Identification of a human estrogen receptor α tetrapeptidic fragment with dual antiproliferative and anti-nociceptive action.

The synthetic peptide ERα17p (sequence: PLMIKRSKKNSLALSLT), which corresponds to the 295-311 region of the human estrogen receptor α (ERα), induces apoptosis in breast cancer cells. In mice and at low doses, it promotes not only the decrease of the size of xenografted triple-negative human breast tumors, but also anti-inflammatory and anti-nociceptive effects. Recently, we have shown that these effects were due to its interaction with the seven-transmembrane G protein-coupled estrogen receptor GPER. Following modeling studies, the C-terminus of this peptide (sequence: NSLALSLT) remains compacted at the entrance of the GPER ligand-binding pocket, whereas its N-terminus (sequence: PLMI) engulfs in the depth of the same pocket. Thus, we have hypothesized that the PLMI motif could support the pharmacological actions of ERα17p. Here, we show that the PLMI peptide is, indeed, responsible for the GPER-dependent antiproliferative and anti-nociceptive effects of ERα17p. By using different biophysical approaches, we demonstrate that the NSLALSLT part of ERα17p is responsible for aggregation. Overall, the tetrapeptide PLMI, which supports the action of the parent peptide ERα17p, should be considered as a hit for the synthesis of new GPER modulators with dual antiproliferative and anti-nociceptive actions. This study highlights also the interest to modulate GPER for the control of pain.

Recent advances of anti-angiogenic inhibitors targeting VEGF/VEGFR axis.

Vascular endothelial growth factors (VEGF), Vascular endothelial growth factor receptors (VEGFR) and their downstream signaling pathways are promising targets in anti-angiogenic therapy. They constitute a crucial system to regulate physiological and pathological angiogenesis. In the last 20 years, many anti-angiogenic drugs have been developed based on VEGF/VEGFR system to treat diverse cancers and retinopathies, and new drugs with improved properties continue to emerge at a fast rate. They consist of different molecular structures and characteristics, which enable them to inhibit the interaction of VEGF/VEGFR, to inhibit the activity of VEGFR tyrosine kinase (TK), or to inhibit VEGFR downstream signaling. In this paper, we reviewed the development of marketed anti-angiogenic drugs involved in the VEGF/VEGFR axis, as well as some important drug candidates in clinical trials. We discuss their mode of action, their clinical benefits, and the current challenges that will need to be addressed by the next-generation of anti-angiogenic drugs. We focus on the molecular structures and characteristics of each drug, including those approved only in China.

A Cyclic Peptide Epitope of an Under-Explored VEGF-B Loop 1 Demonstrated In Vivo Anti-Angiogenic and Anti-Tumor Activities.

Pathological angiogenesis is mainly initiated by the binding of abnormal expressed vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). Blocking the VEGF/VEGFR interaction is a clinically proven treatment in cancer. Our previous work by epitope scan had identified cyclic peptides, mimicking the loop 1 of VEGF-A, VEGF-B and placental growth factor (PlGF), inhibited effectively the VEGF/VEGFR interaction in ELISA. We described here the docking study of these peptides on VEGFR1 to identify their binding sites. The cellular anti-angiogenic activities were examined by inhibition of VEGF-A induced cell proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). The ability of these peptides to inhibit MAPK/ERK1/2 signaling pathway was examined as well. On chick embryo chorioallantoic membrane (CAM) model, a cyclic peptide named B-cL1 with most potent in vitro activity showed important in vivo anti-angiogenic effect. Finally, B-cL1 inhibited VEGF induced human gastric cancer SGC-7901 cells proliferation. It showed anti-tumoral effect on SGC-7901 xenografted BALB/c nude mouse model. The cyclic peptides B-cL1 constitutes an anti-angiogenic peptide drug lead for the design of new and more potent VEGFR antagonists in the treatment of angiogenesis related diseases.

One-pot oxime ligation from peptides bearing thiazolidine and aminooxyacetyl groups.

One-pot oxime ligation under mild conditions using Pd(ii) as a shared catalyst from an aldehyde precursor (Thz) and a protected aminooxyacetyl group (Proc-Aoa) is reported. Two complementary metal-free protocols using unmasked Aoa-peptide are also described. Acetoxime-peptide can proceed to the desired oxime through an additional transoximation step.

