NRPa-308, a new neuropilin-1 antagonist, exerts in vitro anti-angiogenic and anti-proliferative effects and in vivo anti-cancer effects in a mouse xenograft model.

Neuropilin-1 (NRP-1) is an extra-cellular receptor for the main Vascular Endothelial Growth Factor over-expressed in tumour tissues, VEGF-A165. Consequently, NRP-1 is involved in angiogenesis and in tumour growth, and its over-expression is related to a clinical poor prognosis. NRP-1 appears as a major target in oncology, which remains poorly exploited. Herein, we report a new series of 18 small-sized fully organic VEGF-A165/NRP-1 antagonists (NRPas). These compounds share an original scaffold, including two linkers (sulphonamide and amide) and three aromatic cores. Among them, 2a (renamed NRPa-308) emerges as a promising "hit". In vitro,2a exerts not only potent anti-angiogenic activity, but also significant effects on cell viability of large panel of human solid and haematological cancer cell lines. Importantly, 2a is less cytotoxic on healthy tissues than the marketed anti-angiogenic drug sunitinib. Lastly, in a mouse xenograft model (human MDA-MB-231 breast cancer cells), 2a improves the median survival and reduces the tumour growth, but does not exert visible acute toxicity. Altogether, these results highlight its huge potential for a further "hit-to-lead" optimization, leading to new anti-cancer drugs.

Conformational analysis of a polyconjugated protein-binding ligand by joint quantum chemistry and polarizable molecular mechanics. Addressing the issues of anisotropy, conjugation, polarization, and multipole transferability.

We investigate the conformational properties of a potent inhibitor of neuropilin-1, a protein involved in cancer processes and macular degeneration. This inhibitor consists of four aromatic/conjugated fragments: a benzimidazole, a methylbenzene, a carboxythiourea, and a benzene-linker dioxane, and these fragments are all linked together by conjugated bonds. The calculations use the SIBFA polarizable molecular mechanics procedure. Prior to docking simulations, it is essential to ensure that variations in the ligand conformational energy upon rotations around its six main-chain torsional bonds are correctly represented (as compared to high-level ab initio quantum chemistry, QC). This is done in two successive calibration stages and one validation stage. In the latter, the minima identified following independent stepwise variations of each of the six main-chain torsion angles are used as starting points for energy minimization of all the torsion angles simultaneously. Single-point QC calculations of the minimized structures are then done to compare their relative energies ΔE conf to the SIBFA ones. We compare three different methods of deriving the multipoles and polarizabilities of the central, most critical moiety of the inhibitor: carboxythiourea (CTU). The representation that gives the best agreement with QC is the one that includes the effects of the mutual polarization energy E pol between the amide and thioamide moieties. This again highlights the critical role of this contribution. The implications and perspectives of these findings are discussed.

Synthesis and structure-activity relationship of non-peptidic antagonists of neuropilin-1 receptor.

Neuropilins (NRPs) are VEGF-A165 co-receptors over-expressed in tumor cells, and considered as targets in angiogenic-related pathologies. We previously identified compound 1, the first non-peptidic antagonist of the VEGF-A165/NRP binding, which exhibits in vivo anti-angiogenic and anti-tumor activities. We report here the synthesis and biological evaluations of new antagonists structurally-related to compound 1. Among these molecules, 4a, 4c and 4d show cytotoxic effects on HUVEC and MDA-MB-31 cells, and antagonize VEGF-A165/NRP-1 binding. This study confirmed our key structure-activity relationships hypothesis and paved the way to compound 1 'hit to lead' optimization.

Structure-based discovery of a small non-peptidic Neuropilins antagonist exerting in vitro and in vivo anti-tumor activity on breast cancer model.

Neuropilin-1/-2 (+33 NRPs), VEGF-A165 co-receptors, are over-expressed during cancer progression. Thus, NRPs targeted drug development is challenged using a multistep in silico/in vitro screening procedure. The first fully non-peptidic VEGF-A165/NRPs protein-protein interaction antagonist (IC50=34 µM) without effect on pro-angiogenic kinases has been identified (compound-1). This hit showed breast cancer cells anti-proliferative activity (IC50=0.60 µM). Compound-1 treated NOG-xenografted mice significantly exerted tumor growth inhibition, which is correlated with Ki-67(low) expression and apoptosis. Furthermore, CD31(+)/CD34(+) vessels are reduced in accordance with HUVEC-tube formation inhibition (IC50=0.20 µM). Taking together, compound-1 is the first fully organic inhibitor targeting NRPs.

