Antiangiogenic Small Molecule Research Articles

A new anti-angiogenic small molecule, G0811, inhibits angiogenesis via targeting hypoxia inducible factor (HIF)-1α signal transduction

Regulation of hypoxia inducible factor (HIF)-1α stabilization, which in turn contributes to adaptation of tumor cells to hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. We have identified a new small molecule, G0811, as a potent angiogenesis inhibitor that targets HIF-1α signal transduction. G0811 suppressed HIF-1α stability in cancer cells and inhibited in vitro and in vivo angiogenesis, as validated by tube formation, chemoinvasion, and chorioallantoic membrane (CAM) assays. In addition, G0811 effectively decreased the expression of vascular endothelial growth factor (VEGF), which is one of target genes of HIF-1α. However, G0811 did not exhibit anti-proliferative activities or toxicity in human umbilical vein endothelial cells (HUVECs) at effective doses. These results demonstrate that G0811 could be a new angiogenesis inhibitor that acts by targeting HIF-1α signal transduction pathway.

Abstract 3890: Antiangiogenic small molecule antagonists of the anthrax toxin receptor CMG2.

Abstract We have previously shown that the anthrax protective antigen mutant PASSSR can inhibit endothelial cell migration, VEGF and bFGF driven angiogenesis in the corneal micropocket assay, and tumor growth. However anthrax toxin has three reported receptors (CMG2, TEM8, and β1 integrin) that might mediate antiangiogenic effects. Knockdown of β1 integrin in endothelial cells had no effect on PASSSR inhibition of migration and circulating levels of PASSSR in mice are too low for significant integrin occupancy. Using soluble extracellular domain constructs fused to the antibody Fc region to improve pharmacokinetics, we observed that CMG2-Fc inhibits angiogenesis, while TEM8-Fc does not. Similarly, treatment of mice undergoing the corneal micropocket assay with the SR8F7C7 anti-CMG2 antibody resulted in dose-dependent inhibition of bFGF-induced corneal neovascularization, while the L2 anti-TEM8 antibody had no effect at a dose that has potent antitumor activity. Thus, CMG2 blockade, but not TEM8 blockade, can inhibit corneal angiogenesis. To assess the role of CMG2 in angiogenesis in vivo without pharmacologic intervention, we performed the corneal micropocket assay on knockout mice, and observed that knockout mice exhibit a dramatic decrease in angiogenesis vs. wild-type controls. Also, PASSSR does not further inhibit corneal angiogenesis in CMG2−/− mice. This is consistant with the ex vivo observation that CMG2 knockdown reduces the ability of HMVEC to migrate to serum and knockdown cells are no longer inhibited by PASSSR. To allow identification of CMG2 antagonists, we developed a high-throughput FRET-based screening assay. Validation of the assay suggested that tannic acid may inhibit this protein. However, further analysis demonstrated that this activity is attributable not to tannic acid, but instead to a hydrolysis product (PGG) that contaminated our tannic acid sample. We found that PGG inhibits CMG2 with an IC50 of 300 nM, a value consistent with serum levels achieved by antiangiogenic and antitumor dosing. PGG also inhibited endothelial cell migration and angiogenesis in the corneal micropocket assay, demonstrating that this compound can inhibit not only CMG2-PA binding, but also angiogenesis. Further screening of small molecule libraries identified several additional compounds that inhibit endothelial cell migration and angiogenesis in the corneal micropocket assay. In addition, we identified one compound that stimulates both activities. This important result reveals that CMG2 modulators can both stimulate and inhibit angiogenesis, ruling out indirect effects (such as compound toxicity) as a general explanation for the observed effects on angiogenesis. These results demonstrate small molecule modulation of CMG2 and identify potential pharmacoactive leads. Together, these data strongly suggest that small molecule CMG2 antagonists can be used as antiangiogenic therapies. Citation Format: Lorna M. Cryan, P. Christine Ackroyd, Shugeng Cao, Jon Clardy, Kenneth A. Christensen, Michael S. Rogers. Antiangiogenic small molecule antagonists of the anthrax toxin receptor CMG2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2013-3890

Abstract 5292: Identification of antiangiogenic small molecule natural products targeting anthrax toxin receptor 2

