Pharmaceutical compositions for preventing or treating angiogenic diseases comprising inhibitors of NUP153 gene expression or NUP153 activity as active ingredient

#This application claims priority to and the benefit of Korean Patent Application No. 10-2013-0085670, filed on Jul. 19, 2013, the disclosure of which is incorporated herein by reference in its entirety.

#Field of the Invention

#The present invention relates to a pharmaceutical composition for preventing or treating angiogenic diseases including inhibitors of NUP153 gene expression or NUP153 activity as active ingredient and a method for screening an agent for preventing or treating angiogenic diseases.

#FIGS. 1A through 1E represent effect of OMe-Syn on ISV angiogenesis in fli1a:EGFP transgenic zebrafish embryos. FIG. 1A shows light microscopic images of whole embryos are shown at 30 hpf: (a) DMSO-treated, (b) OMe-Syn (2.5 μM)-treated, and (c) OMe-Syn (5 μM)-treated embryos. FIG. 1B shows fluorescent images showing dose-dependent OMe-Syn suppression of ISV formation: (a) DMSO-treated, (b) OMe-Syn (2.5 μM)-treated, and (c) OMe-Syn (5 μM)-treated embryos. The drawings were selected as representative data from three independent experiments. FIG. 1C shows quantitative analysis of complete ISVs and angiogenic sprouts at 30 hpf. *p<0.005 versus control. Each value represents the mean±SE from three independent experiments. FIG. 1D shows effect of OMe-Syn-treated embryos on viability at 30 hpf, showing no difference in the survival rate following OMe-Syn treatment. FIG. 1E shows that the VEGF mRNA level decreases with OMe-Syn treatment as detected by RT-PCR. β-actin was used as an internal control.

#FIGS. 2A through 2E represent identification of OMe-Syn-binding protein using phage display biopanning. FIG. 2A shows the structure of OMe-Syn. FIG. 2B shows the structure of the biotinylated molecular probe of OMe-Syn (biotinyl-OMe-Syn). FIG. 2C shows the effect of OMe-Syn and biotinyl-OMe-Syn on the proliferation of HUVECs. Cells were treated with the compounds (0-50 μM) for 72 hours, and then cell growth was measured using the MTT colorimetric assay. A similar reduction in cell growth was observed in both treatment groups. FIG. 2D shows the result of phage display biopanning. “Wash” indicates non-specific phages bound to biotinyl-OMe-Syn immobilized on a streptavidin-coated well. FIG. 2E shows the OMe-Syn binding domain of NUP153. OMe-Syn interacts with the mRNA binding site in NUP153. The phage sequences were 100% identical to the N-terminus (amino acids 245-326) of human NUP153 (SEQ ID NO:4).

#FIGS. 3A to 3D represent validation of the interaction of OMe-Syn and NUP153. FIG. 3A shows the phage binding assay of OMe-Syn. Among isolated phages, NUP153-expressing phage specifically bound to OMe-Syn and not to biotin. CaM- and ARMET-expressing phages, which were other possible binding phages of OMe-Syn, did not bind to OMe-Syn. FIG. 3B shows the phage competition assay using NUP153-expressing phage. After pre-incubation with OMe-Syn (50, 100 μM) and NUP153-expressing phage for 1 hour, the phage was immobilized in the OMe-Syn well. FIG. 3C shows the SPR analysis of the interaction between OMe-Syn and recombinant NUP153 (10-27 μM). Bovine serum albumin (BSA) was used as a negative control. FIG. 3D shows the pull-down assay of biotinyl-OMe-Syn and NUP153 using HUVEC lysates. After the lysates bound to biotin or biotinyl-OMe-Syn, the binding complex was pulled down using streptavidin-agarose beads. The supernatant (sup) and bead fractions were detected by western blotting using an antibody against NUP153.

#In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating angiogenic diseases including inhibitors of NUP153 (nucleoporin 153 kDa) gene expression or NUP153 activity as active ingredient.

#The present inventors have made intensive efforts to develop a pharmaceutical composition for preventing or treating angiogenic diseases. The present inventors have made intensive efforts to develop a pharmaceutical composition for preventing or treating angiogenic diseases. As a result, the present inventors have found out that NUP153 is a target protein of OMe-Syn inhibiting VEGF-induced angiogenesis, an expression of pro-angiogenic factors is reduced and angiogenesis is inhibited through the mechanism of inhibiting invasion and tube formation when the NUP153 gene expression are down-regulated.

#R-(−)-β-O-Methylsynephrine (OMe-Syn), a natural small molecule isolated from plants of the Rutaceae family, was reported to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo (Kim et al., 2010). The simple and unique chemical structure, biological activity, and good membrane permeability strongly implicate OMe-Syn as a useful seed for chemical genetic probe and drug discovery. Using a biotinylated affinity probe of OMe-Syn and a T7 phage display of human cDNA-expressed proteome library, the present inventors identified nucleoporin 153 kDa (NUP153) as one of the binding proteins of the compound.