Synthesis, 3D-structure and stability analyses of NRPa-308, a new promising anti-cancer agent.

We report herein the synthesis of a newly described anti-cancer agent, NRPa-308. This compound antagonizes Neuropilin-1, a multi-partners transmembrane receptor overexpressed in numerous tumors, and thereby validated as promising target in oncology. The preparation of NRPa-308 proved challenging because of the orthogonality of the amide and sulphonamide bonds formation. Nevertheless, we succeeded a gram scale synthesis, according to an expeditious three steps route, without intermediate purification. This latter point is of utmost interest in reducing the ecologic impact and production costs in the perspective of further scale-up processes. The purity of NRPa-308 has been attested by means of conventional structural analyses and its crystallisation allowed a structural assessment by X-Ray diffraction. We also reported the remarkable chemical stability of this molecule in acidic, neutral and basic aqueous media. Eventually, we observed for the first time the accumulation of NRPa-308 in two types of human breast cancer cells MDA-MB231 and BT549.

Structural studies of the binding of an antagonistic cyclic peptide to the VEGFR1 domain 2.

Physiological and pathological angiogenesis is mainly regulated by the binding of the vascular endothelial growth factor (VEGF) to its receptors (VEGFRs). Antagonists of VEGFR are very attractive for the treatment of diseases related to excessive angiogenesis. Our previously designed C-terminal alkylated cyclic peptides [YKDEGLEE]-NHR (R = alkyl, arylalkyl) disrupt the interaction between VEGF and VEGFRs in biological assays. In this paper, we described the structural studies of the binding of one of these cyclic peptides named Peptide 3 to the VEGFR1 domain 2 (VEGFR1-D2). The molecular docking and NMR mapping identified the binding site on VEGFR1-D2. The anti-angiogenic effect of our peptide was evaluated by an experiment of VEGF-induced tube formation in two cell lines, retinal cell type RF6/A and vascular endothelial cell type HUVEC. Some new peptides were also synthesized and compared by an ELISA-based assay, in order to verify their ability to disrupt the formation of the complex VEGF-A/VEGFR1. In conclusion, the structural studies of Peptide 3 with VEGFR1-D2 will help the design of more efficient VEGFR antagonists. Moreover, Peptide 3, with improved receptor binding affinity, could be more suitable for VEGFR targeting bioimaging studies once labeled.

LC-MS/MS-based quantification of efflux transporter proteins at the BBB.

Targeted protein quantification using tandem mass spectrometry coupled to high performance chromatography (LC-MS/MS) has been used to quantify proteins involved in the absorption, distribution, metabolism and excretion (ADME) of xenobiotics to better understand these processes. At the blood-brain barrier (BBB), these proteins are particularly important for the maintenance of brain homeostasis, but also regulate the distribution of therapeutic drugs. Absolute quantification (AQUA) is achieved by using stable isotope labeled surrogate peptides specific to the target protein and analyzing the digested proteins in a triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode to achieve a high specificity, sensitivity, accuracy and reproducibility. The main objective in this work was to develop and validate an UHPLC-MS/MS method for quantification of the ATP-binding cassette (ABC) transporter proteins Bcrp and P-gp and Na+/K + ATPase pump at the BBB. Three isoforms of the α-subunit from this pump (Atp1a 1, 2 and 3) were quantified to evaluate the presence of non-endothelial cells in the BBB using one common and three isoform-specific peptides; while Bcrp ad P-gp were quantified using 2 and 3 peptides, respectively, to improve the confidence on their quantification. The protein digestion was optimized, and the analytical method was comprehensively validated according to the American Food and Drug Administration Bioanalytical Method Validation Guidance published in 2018. Linearity across four magnitude orders (0.125 to 510 pmol·mL-1) sub-pmol·mL-1 LOD and LOQ, accuracy and precision (deviation < 15% and CV < 15%) were proven for most of the peptides by analyzing calibration curves and four levels of quality controls in both a pure solution and a complex matrix of digested yeast proteins, to mimic the matrix effect. In addition, digestion performance and stability of the peptides was shown using standard peptides spiked in a yeast digest or mouse kidney plasma membrane proteins as a study case. The validated method was used to characterize mouse kidney plasma membrane proteins, mouse brain cortical vessels and rat brain cortical microvessels. Most of the results agree with previously reported values, although some differences are seen due to different sample treatment, heterogeneity of the sample or peptide used. Importantly, the use of three peptides allowed the quantification of P-gp in mouse kidney plasma membrane proteins which was below the limit of quantification of the previously NTTGALTTR peptide. The different levels obtained for each peptide highlight the importance and difficulty of choosing surrogate peptides for protein quantification. In addition, using isoform-specific peptides for the quantification of the Na+/K + ATPase pump, we evaluated the presence of neuronal and glial cells on rat and mouse brain cortical vessels in addition to endothelial cells. In mouse liver and kidney, only the alpha-1 isoform was detected.

