Figure 6. In vitro and in vivo testing of close analogs of the novel active compound, NCGC00117362.

a. Chemical structure of NCGC00117362 and 4 analogs. b. Chemical structure of 3 inert control compounds. c-e. Secondary in vitro screens were performed. The effect of compounds at 4-doses (1, 2.5, 5 and 10 μM) was tested in three ovarian cancer (OvCa) cell lines. c, e. OvCa cell adhesion (2h; c) and proliferation (96h; e) were tested in 384-well plates on the 3D organotypic culture. d. OvCa invasion (24–48h) was tested using a 96-well Boyden chamber lined with collagen type I as ECM and the 3D organotypic culture. f-g. Secondary in vivo screens were performed. f. Adhesion/Invasion Assay. The analog compounds were tested at a 5μM dose. Luciferase-labeled ID8p53−/− cells (5 million) were mixed with the indicated compound and injected into C57BL/6 mice. The mice were sacrificed at 16h and the luciferase signal in the omentum was measured using a luminometer. g. Prevention Metastasis Assay. Five million Ovcar5 cells were injected i.p. with individual compounds (5 μM), and i.p. treatment continued at 48 and 96 h post-cancer cell injection (10 mg/kg or equal volume of dimethyl sulfoxide (DMSO), n=5). Forty-five days post cancer cell injection the weight and number of tumors determined. Mean+/− standard deviation. *, p<0.05. **, p<0.01. ***, p<0.001. ****, p<0.0001, n=4–8.