TX-2152: a conformationally rigid and electron-rich diyne analogue of FTY720 with in vivo antiangiogenic activity

We designed FTY720 analogues with conformationally rigid and electron-rich acetylenic chains as antiangiogenic agents (the monoyne 1: TX-2148, the diyne 2: TX-2152, the triyne 3: TX-2256). Molecular orbital (MO) calculations of our designed acetylenic analogues and FTY720 showed that the localization of the lowest unoccupied MO and the highest occupied MO increased from phenyl ring to acetylenic chain compared with that of FTY720. These acetylenic analogues were synthesized from p-hydroxyphenylethanol as a starting material. The construction of the acetylenic chain was carried out by an iterative strategy using a Sonogashira cross-coupling reaction and desilylative bromination in two steps. The corresponding overall yields of the monoyne 1, the diyne 2, and the triyne 3 were 27% (11 steps), 13% (13 steps), and 10% (15 steps). The in vivo antiangiogenic activities of these acetylenic analogues and FTY720 were evaluated by the chick embryo chorioallantoic membrane (CAM) assay and compared to the activities of the known antiangiogenic agent TNP-470. The diyne 2 showed more potent antiangiogenic activity (90% inhibition) than FTY720 (77% inhibition) and other acetylenic analogues (the monoyne 1: 42% inhibition, the triyne 3: 60% inhibition), and TNP-470 (82% inhibition) at a dose of 10 microg/CAM, without showing toxicity. The diyne 2 also had potent inhibitory activity at a dose of 5 and 2.5 microg/CAM. These results indicate that the flexibility of C8 alkyl chain of FTY720 is not required for its antiangiogenic activity. We suggest that the diyne 2 (TX-2152) may be a promising candidate as an antiangiogenic agent for antineoplastic drug discovery.     

Design,synthesis and biological activities of antiangiogenic hypoxic cytotoxin,triazine-N-oxide derivatives

For cancer therapy, hypoxia represents an important tumor specific target. Therefore we designed and synthesized antiangiogenic hypoxic cytotoxins as 'hypoxia modifiers'. They can be activated bioreductively in hypoxic cells to kill the oxygen-deficient tumor cells selectively and prevent their re-growth. The aromatic heterocycle di-N-oxides, tirapazamine (TPZ), TX-1102, and TX-402 inhibited growth of EMT6/KU cells, SAS/neo cells, and SAS/Trp248 cells (mutant p53 gene transformant) under hypoxic condition. They also induced apoptosis selectively at a dose of 10 microM each under hypoxic condition for 5 h. Their hypoxic cytotoxicities and apoptosis inducing activities were p53-independent because the activities in SAS/neo cells were almost similar to that in SAS/Trp248 cells. In angiogenesis inhibition assay using chick embryo chorioallantoic membrane (CAM), TPZ, TX-1102, TX-402 and TX-1033 showed 40, 25, 60 and 60% inhibition of angiogenesis each at a dose of 10 microg/CAM. On the other hand, the nitrosopyrimidine, TX-1041 had neither antiangiogenic activity nor cytotoxicity. Therefore the di-N-oxide group is thought to be required for the biological activities. TX-1102 was a potent antiangiogenic hypoxic cytotoxin inducing apoptosis p53-independently.     

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.     

Collagen XVIII/endostatin structure and functional role in angiogenesis

The angiogenesis inhibitor endostatin is a 20 kDA C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The endostatin fragment was originally identified in conditioned media from a murine endothelial tumor cell line. Endostatin inhibits endothelial cell migration in vitro and appears to be highly effective in murine in vivo studies. The molecular mechanisms behind the inhibition of angiogenesis have not yet been elucidated. Studies of the crystal structure of endostatin have shown a compact globular fold, with one face particularly rich in arginine residues acting as a heparin-binding epitope. It was initially suggested that zinc binding was essential for the antiangiogenic mechanism but later studies indicate that zinc has a structural rather than a functional role in endostatin. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. The processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis.     

Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity

The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8 microg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p < 0.05). However, when the protein dosage is less than 0.4 microg/ml, there is no significance between their inhibition rates (p > 0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6 microg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p < 0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds.     

The morphogenic properties of oligomeric endostatin are dependent on cell surface heparan sulfate

Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with N- and 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface.     