Colorimetric immunoassays for the screening and specificity evaluation of molecules disturbing VEGFs/VEGFRs interactions.

Angiogenesis and its involved proteins, particularly Vascular Endothelial Growth Factor family (VEGFs) and VEGF receptors (VEGFRs), have been considered as a target of therapeutic interest for numerous inflammatory and vascular diseases. Acting on this biological process through interaction with VEGFs or VEGFRs has received considerable attention. Indeed, VEGFs and VEGFRs are currently targeted by drugs such as monoclonal antibodies. The feasibility of a therapeutic strategy based on blocking the VEGF/VEGFR interaction by using ligands "other-than-biologics" is also explored. To help to the discovery of new molecules, screening assays have been developed, particularly to evaluate the VEGFA/VEGFR1 interaction. Despite the therapeutic importance of VEGFB and PlGF (Placental Growth Factor), no assays have been developed to evaluate molecules against their interactions with VEGFR1. Here, we present new versatile colorimetric immunoassays to screen and evaluate the specific interaction of discovered molecules with different growth factors (VEGFA, VEGFB, PlGF) and receptors (VEGFR1, VEGFR2). These tests, based on competitive immunoassay format, will provide essential information on specificity and selectivity of molecules for their targets and will help to work on the pharmaco-modulation of molecules for targeting one specific interaction.

Synthesis and characterization of water-soluble macrocyclic peptides stabilizing protein α-turn.

Short peptides composed of naturally occurring amino acids are usually unstructured in aqueous media. The installation of covalent constraints within their side chains or backbones, resulting in the formation of macrocyclic peptides, is an appealing approach to stabilize them in defined secondary structures. Therefore, with the objective to stabilize α-turn conformation, we designed, synthesized and characterized constrained 13-membered macrocyclic peptides. Their design was inspired by previous work using the replacement of a hydrogen bond by a covalent bond, for the stabilization of α-helical secondary structures. Their synthesis employed our recently published solid-phase method based on Fukuyama-Mitsunobu alkylation reactions. We report herein an optimized synthesis leading to three water-soluble 13-membered macrocyclic peptides 10a-c, including respectively two, one and zero glycine residues. They were characterized by CD and NMR, which indicated the presence of equilibrating conformers. The detailed conformational analysis was based on extensive NMR and molecular dynamics studies. We found that the peptide without glycine residues 10c was mostly present as slowly interconverting conformers whereas the peptide with two glycine residues 10a was mostly present as rapidly interconverting conformers. We did not find a good match between the conformers of 10a and α-turns occurring in proteins, due to the high flexibility of the glycine backbone. Interestingly, we found that the major conformer of 10c accurately matched the "non-classical" or "tight" α-turn of type II-αLS, with a RMSD value of 0.42 Å for heavy atoms constituting the macrocycle. This is, to the best of our knowledge, the first molecule reported to mimic this type of α-turn found in proteins.

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.

Identification of Peptidic Antagonists of Vascular Endothelial Growth Factor Receptor 1 by Scanning the Binding Epitopes of Its Ligands.