Inhibition of both focal adhesion kinase and fibroblast growth factor receptor 2 pathways induces anti-tumor and anti-angiogenic activities.

FAK and FGFR2 signaling pathways play important roles in cancer development, progression and tumor angiogenesis. PHM16 is a novel ATP competitive inhibitor of FAK and FGFR2. To evaluate the therapeutic efficacy of this agent, we examined its anti-angiogenic effect in HUVEC and its anti-tumor effect in different cancer cell lines. We showed PHM16 inhibited endothelial cell viability, adherence and tube formation along with the added ability to induce endothelial cell apoptosis. This compound significantly delayed tumor cell growth. Together, these data showed that inhibition of both FAK and FGFR2 signaling pathways can enhance anti-tumor and anti-angiogenic activities.

Synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK inhibitors with anti-angiogenic activity.

We report herein the synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK (focal adhesion kinase) inhibitors and the evaluation of their anti-angiogenic activity on HUVEC cells. Generally, the effects of these compounds on endothelial cells could be correlated with their kinase inhibitory activity. The most efficient compounds displayed inhibition of viability against HUVEC cells in the micromolar range, as observed with TAE-226, which was designed by Novartis Pharma AG. X-ray crystallographic analysis of the co-crystal structure for compound 34 revealed that the mode of interaction with the FAK kinase domain is highly similar to that observed in the complex of TAE-226.

Human Muscle Progenitor Cells Displayed Immunosuppressive Effect through Galectin-1 and Semaphorin-3A.

In human skeletal muscle, myoblasts represent the main population of myogenic progenitors. We previously showed that, beside their myogenic differentiation capacities, myoblasts also differentiate towards osteogenic and chondrogenic lineages, some properties generally considered being hallmarks of mesenchymal stem cells (MSCs). MSCs are also characterized by their immunosuppressive potential, through cell-cell contacts and soluble factors, including prostaglandin E-2 (PGE-2), transforming growth factor-ß1 (TGF-ß1), interleukine-10, or indoleamine 2,3-dioxygenase. We and others also reported that Galectin-1 (Gal-1) and Semaphorin-3A (Sema-3A) were involved in MSCs-mediated immunosuppression. Here, we show that human myoblasts induce a significant and dose-dependant proliferation inhibition, independently of PGE-2 and TGF-ß1. Our experiments revealed that myoblasts, in culture or in situ in human muscles, expressed and secreted Gal-1 and Sema-3A. Furthermore, myoblasts immunosuppressive functions were reverted by using blocking antibodies against Gal-1 or Sema-3A. Together, these results demonstrate an unsuspected immunosuppressive effect of myoblasts that may open new therapeutic perspectives.

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells.

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A(165)-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A(165) to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A(165). The specific inhibition of VEGF-A(165) binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A(165)-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA(165)-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.

Inhibitory effect of semaphorin-3A, a known axon guidance molecule, in the human thymocyte migration induced by CXCL12.

Intrathymic T cell differentiation takes place within the thymic lobules and depends on interactions between developing thymocytes and cells of the thymic microenvironment. Along with differentiation, thymocytes migrate in an oriented progression, which is tightly regulated by a number of interactions, including one mediated by the chemokine CXCL12. It has been shown recently that SEMA-3A, a soluble member of the semaphorin family, is also involved in this human thymocyte migration and can have a chemorepulsive and de-adhesive role. Herein, we study the role of SEMA-3A on the CXCL12-driven migration of human thymocytes. We have shown that SEMA-3A is able to inhibit the chemotaxis triggered by CXCL12. Such an inhibition was seen in respect to immature and mature CD4/CD8-defined thymocyte subsets and can be reverted specifically by neutralizing anti-SEMA-3A mAb. We have also shown that SEMA-3A consistently down-regulates CXCR4 membrane expression in all CD4/CD8-defined thymocyte subsets, and this down-regulation is accompanied by a decrease in the phosphorylation of FAK and ZAP-70 protein kinases. Taken together, these results demonstrate the involvement of SEMA-3A in the regulation of CXCL12-driven human thymocyte migration, where it acts as a physiological antagonist.

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis.

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A(165)). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.

Toll-like receptor control of glucocorticoid-induced apoptosis in human plasmacytoid predendritic cells (pDCs).