Structural analysis of the N‐terminal fragment of the antiangiogenic protein endostatin: A molecular dynamics study

Endostatin is a potent antiangiogenic protein derived from the noncollagenous domain 1 (NC1) of collagen XVIII. The mechanism by which endostatin exerts its antiangiogenic effect is still incompletely understood. It has been shown that the 27 amino acid N‐terminal fragment of murine endostatin has antitumor, antimigration, and antipermeability activities comparable to the full soluble protein. To understand how this peptide can exert such elaborate function, we performed structural analysis using molecular dynamics to evaluate the behavior of this fragment in aqueous environment. Here, we show that the N‐terminal peptide of murine endostatin is able to assume a well‐defined structure, folding into a zinc‐dependent β‐hairpin conformation. Analyzing the folding mechanism, we were able to understand why the N‐terminal peptide of human endostatin with the same length failed to acquire a stable conformation. Conversely, we were able to predict the successful folding of the R4Q mutant and of a shorter form of the human peptide with 25 residues. Finally, we show that the β‐hairpin conformation assumed by the zinc‐bound peptide of murine endostatin has a high structural similarity with fragments of another family of angiogenesis inhibitors: the integrin‐binding portion of the NC1 domain of collagen IV. Indeed, our docking simulations show that arresten, canstatin, and the endostatin peptide bind to the same spot of αVβ3 integrin, suggesting similar interactions via a common binding site on this receptor. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay

Endostatin has demonstrated potent antiangiogenic and antitumor activity in mouse models. We have investigated the ex vivo rat aortic ring assay and a human vein model to assess the biological activity of murine and human endostatin. Rat aortic rings were exposed to recombinant murine endostatin (Spodoptera frugipera; Calbiochem, San Diego, CA) or recombinant human endostatin (Pichia pastoris; EntreMed, Rockville, MD). After 5 days, murine endostatin (500 microgram/ml) demonstrated inhibition of microvessel outgrowth with dose-dependent effects (down to 16 microgram/ml). No significant inhibition was observed with human endostatin in the rat assay. Human endostatin at 250 and 500 microgram/ml inhibited outgrowths from human saphenous vein rings after a 14-day incubation. Electron microscopy assessed the formation of basal lamina, confirming that the microvessels were progenitors of patent vessels. Immunostaining for Factor VIII or CD34 demonstrated that the microvessel cells were endothelial. BrdU incorporation assays supported the presence of proliferating endothelial cells, correlating with neovascularization from the aortic wall. We conclude that the rat aortic ring assay confirms the antiangiogenic activity of murine but not human endostatin, suggesting that the model may have species specificity. However, the human form shows biological activity against human vascular tissue.     

Calmodulin structure and function: Implication of arginine in the compaction related to ligand binding

Calmodulin (CAM) is a modulatory protein that regulates cellular activity by binding to a large number of proteins. Key elements in the Ca²⁺-dependent mechanism of interaction between CAM and the proteins it activates are the selectivity for Ca²⁺ ions and the requirement for Ca²⁺-dependent conformational changes. We report on results from a series of molecular dynamics simulations that identified discrete steps in the mechanism of structural rearrangement of CAM. The findings implicate the side chains of arginine residues in the bending of the central alpha helix. Structural and energetic considerations point to a dynamic hydrogen bonding pattern around the arginine residues as a ratcheting-type mechanism, causing the kinking of the central helix in consecutive steps stabilized by each new pattern of hydrogen bonds. Initial model building studies to locate potential binding sites of ligands such as trifluoperazine (TFP) indicate that the compaction of CAM results in several structural changes, that explain the selective binding of molecules such as TFP in the N-terminal domain. The present studies identify specific residues involved in the process of compaction and point to specific CAM residues involved in the binding of the ligand. These insights lead directly to propositions for experimental engineering of the molecular structure of CAM in order to probe the hypotheses and their consequences for the function of this important protein.     

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.     

Canavanine inhibits vimentin assembly but not its synthesis in chicken embryo erythroid cells

In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine- vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo.     

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution.

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C-terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 A resolution. The structure reveals a compact fold distantly related to the C-type lectin carbohydrate recognition domain and the hyaluronan-binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.     

Human endostatin-derived synthetic peptides possess potent antiangiogenic properties in vitro and in vivo

Pharmacological control of the angiogenic process (i.e., the neovascularization necessary for the growth and progression of tumors and metastases) is considered to be one of the most promising approaches to antineoplastic therapy. Endostatin, a 20-kDa protein derived from collagen XVIII, is one of the first recently discovered endogeneous antiangiogenic substances, but its cell targets and mechanism(s) of action are still unknown. We thought it would be interesting to test whether shorter peptides derived from endostatin might preserve its antiangiogenic activity. Four synthetic peptides corresponding to the sequences 6-49 (I), 50-92 (II), 93-133 (III), and 134-178 (IV) of human endostatin were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis. Fragment I (and fragment IV in the tests performed) was found to be fully biologically active in all of the angiogenesis assays, and sometimes showed even greater potency and efficacy than full-length human endostatin itself.     

