New WS9326A Congeners from <i>Streptomyces</i> sp. 9078 Inhibiting <i>Brugia malayi</i> Asparaginyl-tRNA Synthetase

Lymphatic filariasis is caused by the <i>Brugia malayi</i> parasite. Three new congeners of the depsipeptide WS9326A (<b>1</b>), WS9326C (<b>2</b>), WS9326D (<b>3</b>), and WS9326E (<b>4</b>), were isolated from <i>Streptomyces</i> sp. 9078 by using a <i>B. malayi</i> asparaginyl-tRNA synthetase (<i>Bm</i>AsnRS) inhibition assay. WS9326D specifically inhibits the <i>Bm</i>AsnRS, kills the adult <i>B. malayi</i> parasite, and does not exhibit significant general cytotoxicity to human hepatic cells, representing a new lead scaffold for antifilarial drug discovery

The effect of rBmAsnRS and VEGF on EA.hy926 cell migration was determined in Boyden chamber experiments.

<p>Data are presented as means ± SEM. The number of cells migrated in response to 1 ng/ml rBmAsnRS was 14, and 10 ng/ml rBmAsnRS caused the greatest migration (72.75) similar to 10 ng/ml VEGF. Asterisk (*) denotes significant differences (p<0.0001) for negative control vs 10 ng/ml rBmAsnRS and VEGF.</p

Hexa-histidine tagged rBmAsnRS was purified from the sonicated supernatant of IPTG induced <i>E</i>. <i>coli</i> cultures expressing the full length wild type BmAsnRS cDNA.

<p>Purified rBmAsnRS was subjected to electrophoresis in a 12%SDS-polyacrylamide gel and visualized by staining with Coomassie blue R250. Lane 1: molecular weight markers. Lane 2: purified rBmAsnRS.</p

The effect of rBmAsnRS and VEGF in endothelial ring formation.

<p>Fig 4A. Photomicrographs show the morphology of endothelial rings (arrows) formed by EA.hy926 cells in response to stimulation with 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Fig 4B. Graphical representation of the induction of endothelial rings in response to rBmAsnRS and VEGF. Data are means ± SEM. The percentage of cells forming endothelial rings in 10 ng/ml rBmAsnRS and 10 ng/ml VEGF were similar and both were significantly greater than the PBS control. Asterisk (*) denotes p<0.0001.</p

The effect of rBmAsnRS and VEGF <i>in ex vivo</i> angiogenesis.

<p>Fig 6A. Photomicrographs denote areas with angiogenesis induced by 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Angiogenesis was induced by placing sterile paper discs containing rBmAsnRS, VEGF or negative control (PBS) on the egg yolk vascular bed for 30 min, then incubated for another 24 hours. Images are representative of five different experiments. Fig 6B. Graphical representation of the fold increase in vessel length induced by 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Data are presented as means ± SEM of five experiments. The fold increase in length of blood vessel in 10 ng/ml rBmAsnRS and 10 ng/ml VEGF were significantly greater than negative controls. Asterisk (*) denotes statistically significant difference (p<0.005).</p

The effect of rBmAsnRS and VEGF on vasodilation of capillary blood vessels.

<p>Fig 7A. Images represent the change in capillary width induced by 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Images were captured after 20 minutes observation and are representative of 3 sets of experiments. Fig 7B. Graphical representation of changes in vessel width induced by rBmAsnRS and VEGF. Data are presented as means ± SEM. rBmAsnRS and VEGF treated blood vessels were both significantly dilated compared to controls. Asterisk (*) indicates statistically significant difference between control vs rBmAsnRS or VEGF (p < 0.005.).</p

The effect of rBmAsnRS and VEGF on tube formation.

<p>Fig 5A. Images indicate with arrows the endothelial tube formation in negative control, 10 ng/ml rBmAsnRS and 10 ng/ml VEGF groups. Photomicrographs were taken with 20X magnification under an inverted bright field microscope. Fig 5B. Graphical representation of the number of tubes formed by EA.hy926 cells exposed to negative control, 10 ng/ml rBmAsnRS or 10 ng/ml VEGF for 24 hours in Matrigel-coated 24-well plates. Data are presented as means ± SEM. Asterisk (*) denotes statistically significant differences compared to negative control.</p