Abstract Bacillus anthracis protective antigen (PA), the B-subunit of the binary anthrax toxin, binds to the cellular receptors capillary morphogenesis gene protein 2 (CMG2) and tumor endothelial marker 8 (TEM8) with high affinity. Both CMG2 and TEM8 are expressed on endothelial cells during angiogenesis. We have shown that wild-type PA inhibits both VEGF-induced and bFGF-induced angiogenesis at moderate but statistically significant levels. Structure-activity studies identified a PA mutant that exhibited markedly enhanced inhibition of angiogenesis and also inhibited tumor growth in vivo. This mutant, PASSSR, is unable to undergo normal cellular processing and thus, remains bound to the surface receptor. Further mutation of PASSSR so that it does not bind to these cell surface receptors abolished its ability to inhibit angiogenesis. We conclude that high affinity anthrax receptor ligands, such as PA and PASSSR, are angiogenesis inhibitors, and that anthrax toxin receptors are useful targets for anti-angiogenic therapy. The long-term goal of our research is to determine the extent to which antrax toxin receptor directed therapeutics can control tumor growth and metastasis. Here we show that in addition to inhibition of subcutaneous lung tumors, PASSSR can inhibit the growth of orthotopically implanted breast cancer cells and extend survival in this mouse model. We also sought to identify small molecule natural products that can inhibit the interaction with the anthrax toxin receptor CMG2 and to determine their antiangiogenic and antitumor capacity in vivo. This work was based on the hypothesis that small molecule natural products can inhibit CMG2-ECM interactions required for breast cancer angiogenesis. This work leverages the wide variety of chemical entities generated by endophytic fungi, and the ability of other fungal compounds such as fumagillin to provide interesting insights into angiogenesis inhibitors. Following a high-throughput screen of >10,000 natural product extracts, we identified 48 extracts with significant inhibitory activity, of which ∼0.5% exhibited measurable activity. Of these, 9 exhibited 1:1 binding to CMG2 with IC50 <100 ug/ml total solids. Purification and chemical and in vitro characterization resulting in the identification of six compounds with anti-CMG2 and endothelial cell inhibitory, one of which exhibits antiangiogenic activity. Thus, we have identified several small molecule natural product inhibitors of CMG2 and demonstrated that CMG2 inhibitors can inhibit angiogenesis and breast cancer growth. While none of these molecules is currently suitable as a lead for the development of a breast cancer therapeutic, we believe that these studies confirm that the approach that we have taken is suitable for the development of such a therapeutic and propose to dramatically expand the number of molecules screened for such activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5292. doi:1538-7445.AM2012-5292

Currently Applied Clinical Naturopathy and Phytotherapy?Keystone for Disease Prevention and Therapy

Cancer is a major cause of death worldwide and angiogenesis is critical in cancer progression. Development of new blood vessels and nutrition of tumor cells are heavily dependent on angiogenesis. Thus, angiogenesis inhibition might be a promising approach for anticancer therapy. Anti-angiogenic small molecule and phytochemicals as a cancer treatment approach are focused in these main points; modes of action, adverse effects, mechanisms of resistance and new developments. Treatment with anti-angiogenic compounds might be advantageous over conventional chemotherapy due to the fact that those compounds mainly act on endothelial cells, which are genetically more stable and homogenous compared to tumor cells and they show lower susceptibility to acquired drug resistance (ADR). Targeting the VEGF (vascular endothelial growth factor) signalling pathway with synthetic small molecules inhibiting Receptor Tyrosine Kinases (RTKs) in addition to antagonizing VEGF might be a promising approach. Moreover, beneficial effect of phytochemicals were proven on cancer-related pathways especially concerning anti-angiogenesis. Plant phenolics being an important category of prominent phytochemicals affect different pathways of angiogenesis. Green tea polyphenols (epigallocatechin gallate) and soy bean isoflavones (genistein) are two examples involving an anti-angiogenic effect.

Targeting Angiogenesis By Phytochemicals

Cancer is a major cause of death worldwide and angiogenesis is critical in cancer progression. Development of new blood vessels and nutrition of tumor cells are heavily dependent on angiogenesis. Thus, angiogenesis inhibition might be a promising approach for anticancer therapy. Anti-angiogenic small molecule and phytochemicals as a cancer treatment approach are focused in these main points; modes of action, adverse effects, mechanisms of resistance and new developments. Treatment with anti-angiogenic compounds might be advantageous over conventional chemotherapy due to the fact that those compounds mainly act on endothelial cells, which are genetically more stable and homogenous compared to tumor cells and they show lower susceptibility to acquired drug resistance (ADR). Targeting the VEGF (vascular endothelial growth factor) signalling pathway with synthetic small molecules inhibiting Receptor Tyrosine Kinases (RTKs) in addition to antagonizing VEGF might be a promising approach. Moreover, beneficial effect of phytochemicals were proven on cancer-related pathways especially concerning anti-angiogenesis. Plant phenolics being an important category of prominent phytochemicals affect different pathways of angiogenesis. Green tea polyphenols (epigallocatechin gallate) and soy bean isoflavones (genistein) are two examples involving an anti-angiogenic effect.

