Discovery of novel inhibitors of vascular endothelial growth factor-A-Neuropilin-1 interaction by structure-based virtual screening.

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A165 to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A165 binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A165 binding to recombinant NRP-1 with Ki of about 10 µM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.

New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation.

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC).

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.

The importance of monochromatic lights in the production of phenolic acids and flavonoids in shoot cultures of Aronia melanocarpa, Aronia arbutifolia and Aroniaâ€¯× prunifolia.

Shoot cultures of Aronia melanocarpa, A. arbutifolia and A. × prunifolia were maintained on Murashige and Skoog medium with 1 mg/l each of BA and NAA under monochromatic lights (far-red, red, blue lights, UV-A-irradiation), in darkness, and under white light (control). HPLC-DAD analyses of 19 phenolic acids and 11 flavonoids in methanolic extracts from the shoots revealed in all of them the presence of three depsides (chlorogenic, neochlorogenic and rosmarinic acids), protocatechuic acid, four flavonoid glycosides (cynaroside, quercitrin, hyperoside and rutoside), and additionally, in A. arbutifolia, 3,4-dihydroxyphenylacetic acid. Depending on light quality, the total amounts of these metabolites increased 1.8-5.9 times, reaching maximum values under blue light: 527.40 and 144.61 mg 100 g-1 DW (A. melanocarpa), 543.27 and 85.82 mg 100 g-1 DW (A. arbutifolia) and 1615.18 and 220.65 mg 100 g-1 DW (A. × prunifolia), respectively. The maximum total amounts were 1.3-3.6 times higher than under white light. The quantities of individual metabolites changed from 1.2 to 11.0 times, with high amounts of neochlorogenic acid and quercitrin in A. melanocarpa (243.35 and 75.64 mg 100 g-1 DW), and of chlorogenic and rosmarinic acids and quercitrin in A. arbutifolia (236.52, 219.35 and 51.01 mg 100 g-1 DW). Extremely high amounts of depsides (418.83, 644.68, 548.86 mg 100 g-1 DW) and quercitrin (165.88 mg 100 g-1 DW) were produced in cultures of the hybrid - A. × prunifolia. The results are potentially useful for practical applications. This is the first report documented the importance of light quality on the production of phenolic acids and flavonoids in three aronia in vitro cultures.

Structure-function analysis of the antiangiogenic ATWLPPR peptide inhibiting VEGF(165) binding to neuropilin-1 and molecular dynamics simulations of the ATWLPPR/neuropilin-1 complex.

Heptapeptide ATWLPPR (A7R), identified in our laboratory by screening a mutated phage library, was shown to bind specifically to neuropilin-1 (NRP-1) and then to selectively inhibit VEGF(165) binding to this receptor. In vivo, treatment with A7R resulted in decreasing breast cancer angiogenesis and growth. The present work is focused on structural characterization of A7R. Analogs of the peptide, obtained by substitution of each amino acid with alanine (alanine-scanning) or by amino acid deletion, have been systematically assayed to determine the relative importance of the side chains of each residue with respect to the inhibitory effect of A7R on VEGF(165) binding to NRP-1. We show here the importance of the C-terminal sequence LPPR and particularly the key role of C-terminal arginine. In solution, A7R displays significant secondary structure of the backbone adopting an extended conformation. However, the functional groups of arginine are very flexible in the absence of NRP-1 pointing to an induced fit upon binding to the receptor. A MD trajectory of the A7R/NRP-1 complex in explicit water, based on the recent tuftsin/NRP-1 crystal structure, has revealed the hydrogen-bonding network that contributes to A7R's binding activity.

Effects of maternal deprivation on the adrenocorticotrophic and gonadotrophic axes in the hypothalamo-pituitary unit of preweanling female sheep: the histomorphometric approach.