Cancer angiogenesis is mainly initiated by vascular endothelial growth factors (VEGFs). On the basis of the reported crystal structures of three natural ligands (VEGF-A, -B, and PlGF) with the major receptors VEGFR-1 and VEGFR-2, we scanned receptor-binding epitopes of these ligands by designing linear and cyclic peptides with the aim to disrupt the VEGF-A/VEGFR-1 interaction, which is implicated in cancer development. The ability of peptides to inhibit this interaction was evaluated by an ELISA-based assay. Several peptides, especially those mimicking loop 1 (L1) of these ligands that binds primarily to domain D3 of VEGFRs, have demonstrated higher inhibition for VEGF-A/VEGFR-1 binding. They have also shown inhibitory effects on VEGF-induced tube formation in HUVECs (human umbilical vein endothelial cells). These results validate the domain D3 of VEGFRs as an efficient target for the design of VEGFR antagonists.

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels.

Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per µg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels. Peptide peak identities were evaluated using a spectral library and chromatographic parameters. Sprague-Dawley rat microvessels obtained on three different days were analyzed with this method complemented by an absolute quantification multiple reaction monitoring method for transporter proteins P-gp, Bcrp, and Na+ /K+ ATPase pump using stable isotope labeled peptides as internal standard. Inter-day differences in the cell markers and BSA contamination were observed. Levels of cell markers correlated positively between each other. Then, the correlation between cell marker proteins and transporter proteins was evaluated to choose the best EC marker protein for protein quantification normalization. The membrane protein Pecam-1 showed a very high correlation with the EC-specific transporter P-gp (Pearson product-moment correlation coefficient (r) > 0.89) and moderate to high with Bcrp (r ≥ 0.77), that can be found also in pericytes and astrocytes. Therefore, Pecam-1 was selected as a marker for the normalization of the quantification of the proteins of endothelial cells.

[18F]MEL050 as a melanin-targeted PET tracer: Fully automated radiosynthesis and comparison to 18F-FDG for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases.

INTRODUCTION: Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [18F]MEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of [18F]MEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of [18F]FDG. METHODS: Automated radiosynthesis of [18F]MEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module. [18F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of [18F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to [18F]FDG and correlated to in vivo bioluminescence imaging. RESULTS: The automated radiosynthesis of [18F]MEL050 required an overall radiosynthesis time of 48min, with a yield of 13-18% (not-decay corrected) and radiochemical purity higher than 99%. [18F]MEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with [18F]MEL050 and [18F]FDG in subcutaneous tumors and higher TBR with [18F]MEL050 than with [18F]FDG in pulmonary metastases. CONCLUSION: We successfully implemented the radiosynthesis of [18F]MEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of [18F]MEL050 was obtained in mouse models of pigmented melanoma, where higher [18F]MEL050 uptake was observed in sub-millimetric pulmonary metastases, comparatively to [18F]FDG.

TGF-ß2 induces Grb2 to recruit PI3-K to TGF-RII that activates JNK/AP-1-signaling and augments invasiveness of Theileria-transformed macrophages.

Theileria-infected macrophages display many features of cancer cells such as heightened invasive capacity; however, the tumor-like phenotype is reversible by killing the parasite. Moreover, virulent macrophages can be attenuated by multiple in vitro passages and so provide a powerful model to elucidate mechanisms related to transformed macrophage virulence. Here, we demonstrate that in two independent Theileria-transformed macrophage cell lines Grb2 expression is down-regulated concomitant with loss of tumor virulence. Using peptidimer-c to ablate SH2 and SH3 interactions of Grb2 we identify TGF-receptor II and the p85 subunit of PI3-K, as Grb2 partners in virulent macrophages. Ablation of Grb2 interactions reduces PI3-K recruitment to TGF-RII and decreases PIP3 production, and dampens JNK phosphorylation and AP-1-driven transcriptional activity down to levels characteristic of attenuated macrophages. Loss of TGF-R>PI3-K>JNK>AP-1 signaling negatively impacts on virulence traits such as reduced JAM-L/ITG4A and Fos-B/MMP9 expression that contribute to virulent macrophage adhesion and invasiveness.

Disruption of phactr-1 pathway triggers pro-inflammatory and pro-atherogenic factors: New insights in atherosclerosis development.