Structural interpretation of reduced insulin activity as seen in the crystal structure of human Arg-insulin

The N-terminal glycine of the A-chain in insulin is reported to be one of the residues that binds to the insulin receptor. Modifications near this region lead to variations in the biological activity of insulin. One such modification viz., an addition of an arginine at the N-terminal A-chain, was reported to possess two-thirds the activity of native insulin. The crystal structure of 2 zinc human arg (A0) insulin has been elucidated to 2A resolution to understand the mechanism of reduction in insulin activity. A conformational transition from T6 to T3R3(f) and a decrease in the surface accessibility of residues in the so called receptor binding region have been observed. The presence of arginine has also induced distortions in the A chain N-terminal helix. The subtle conformational alterations like decrease in surface accessibility, alterations in the charge surface and changes in the relative orientation of the two helices in the A chain may be responsible for the reduction in activity.     

Structural requirements for high-affinity heparin binding: alanine scanning analysis of charged residues in the C-terminal domain of human extracellular superoxide dismutase

An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.     

人内皮抑素基因的克隆以及在大肠杆菌中的可溶性表达研究

用PCR的方法从人胎肝cDNA文库中得到人内皮抑素基因 ,克隆测序正确后连接到硫氧还蛋白融合表达载体上 ,转化大肠杆菌BL2 1 (DE3)得到表达人内皮抑素的工程菌。用IPTG诱导表达 ,表达量达到全菌蛋白的 64%。经分析硫氧还蛋白可以辅助内皮抑素可溶性表达 ,表达的融合蛋白保持了天然蛋白的免疫学特性。而且表面带有多聚组氨酸的突变的硫氧还蛋白还简化了蛋白纯化的步骤 ,使融合蛋白可以通过固相金属螯和层析 (IMAC)的方法纯化。纯化后的融合蛋白经IgA蛋白酶的切割可得到大小正确的重组人内皮抑素 ,用此方法获得的重组人内皮抑素可以在CAM试验中抑制新生血管的形成。高效可溶型表达内皮抑素的工程菌的构建成功 ,为内皮抑素的生产应用打下了良好的基础。     

Mapping the tRNA Binding Site on the Surface of Human DNMT2 Methyltransferase

The DNMT2 enzyme methylates tRNA-Asp at position C38. Because there is no tRNA-Dnmt2 cocrystal structure available, we have mapped the tRNA binding site of DNMT2 by systematically mutating surface-exposed lysine and arginine residues to alanine and studying the tRNA methylation activity and binding of the corresponding variants. After mutating 20 lysine and arginine residues, we identified eight of them that caused large (>4-fold) decreases in catalytic activity. These residues cluster within and next to a surface cleft in the protein, which is large enough to accommodate the tRNA anticodon loop and stem. This cleft is located next to the binding pocket for the cofactor S-adenosyl-l-methionine, and the catalytic residues of DNMT2 are positioned at its walls or bottom. Many of the variants with strongly reduced catalytic activity showed only a weak loss of tRNA binding or even bound better to tRNA than wild-type DNMT2, which suggests that the enzyme induces some conformational changes in the tRNA in the transition state of the methyl group transfer reaction. Manual placement of tRNA into the structure suggests that DNMT2 mainly interacts with the anticodon stem and loop.     

Sulfate stabilizes the folding intermediate more than the native structure of endostatin

Endostatin, a potent angiogenesis inhibitor, is an acid resistant protein with compact tertiary structure. Nuclear magnetic resonance, circular dichroism, and tryptophan emission fluorescence were used to monitor the structural changes of endostatin during acid-, heat-, and urea-induced unfolding processes. Results show that sulfate anions sensitize endostatin to acid, but specifically stabilize it against heat or urea. Moreover, the disappearance of the tertiary structure and the formation of the folding intermediate of endostatin at pH 3.0 are sulfate concentration dependent. These phenomena indicate that sulfate anions stabilize the folding intermediate more than the native structure of endostatin. In addition, heparin shows stronger effect than sodium sulfate on sensitizing endostatin against acid, and very limited stabilizing effect against urea. The loose structure of endostatin upon heparin binding may imply that the physiologically favorable structure for endostatin exerting its biological functions is not as compact as what was reported.