Fig 1.
Identification of small molecule inhibitors of VEGF UTRs-mediated reporter expression via GEMS™ platform.
(A) Schematic representation of the VEGF GEMS™ vector. The firefly luciferase reporter flanked with the VEGF 5’- and 3’-UTR was cloned into the pCDNA3.1 between the unique restriction sites BamH1 and Not I. (B) A scattergram for results from a typical HTS plate. HTS was conducted in 384-well plates, and compounds were assayed at a concentration of 7.5 μM in duplicate. The red dots represent the dose-response of puromycin control, while the purple dots represent the blank control. Two hits (> 52% inhibition of luciferase expression) identified in this plate are shown in the green circles. (C) Statistic summary of the VEGF high throughput screening. A total of approximately 150,000 compounds was screened, of which 756 hits were identified using a mean inhibition of + 3 times of standard deviation as cut-off. CMV P: Human cytomegalovirus promoter; BGH-pA: Bovine Growth Hormone Polyadenylation Signal.
Fig 2.
Structure and biological activity of a representative HTS hit, PTC-858.
(A) Chemical structure of PTC-858 (6-bromo-1-(1H-pyrrol-1-yl)-2,3,4,9-tetrahydro-1H-carbazole). (B) Inhibition of VEGF production in HeLa cells by PTC-858. Results are expressed as percent inhibition of hypoxia-induced VEGF production relative to vehicle-treated controls from a representative study. Cytotoxicity was performed in parallel to the VEGF ELISA using a Celltiter Glo assay kit (Promega). (C) Western blot analysis of effects of PTC-858 on membrane bound VEGF expression in HT-1080 cells. The same amount of protein for each cell lysate was loaded for western blot analysis. β-actin was used as an internal loading control. (D) PTC-858 preferentially inhibits reporter gene expression under the control of VEGF UTRs compared to the control UTRs derived from the vector. The stable cell lines B9 and B12 used in this study were generated in HEK293 cells transfected with the constructs shown in the diagrams on the top of the graphs. The activity of luciferase was measured with the substrate Bright-Glow (Promega). (E) PTC-858 has no dose-dependent effects on VEGF mRNA expression. Endogenous VEGF and β-actin message levels in the HeLa cells were determined using real-time PCR (pre-designed primer sets purchased from Applied Biosystems). Results shown in the graph from a typical study were normalized to the internal control of β-actin. Assay was done in triplicate and average VEGF mRNA levels from DMSO treated samples were arbitrarily set at 1.
Table 1.
Selectivity and cytotoxicity of two HTS hits.
Fig 3.
PTC-858 effectively reduced tumor growth in a chick embryo model.
A375 human melanoma cells (5 x 106) were inoculated into the CAM of 10-days-old chick embryos. After 24 h, the embryos received a single intravenous injection of test compound or vehicle control (5% Cremaphor and 5% ethanol in PBS). At 6 days after the single dose, tumors were excised and weighed. Data represent means ± SD, n = 7. * indicates P < 0.05, unpaired t-test vs vehicle.
Table 2.
PTC-858 selectively reduces hVEGF in an HT1080 pharmacodynamic model.
Fig 4.
PTC-510 is an improved analog of the HTS hit PTC-031 and effectively controls growth in HT1080 tumor xenografts in mice.
(A) Chemical structures of the HTS hit PTC-031 and its improved analog PTC-510. (B) Inhibition of VEGF production in vitro by PTC-510 is significantly improved compared to PTC-031. PTC-510 and PTC-031 were tested side-by-side in HeLa cells under hypoxia. Each data point represents average % inhibition of VEGF expression +/- SD. (C) PTC-510 reduces levels of high molecular weight VEGF. (D) Oral administration of PTC-510 delays tumor growth of HT1080 human tumor xenografts in athymic nude mice. * P < 0.01, One-way ANOVA test, n = 10.