirectly through the inhibition of angiogenesis.

Topics: Animals; Antibiotics, Antineoplastic; Cell Division; Cells, Cultured; Cyclohexanes; Diethylstilbestrol; Fatty Acids, Unsaturated; Injections, Subcutaneous; Male; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Prolactinoma; Rats; Rats, Inbred F344; Sesquiterpenes

The anti-angiogenic activity of AGM-1470, a new synthetic analog of fumagillin isolated from Aspergillus fumigatus, was extensively examined both in vitro and in vivo using four different types of assay and compared to that of the fumagillin parent. Locally administered AGM-1470 inhibited the angiogenesis in the chick embryo chorioallantoic membrane assay and the rat corneal assay. In the rat sponge implantation assay, systemically administered AGM-1470 inhibited angiogenesis induced by basic fibroblast growth factor. Furthermore, in the rat blood vessel organ culture assay, AGM-1470 (1-1,000 ng/ml) was found to selectively inhibit the capillary-like tube formation of endothelial cells with a minimal effect on the non-endothelial cell growth. AGM-1470 showed more potent anti-angiogenic activity and less toxicity than the fumagillin parent. Therefore, AGM-1470 is much better than the fumagillin parent as anti-angiogenic compound.

Topics: Animals; Antibiotics, Antineoplastic; Cornea; Cyclohexanes; Fatty Acids, Unsaturated; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Organ Culture Techniques; Rats; Rats, Inbred Strains; Sesquiterpenes; Veins

Neovascularization is critical for the growth of tumours and is a dominant feature in a variety of angiogenic diseases such as diabetic retinopathy, haemangiomas, arthritis and psoriasis. Recognition of the potential therapeutic benefit of controlling unabated capillary growth has led to a search for safe and effective angiogenesis inhibitors. We report here the synthesis of a family of novel inhibitors that are analogues of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius. We first isolated this fungus from a contaminated culture of capillary endothelial cells. Purified fumagillin inhibited endothelial cell proliferation in vitro and tumour-induced angiogenesis in vivo; it also inhibited tumour growth in mice, but prolonged administration was limited because it caused severe weight loss. Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.

Topics: Animals; Antibiotics, Antineoplastic; Aspergillus fumigatus; Cattle; Cells, Cultured; Chick Embryo; Cyclohexanes; Fatty Acids, Unsaturated; In Vitro Techniques; Mice; Neoplasms, Experimental; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Time Factors