Identification of target protein of anti-angiogenic small molecule via chemical proteomics

Identification of target protein of anti-angiogenic small molecule via chemical proteomics

E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models

Tumor angiogenesis is a degenerate process regulated by a complex network of proangiogenic factors. Existing antiangiogenic drugs used in clinic are characterized by selectivity for specific factors. Antiangiogenic properties might be improved in drugs that target multiple factors and thereby address the inherent mechanistic degeneracy in angiogenesis. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family members and their cognate receptors are key players in promoting tumor angiogenesis. Here we report the pharmacologic profile of E-3810, a novel dual inhibitor of the VEGF and FGF receptors. E-3810 potently and selectively inhibited VEGF receptor (VEGFR)-1, -2, and -3 and FGF receptor (FGFR)-1 and -2 kinases in the nanomolar range. Ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 was suppressed along with human vascular endothelial cell growth at nanomolar concentrations. In contrast, E-3810 lacked cytotoxic effects on cancer cell lines under millimolar concentrations. In a variety of tumor xenograft models, including early- or late-stage subcutaneous and orthotopic models, E-3810 exhibited striking antitumor properties at well-tolerated oral doses administered daily. We found that E-3810 remained active in tumors rendered nonresponsive to the general kinase inhibitor sunitinib resulting from a previous cycle of sunitinib treatment. In Matrigel plug assays performed in nude mice, E-3810 inhibited basic FGF-induced angiogenesis and reduced blood vessel density as assessed by histologic analysis. Dynamic contrast-enhanced magnetic resonance imaging analysis confirmed that E-3810 reduced the distribution of angiogenesis-sensitive contrast agents after only 5 days of treatment. Taken together, our findings identify E-3810 as a potent antiangiogenic small molecule with a favorable pharmacokinetic profile and broad spectrum antitumor activity, providing a strong rationale for its clinical evaluation.

Identification and biological activities of a new antiangiogenic small molecule that suppresses mitochondrial reactive oxygen species

Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1α and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

Cilengitide: The First Anti-Angiogenic Small Molecule Drug Candidate. Design, Synthesis and Clinical Evaluation

Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins αvβ3, αvβ5 and α5β1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive αvβ3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor αIIbβ3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany). This article describes the chemical development of Cilengitide, the biochemical background of its activity and a short review about the present clinical trials. The positive anti-angiogenic effects in cancer treatment can be further increased by combination with “classical” anti-cancer therapies. Several clinical trials in this direction are under investigation.

Targeting tumor angiogenesis with TSP-1-based compounds: rational design of antiangiogenic mimetics of endogenous inhibitors.

Inhibitors of angiogenesis are an important addition to conventional chemotherapy. Among different "druggable" angiogenic factors, fibroblast growth factor-2 (FGF-2) is an attractive target for novel therapies because of its intricated involvement in tumor neovascularization, tumor cell proliferation and migration, and the acquisition of resistance to antiangiogenic therapies. FGF-2 bioavailability and activity is affected by several natural ligands, including the endogenous inhibitor of angiogenesis thrombospondin-1 (TSP-1). We hypothesized that the FGF-2-binding sequence of TSP-1 might serve as a template for the development of non-peptide inhibitors of angiogenesis. Computational biology and nuclear magnetic resonance spectroscopy approaches, major investigative tools in the characterizations of protein-protein interaction (PPI), were used to map the residues at the TSP-1/FGF-2 interface. The translation of this three-dimensional information into a pharmacophore model allowed screening a small molecule databases, identifying three FGF-2-binding, antiangiogenic small molecules, mimetic of TSP-1. Pharmacophore-based approaches are thus feasible tools to exploit naturally occurring PPI, by generating a set of lead compounds mimetic of endogenous proteins, as a starting point for the development of novel therapeutic agents.

Targeting tumor angiogenesis with TSP-1-based compounds: rational design of antiangiogenic mimetics of endogenous inhibitors.