Significant interest has recently emerged for phosphatase and actin regulatory protein (PHACTR1) gene in heart diseases prognosis. However, the functional role of phactr-1 protein remains elusive in heart related-diseases such as atherosclerosis, coronary artery calcification, ischaemic stroke, coronary artery stenosis and early-onset myocardial infarction. Phactr-1 is directly regulated by vascular endothelial growth factor A165 (VEGF-A165) through VEGF receptor 1 (VEGR-1) and Neuropilin-1 (NRP-1). Using an antagonist peptide approach to inhibit the interaction of VEGF-A165 to NRP-1 and VEGF-R1, we highlighted the importance of both cysteine residues located at the end of VEGF-A165 exon-7 and at the exon-8 to generate functional peptides, which decreased Phactr-1 expression. Here, we report original data showing Phactr-1 down-expression induces the expression of Matrix Metalloproteinase (MMP) regulators such as Tissue inhibitor of metalloproteinase (TIMP-1/-2) and Reversion-inducing-cysteine-rich protein with kazal motifs (RECK). Furthermore, focal adhesion kinases (FAK/PYK2/PAXILLIN) and metabolic stress (AMPK/CREB/eNOS) pathways were inhibited in endothelial cells. Moreover, the decrease of phactr-1 expression induced several factors implicated in atherosclerotic events such as oxidized low-density lipoprotein receptors (CD36, Clusterin, Cadherin-13), pro-inflammatory proteins including Thrombin, Thrombin receptor 1 (PAR-1), A Disintegrin And Metalloprotease domain-9/-17 (ADAM-9/-17), Trombospondin-2 and Galectin-3. Besides, Phactr-1 down-expression also induces emerging atherosclerosis biomarkers such as semicarbazide-sensitive amine oxidase (SSAO) and TGF-beta-inducible gene h3 (ßIG-H3). In this report, we show for the first time the direct evidence of the phactr-1 biological function in the regulation of pro-atherosclerotic molecules. This intriguing result strengthened heart diseases PHACTR-1 single-nucleotide polymorphisms (SNP) correlation. Taken together, our result highlighted the pivotal role of phactr-1 protein in the pathogenesis of atherosclerosis.

Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards.

BACKGROUND: For the last 40 years, the technique of extracorporeal photopheresis (ECP) has constantly developed. Among irradiation systems, those called "off-line" allow the validation of the quality of the cell therapy product. The inhibition of the proliferation of lymphocytes after ultraviolet irradiation (UVA) is usually verified by the tritiated thymidine assay as in vitro proliferation assay. The document presented here describes the results obtained while performing the setting up of an alternative proliferation assay using flow cytometry according to ISO 15189:2007 Standard. METHODS: Cells samples taken before and after UVA irradiation were labeled with CarboxyFluorescein Succinimidyl Ester (CFSE) and then cultured with phytohemagglutinin-A (PHA). After 3 days, an analysis of the CFSE staining was realized by flow cytometry. In order to validate the shift in the method used according to Standard, the following tests were performed: 1) comparison with the reference method, 2) robustness test, 3) reagents stability. RESULTS: Comparison method demonstrated that the sensitivity of the CFSE test is 100%, the specificity is 89%, and the concordance is almost complete. The CFSE test is robust regarding parameters like cell concentration or PHA concentration. PHA and CFSE are stable for 6 months and one year, respectively. CONCLUSION: Validation of this alternative test, according to the ISO 15189:2007 Standard, has demonstrated good concordance with reference method. The results of the robustness and stability of reagents are appropriate for its routine use. Thus, the benefits of alternative technique make it a wise choice for the quality control of ECP in a cell therapy laboratory.

Design and synthesis of C-terminal modified cyclic peptides as VEGFR1 antagonists.

Previously designed cyclic peptide antagonist c[YYDEGLEE]-NH2 disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). It represents a promising tool in the fight against cancer and age-related macular degeneration. We described in this paper the optimization of the lead peptide by C-terminal modification. A new strategy for the synthesis of cyclic peptides is developed, improving the cyclisation efficiency. At 100 µM, several new peptides with an aromatic group flexibly linked at C-terminal end showed significantly increased receptor binding affinities in competition ELISA test. The most active peptide carrying a coumarin group may be a useful tool in anti-angiogenic biological studies.

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.

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.