Microbial infection triggers the endogenous production of immunosuppressive glucocorticoid (GC) hormones and simultaneously activates innate immunity through toll-like receptors (TLRs). How innate immune cells integrate these 2 opposing signals in dictating immunity or tolerance to infection is not known. In this study, we show that human plasmacytoid predendritic cells (pDCs) were highly sensitive to GC-induced apoptosis. Strikingly, they were protected by microbial stimulation through TLR-7 and TLR-9, but not by microbial-independent stimuli, such as interleukin-3, granulocyte macrophage colony-stimulating factor, or CD40-ligand. This protection was dependent on TLR-induced autocrine tumor necrosis factor-α and interferon-α, which collectively increased the expression ratio between antiapoptotic genes (Bcl-2, Bcl-xL, BIRC3, CFLAR) versus proapoptotic genes (Caspase-8, BID, BAD, BAX). In particular, virus-induced Bcl-2 up-regulation was dependent on autocrine interferon-α. Using small interfering RNA technology, we demonstrated that Bcl-2 and CFLAR/c-flip were essential for TLR-induced protection of pDCs from GC-induced caspase-8-mediated apoptosis. Our results demonstrate a novel property of the TLR pathway in regulating the interface between GC and innate immunity and reveal a previously undescribed mechanism of GC resistance.

RasV12 induces Survivin/AuroraB pathway conferring tumor cell apoptosis resistance.

Several cancers are treated by interferons alpha and gamma in association with conventional chemotherapy due to the resistance observed with interferon treatment alone. The frequency of un-sensitive cancer depends on tumor origin and oncogenic genes. Preclinical studies have highlighted interferon resistance in many cancers such as colon carcinoma due to oncogenic Ras. However, the resistance mechanism remains elusive. Apoptosis and proliferation of Ras(wt) and mutated Ras(V12) transformed colon carcinoma cells treated with several recombinant interferon combinations were analyzed by flow cytometer and immunoblot. Apoptotic pathways of resistant Ras(V12) cells were investigated using siRNA strategy to determine key proteins involved in this process. We show that interferons alpha and gamma synergized to induce human Ras(wt) colon carcinoma cell (HT29) apoptosis by caspases and PARP-1 cleavages in contrast to Ras(V12) mutated colon carcinoma cells (SW480, HT29 clone). However, Ras(V12) siRNA restored interferon sensitivity of Ras(V12)-HT29 clone to apoptosis. Survivin siRNA increased interferon apoptosis in Ras(wt) cells demonstrating the key role of this protein in cell survival. Ras(V12) mutation in HT29 clone neutralized the interferon effect on Survivin suppression and maintained high level of phospho-Aurora-B/Histone H3, which protected cells from apoptosis. SiRNA strategy against both Aurora-B and Survivin in Ras(V12) cells synergized to restore interferon -induced apoptosis. Ras(V12) cells are less sensitive than Ras(wt) cells to interferon induced cell apoptosis due to a Survivin/Aurora-B survival alternative pathway. Taken together, these results may provide interest in siRNA-therapeutic strategy and diagnostic relevance for therapy.

Free HTLV-1 induces TLR7-dependent innate immune response and TRAIL relocalization in killer plasmacytoid dendritic cells.

A recent report demonstrated that free human T-cell leukemia virus 1 (HTLV-1) could infect plasmacytoid dendritic cells (pDCs). The major role of pDCs is to secrete massive levels of interferon-alpha (IFN-alpha) upon virus exposure; however, the induction of IFN-alpha by HTLV-1 remains unknown. We demonstrate here that cell-free HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-alpha that were inhibited by anti-HTLV-1 antibodies. HTLV-1 induced costimulatory molecules and rapid expression of the apoptotic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, HTLV-1 stimulated pDC-induced apoptosis of CD4(+) T cells expressing DR5, transforming pDCs into IFN-producing killer pDCs. We also observed that an endosomal acidification inhibitor and a Toll-like receptor-7 (TLR7)-specific blocker drastically inhibited pDC response to HTLV-1. Three-dimensional microscopy analysis revealed that unstimulated pDCs were "dormant" IFN-producing killer pDCs with high levels of intracellular TRAIL that could be rapidly mobilized to the surface in response to TLR7 activation. Inhibition of viral degradation in endosomes by chloroquine maintained viral integrity, allowing virus detection by 3-dimensional microscopy. We demonstrate that pDCs respond to cell-free HTLV-1 by producing high levels of IFN-alpha and by mobilizing TRAIL on cell surface after TLR7 triggering. This is the first demonstration of an innate immune response induced by free HTLV-1.

p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein-protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

Short interfering RNA (siRNA), a novel therapeutic tool acting on angiogenesis.

