Discovery of novel small molecule inhibitors of VEGF expression in tumor cells using a cell-based high throughput screening platform.

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal homeostatic state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5\u27 and 3\u27 untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies

Genomic and transcriptomic analysis of the Asian honeybee Apis cerana provides novel insights into honeybee biology

The Asian honeybee Apis cerana is one of two bee species that have been commercially kept with immense economic value. Here we present the analysis of genomic sequence and transcriptomic exploration for A. cerana as well as the comparative genomic analysis of the Asian honeybee and the European honeybee A. mellifera. The genome and RNA-seq data yield new insights into the behavioral and physiological resistance to the parasitic mite Varroa the evolution of antimicrobial peptides, and the genetic basis for labor division in A. cerana. Comparison of genes between the two sister species revealed genes specific to A. cerana, 54.5% of which have no homology to any known proteins. The observation that A. cerana displayed significantly more vigilant grooming behaviors to the presence of Varroa than A. mellifera in conjunction with gene expression analysis suggests that parasite-defensive grooming in A. cerana is likely triggered not only by exogenous stimuli through visual and olfactory detection of the parasite, but also by genetically endogenous processes that periodically activates a bout of grooming to remove the ectoparasite. This information provides a valuable platform to facilitate the traits unique to A. cerana as well as those shared with other social bees for health improvement

Discovery of Novel Small Molecule Inhibitors of VEGF Expression in Tumor Cells Using a Cell-Based High Throughput Screening Platform

<div><p>Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal “homeostatic” state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5’ and 3’ untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS^™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies.</p></div

PTC-858 selectively reduces hVEGF in an HT1080 pharmacodynamic model.

<p>PTC-858 selectively reduces hVEGF in an HT1080 pharmacodynamic model.</p

Structure and biological activity of a representative HTS hit, PTC-858.

<p>(<b>A</b>) Chemical structure of PTC-858 (6-bromo-1-(1H-pyrrol-1-yl)-2,3,4,9-tetrahydro-1H-carbazole). (<b>B</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). (<b>C</b>) 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. (<b>D</b>) 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). (<b>E</b>) 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.</p

PTC-858 effectively reduced tumor growth in a chick embryo model.

<p>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 <i>P</i> < 0.05, unpaired t-test vs vehicle.</p

Selectivity and cytotoxicity of two HTS hits.

<p>Selectivity and cytotoxicity of two HTS hits.</p

PTC-510 is an improved analog of the HTS hit PTC-031 and effectively controls growth in HT1080 tumor xenografts in mice.

<p>(<b>A</b>) Chemical structures of the HTS hit PTC-031 and its improved analog PTC-510. (<b>B</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. (<b>C</b>) PTC-510 reduces levels of high molecular weight VEGF. (<b>D</b>) Oral administration of PTC-510 delays tumor growth of HT1080 human tumor xenografts in athymic nude mice. * <i>P</i> < 0.01, One-way ANOVA test, n = 10.</p