This study was designed to investigate the histochemical effects of maternal deprivation on the adrenocorticotrophic and gonadotrophic axes in the hypothalamo-pituitary unit of preweanling lambs. Twelve-week-old female lambs were divided into either the control (lambs reared under undisturbed maternal conditions; n=3) or the maternally deprived group (lambs separated for three days from their dams; n=3). The corticotrophin-releasing hormone (CRH) and gonadotrophin-releasing hormone (GnRH) in the median eminence and the adenohypophyseal adrenocorticotrophin (ACTH), gonadotrophins (LH and FSH) and mRNAs for their beta-subunits were investigated using the immunohistochemistry or hybridohistochemistry. In maternally deprived lambs, the percentage of the area occupied by immunoreactive (ir)-CRH nerve terminals was lower (P<0.05) and the percentage of the adenohypophyseal area (PAA) occupied by ir-ACTH cells was higher (P<0.05) compared with the control lambs. In the hypothalamo-gonadotrophic axis of maternally deprived lambs the percentage of area occupied by ir-GnRH nerve terminals was higher (P<0.05) and the PAA occupied by ir-FSHbeta cells was lower (P<0.05) in comparison with controls. The PAA occupied by gonadotrophs detected using hybridohistochemistry was higher (P<0.05) for LHbeta-mRNA in contrast to a lower (P<0.05) percentage for FSHbeta-mRNA in maternally deprived lambs compared with those staying with dams. In conclusion, maternal deprivation affected the accumulation of CRH and ACTH. The different and more striking alterations in FSH synthesis and storage in comparison with those concerning LH were observed in maternally deprived lambs. Thus, rupture of the preweanling young-mother social contact can affect the gonadotroph population activity, especially that relating to FSH-producing cells in the infantile female sheep.

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.

Lipopeptide-based liposomes for DNA delivery into cells expressing neuropilin-1.

In this paper, liposomes containing a lipopeptide bearing a ligand specifically recognized by neuropilin-1 (NRP-1) have been used to target a human breast cancer cell line overexpressing this receptor. The synthesis of this lipopeptide, C16-A7R, formed by the sequence of 7 amino acids ATWLPPR, linked to a palmitoyl fatty chain by an amide bond was described. After the characterisation of cationic liposomes formulated with the lipopeptide, the results obtained using various techniques showed that the lipopeptide-based liposomes were well accumulated in cells of the human breast cancer line MDA-MB-231 overexpressing NRP-1. Delivery of reporter genes expressing either beta-galactosidase (beta-gal) or green fluorescent protein (GFP) was selectively enhanced in these cells when compared with NRP-1-negative cells. In MDA-MB-231 cells, an increase by 250% in beta-gal activity was observed when delivered by lipopeptide-based liposomes compared to cationic liposomes alone.

Aponecrotic, antiangiogenic and antiproliferative effects of a novel dextran derivative on breast cancer growth in vitro and in vivo.

1. Since the sodium phenylacetate (NaPa) was reported to enhance the inhibitory effect of carboxymethyl benzylamide dextran (CMDB) on the breast cancer growth, we performed the esterification of CMDB with NaPa to obtain a new drug carrying the characteristics of these two components. A new molecule, phenylacetate carboxymethyl benzylamide dextran, was named NaPaC. 2. We investigated in vitro and in vivo the effects of NaPaC on MCF-7ras cell growth as well as its apoptotic and antiangiogenic effects in comparison to NaPa and CMDB. In addition, we assessed in vitro the antiproliferative effects of these drugs on other breast cancer cells, including MDA-MB-231, MDA-MB-435 and MCF-7. 3. In vitro, NaPaC inhibited MCF-7ras cell proliferation by 40% at concentration lower than that of CMDB and NaPa (12 microM vs 73 microM and 10 mM). IC(50)s were 6 and 28 microM for NaPaC and CMDB, respectively. The similar results were obtained for three other breast cancer cell lines. NaPaC reduced the DNA replication and induced cell recruitment in G(0)/G(1) phase more efficiently than its components. Moreover, it induced a cell death at concentration 1000-fold lower than NaPa. 4. In vivo, CMDB (150 mg kg(-1)) and NaPa (40 mg kg(-1)) inhibited the MCF-7ras tumour growth by 37 and 57%, respectively, whereas NaPaC (15 mg kg(-1)) decreased tumour growth by 66% without toxicity. 5. NaPa or CMDB reduced the microvessel number in tumour by 50% after 7 weeks of treatment. NaPaC had the same effect after only 2 weeks. After 7 weeks, it generated a large necrosis area without detectable microvessels. In vitro, NaPaC inhibited human endothelial cell proliferation more efficiently than CMDB or NaPa. NaPaC interacts with vascular endothelial growth factor as observed by affinity electrophoresis. 6. NaPaC acts like NaPa and CMDB but in more potent manner than components used separately. Its antiproliferative, aponecrotic and anti-angiogenic actions make it a good candidate for a new anti-cancer drug.

