Figure 3

VLX40 is a tubulin active agent. (A) Microarray based mechanistic evaluation using Connectivity Map (cmap). MCF-7 cells were exposed to VLX40 for 6 h as described in experimental procedures. Out of the 6100 drug specific profiles in the data base, the eight most similar were all derived from compounds known to be tubulin inhibitors. 5252917 corresponds to N-(2-benzooxazol-2-yl-phenyl)-4-methyl-benzenesulfonamide. Score according to cmap data base. (B) Gene Set Enrichment Analysis (GSEA) shows significant up-regulation of genes involved in mitosis. The pre-ranked gene list (VLX40 exposed MCF-7 cells vs. untreated control) was compared to a priori defined and curated gene sets. The purpose of GSEA is to find out whether the a priori defined gene sets are significantly enriched towards the upper or lower end of the pre-ranked list. The p-value refers to the nominal p-value after 1000 permutations. (C) Phospho-histone H3 staining using Arrayscan VTI (total intensity) after exposure to VLX40 for 24 hrs in HCT 116 cells. (D) Analysis of cell cycle distribution after 24 hrs exposure to VLX40 in DAPI stained RPMI 8226, 8226/Dox40, U-937 and HL-60 cells. (E) Confirmation of tubulin inhibition as the mechanism of action of VLX40 using a cell free assay for tubulin polymerization. Vincristine (3 μM) and paclitaxel (3 μM) were used as reference compounds.