Inhibitors of angiogenesis are an important addition to conventional chemotherapy. Among different "druggable" angiogenic factors, fibroblast growth factor-2 (FGF-2) is an attractive target for novel therapies because of its intricated involvement in tumor neovascularization, tumor cell proliferation and migration, and the acquisition of resistance to antiangiogenic therapies. FGF-2 bioavailability and activity is affected by several natural ligands, including the endogenous inhibitor of angiogenesis thrombospondin-1 (TSP-1). We hypothesized that the FGF-2-binding sequence of TSP-1 might serve as a template for the development of non-peptide inhibitors of angiogenesis. Computational biology and nuclear magnetic resonance spectroscopy approaches, major investigative tools in the characterizations of protein-protein interaction (PPI), were used to map the residues at the TSP-1/FGF-2 interface. The translation of this three-dimensional information into a pharmacophore model allowed screening a small molecule databases, identifying three FGF-2-binding, antiangiogenic small molecules, mimetic of TSP-1. Pharmacophore-based approaches are thus feasible tools to exploit naturally occurring PPI, by generating a set of lead compounds mimetic of endogenous proteins, as a starting point for the development of novel therapeutic agents.

NBBA, a synthetic small molecule, inhibits TNF-α-induced angiogenesis by suppressing the NF-κB signaling pathway

Nuclear factor-κB (NF-κB) is a crucial transcription factor that contributes to cancer development by regulating a number of genes involved in angiogenesis and tumorigenesis. Here, we describe ( Z)- N-(3-(7-nitro-3-oxobenzo[ d][1,2]selenazol-2(3 H)-yl)benzylidene)propan-2-amine oxide (NBBA) as a new anti-angiogenic small molecule that targets NF-κB activity. NBBA showed stronger growth inhibition on human umbilical vein endothelial cells (HUVECs) than on the cancer cell lines we tested. Moreover, NBBA inhibited tumor necrosis factor-alpha (TNF-α)-induced tube formation and invasion of HUVECs. In addition, NBBA suppressed the neovascularization of chorioallantonic membrane from growing chick embryos in vivo. To address the mode of action of the compound, the effect of NBBA on TNF-α-induced NF-κB transcription activity was investigated. NBBA suppressed TNF-α-induced c-Jun N-terminal kinase phosphorylation, which resulted in suppression of transcription of NF-κB and its target genes, including interleukin-8, interleukin-1α, and epidermal growth factor. Collectively, these results demonstrated that NBBA is a new anti-angiogenic small molecule that targets the NF-κB signaling pathway.

90 POSTER Inhibition of VEGF (vascular endothelial growth factor)/VEGF receptor system activation and in vivo tumor-induced angiogenesis by an anti-angiogenic small molecule epoxyquinol B

90 POSTER Inhibition of VEGF (vascular endothelial growth factor)/VEGF receptor system activation and in vivo tumor-induced angiogenesis by an anti-angiogenic small molecule epoxyquinol B

Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment

We have recently generated a monoclonal antibody (mAb), Phy-V002, which specifically labels activated vascular endothelial cells (EC) in zebrafish. Here, we show that this mAb labels activated EC in newly formed vessels in vivo without staining mature vessels or other tissues. Using this mAb, drug effects on in vivo EC migration and vessel formation were visually assessed by whole-mount immunochemical staining in the transparent embryo. In addition, we have developed a quantitative microplate-based ELISA that measures EC proliferation in vivo after drug treatment. We have validated the quantitative in vivo ELISA using several antiangiogenic small molecules with different mechanisms of action which were added directly to the fish water. Some of these drugs, including: 2-methoxyestradiol, flavopiridol, paclitaxel, and genistein, are currently in clinical trials. We also injected large molecule drugs, including 3TSR and TSR2+KRFK, recombinant human antiangiogenic peptides of thrombospondin-1, a natural protein. To demonstrate that proangiogenic effects can also be assessed in zebrafish, we assessed effects of penicillamine and simvastatin, two proangiogenic compounds shown to stimulate vessel formation in rodents. Using whole-mount immunochemical staining with Phy-V002, inhibition of EC migration and inhibition or stimulation of vessel formation were visually observed for each compound. Next, using the quantitative in vivo angiogenesis ELISA, we generated dose-response curves for each compound. Compared to conventional assays, advantages of using zebrafish to assess drug effects on angiogenesis include: (1) a short assay time; (2) easy animal maintenance; (3) use of small quantities of drug; (4) single dosing; (5) a quantitative assay format; and (6) use of statistically significant number of animals per test.

A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases.

TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.

Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis.

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.