The formation of new blood vessels, uncontrolled cell expansions and invasions are the common feature of cancer, neovascular inflammatory and ocular diseases, such as age-related macular degeneration (AMD). Short interfering RNA (siRNA) and short-hairpin RNA (shRNA) have recently helped extend our understanding of the mechanisms regulating angiogenesis and tumor developments. Moreover, the early success of these tools has reinforced the therapeutic hopes of preventing endogenous or exogenous gene translation. In vivo experiments using several animal tumor models and human pre-clinical trials augured many benefits to control protein expression and cell signaling. The high specificity of siRNA and shRNA to target a protein is crucial to design a new generation of therapeutic agents. At the present, several investigations are focused on the understanding of both gene function and the proof-of-concept for siRNA-mediated anti-angiogenesis. Taken together, in vitro and in vivo studies shed light on the efficiency of siRNA as a new alternative therapeutic agent.

Control of human thymocyte migration by Neuropilin-1/Semaphorin-3A-mediated interactions.

It is largely established that molecules first discovered in the nervous system are also found in the immune system. Neuropilin-1 (NP-1) was initially identified to mediate semaphorin-induced chemorepulsion during brain development and is also involved in peripheral T cell/dendritic cell interactions. Herein, we studied NP-1 during T cell development in the human thymus. NP-1 is expressed in both cortex and medulla of thymic lobules, being found in distinct CD4/CD8-defined thymocyte subsets. NP-1 is also found in thymic epithelial cells (TEC) in situ and in vitro, and is recruited at the site of TEC-thymocyte contact. Moreover, NP-1 was rapidly up-regulated during thymocyte stimulation by T cell receptor (TCR) and IL-7 or after adhesion to TEC. Semaphorin-3A (Sema-3A), a natural ligand of NP-1, is also present in human thymus, both in TEC and thymocytes, being up-regulated in thymocytes after TCR engagement. Functionally, Sema-3A decreases the adhesion capacity of NP-1(+) thymocytes and induces their migration by a repulsive effect. In conclusion, we show here that NP-1/Sema-3A-mediated interactions participate in the control of human thymocyte development.

The histone variant mH2A1.1 interferes with transcription by down-regulating PARP-1 enzymatic activity.

The histone variant mH2A is believed to be involved in transcriptional repression, but how it exerts its function remains elusive. By using chromatin immunoprecipitation and tandem affinity immunopurification of the mH2A1.1 nucleosome complex, we identified numerous genes with promoters containing mH2A1.1 nucleosomes. In particular, the promoters of the inducible Hsp70.1 and Hsp70.2 genes, but not that of the constitutively expressed Hsp70.8, were highly enriched in mH2A1.1. PARP-1 was identified as a part of the mH2A1.1 nucleosome complex and was found to be associated with the Hsp70.1 promoter. A specific interaction between mH2A1.1 and PARP-1 was demonstrated and found to be associated with inactivation of PARP-1 enzymatic activity. Heat shock released both mH2A1.1 and PARP-1 from the Hsp70.1 promoter and activated PARP-1 automodification activity. The data we present point to a novel mechanism for control of Hsp70.1 gene transcription. mH2A1.1 recruits PARP-1 to the promoter, thereby inactivating it. Upon heat shock, the Hsp70.1 promoter-bound PARP-1 is released to activate transcription through ADP-ribosylation of other Hsp70.1 promoter-bound proteins.

Immunosuppressive role of semaphorin-3A on T cell proliferation is mediated by inhibition of actin cytoskeleton reorganization.

Timely negative regulation of the immune system is critical to allow it to perform its duty while maintaining it under tight control to avoid overactivation. We previously reported that the neuronal receptor neuropilin-1 (NP-1) is expressed in human lymph nodes. However, the role of NP-1 interaction with its physiological ligand semaphorin-3A (Sema-3A) on immune cells remains elusive. Here we show that Sema-3A is expressed by activated DC and T cells, and that its secretion in DC/T cell cocultures is delayed. Sema-3A/NP-1 interaction down-modulated T cell activation since addition of Sema-3A in DC/T cell cocultures dramatically inhibited allogeneic T cell proliferation. More importantly, neutralization by blocking antibodies or by antagonist peptide of endogenous Sema-3A produced by DC/T cell cocultures resulted in a 130% increase in T cell proliferation. Sema-3A acted directly on T cells, since it could block anti-CD3/CD28-stimulated proliferation of T cells. Finally, immunomodulatory functions of Sema-3A relied on the blockage of actin cytoskeleton reorganization, affecting TCR polarization and interfering with early TCR signal transduction events such as ZAP-70 or focal adhesion kinase phosphorylation. Therefore, we propose that Sema-3A secretion and the resulting NP-1/Sema-3A interaction are involved in a late negative feedback loop controlling DC-induced T cell proliferation.