Intracerebroventricular infusion of neuropeptide Y up-regulates synthesis and accumulation of luteinizing hormone but not follicle stimulating hormone in the pituitary cells of prepubertal female lambs.

Neuropeptide Y (NPY) is a putative neuroregulator of the reproductive axis in the central nervous system. In this study we evaluated the effects of central infusion of exogenous NPY on the secretory activity of pituitary gonadotrophic cells in prepubertal lambs. Immature female Merino sheep (n=12) were infused of Ringer solution (control) or 50 microg of NPY to the third ventricle for 5 min and then slaughtered 3 h later. Immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) cells were localised by immunohistochemistry using antibody raised against LHbeta and FSHbeta. Messenger RNA analyses were performed by in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. The results were generated by computer image analysis to determine the area fraction occupied by immunoreactive and/or hybridising cells and optical density for immunostaining and hybridisation signal. LH in the blood plasma was determined by radioimmunoassay. It was found, that in the lambs infused with NPY the area fraction and optical density for immunoreactive LH cells and mRNA LHbeta-expressing cells increased significantly (P<0.001), compared to the vehicle-infused animals. The concentration of LH in the blood plasma did not differ between control and treated groups. The NPY infusions had no effect on the immunoreactivity of FSH cells or on expression of mRNA for FSHbeta. In conclusion we suggest that NPY may be an important component of mechanisms stimulating the synthesis and storage but not the release of LH in the pituitary gonadotrophs from prepubertal female sheep. In addition, this effect is specific for LH, no such effect was apparent on FSH.

In vitro evaluation and biodistribution of a 99mTc-labeled anti-VEGF peptide targeting neuropilin-1.

Neuropilins (NRP) are receptors of angiogenic vascular endothelial growth factor (VEGF). Their overexpression was correlated with tumor angiogenesis and growth suggesting that their specific targeting could provide a new marker of tumor progression. Here, we observed in vitro that new (99m)Tc-labeled derivative of anti-VEGF heptapeptide, ATWLPPR, binds to NRP1 but not to NRP2. Our radiotracer is stable up to 24 h in human serum and in cysteine challenge. But, its too low affinity and too fast extraction indicate further improvement to give a successful imaging of tumor in vivo.

Invading basement membrane matrix is sufficient for MDA-MB-231 breast cancer cells to develop a stable in vivo metastatic phenotype.

INTRODUCTION: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. METHODS: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. RESULTS: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. CONCLUSION: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of pro-angiogenic and survival factors.

Caveolin-1 and doxorubicin-induced P-glycoprotein modulate plasma cholesterol membrane accessibility in erythrolymphoblastic cell line.

UNLABELLED: AIM/ BACKGROUND: Various interactions between Caveolae membrane domains, multidrug resistance transporter P-glycoprotein (P-gp) and cholesterol have been suggested. We tested the assumption that anthracycline-induced P-gp and Caveolin-1 have correlated effects on cholesterol distribution in plasma membrane. MATERIALS AND METHODS: The present study was performed in four lymphoblastic K562 cell lines expressing none (KS), one (Cav and KR cells) or both P-gp and caveolin-1 proteins (CavKR cells). RESULTS: The CavKR cell line exhibits a significantly higher free cholesterol content than the other cell lines. Cholesterol distribution at the outer leaflet was distinct from the total cellular cholesterol by its accessibility to cholesterol oxidase (COase). When cells were ATP-deprived, cholesterol accessibility to oxidation was significantly delayed in CavKR cells. Caveolin-1 or P-gp expression did not induce detectable changes in membrane cholesterol accessibility to COase. CONCLUSION: Combination of functional P-gp, caveolae presence and lasting effect of anthracycline treatment appear determinant in free membrane cholesterol homeostasis and likely modulate cholesterol membrane order.

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1).

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.