Network-based elucidation of a novel tubulin-targeting chemotype.

<p>(A) <i>In-vitro</i> tubulin polymerization assays were used to test the effect of the 212 mitotic compounds on microtubule assembly at 50μM concentration. The end-point absorbance, based on change in OD (dOD), was used to quantify the degree of microtubule polymerization and was converted to percentage fold change relative to DMSO (0%). Among the tested compounds, 134 compounds (63%) had an effect (>20% fold change) on tubulin polymerization. (B) Chemical similarity sub-network consisting of 7 novel anti-tubulin ligands based on a phenyl-sulfanyl-thiazol-acetamide privilege scaffold. The connected analogues within the network showed a consensus tubulin destabilization effect where each step in the path (red) of the sub-network corresponded to a minimum structural change correlating with the observed structure-activity-relationship (SAR). (C) Docking of compound <b>6</b> into the β-tubulin colchicine-binding site based on the crystal structure (PDB: 1AS0) exhibited a similar predicted binding mode to colchicine. (D) Ligand alignment between compound <b>6</b> and colchicine identified a conserved pharmacophore critical for ligand binding, including the 2 and 10-methoxy groups and a 9-keto group that interacts with Cys-241 of beta tubulin and Val-181 (not shown) of alpha tubulin respectively. (E) Hydrophobicity map of docked compound <b>6</b> within the colchicine-binding site revealed a hydrophobic sub-pocket enclosed by Leu-248 and Lys-352. The model showed that compounds <b>7</b> and <b>8</b> enhance binding affinity by fitting the N-propyl and N-phenyl group in the hydrophobic cavity, consistent with the SAR analysis. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004153#pcbi.1004153.s011" target="_blank">S11 Fig</a> for molecular modeling of compounds <b>6–12</b>. (F) The most potent compound <b>8</b> was tested for direct colchicine site binding using mass spectrometry competitive binding assays. Compound <b>8</b> competed strongly with colchicine for the colchicine-binding site, similar to the colchicine-site binder podophyllotoxin. Note that the negative control vincristine did not compete. (G) Immunofluorescence microscopy images of HeLa cells treated with DMSO, taxol, colchicine or compounds <b>6–8</b> for 20 hours. Cells were fixed and stained for DNA (Hoechst 33342) and tubulin (primary rat anti-tubulin antibodies and secondary anti-rat Cy3 antibodies). Scale = 5 μm. Note that colchicine, and compounds <b>6–8</b> depolymerize microtubules. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004153#pcbi.1004153.s010" target="_blank">S10 Fig</a> for compound <b>6–12</b> induced phenotypes.