Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens
Fig 5
Network-based elucidation of a novel tubulin-targeting chemotype.
(A) In-vitro 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 6 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 6 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 6 within the colchicine-binding site revealed a hydrophobic sub-pocket enclosed by Leu-248 and Lys-352. The model showed that compounds 7 and 8 enhance binding affinity by fitting the N-propyl and N-phenyl group in the hydrophobic cavity, consistent with the SAR analysis. See S11 Fig for molecular modeling of compounds 6–12. (F) The most potent compound 8 was tested for direct colchicine site binding using mass spectrometry competitive binding assays. Compound 8 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 6–8 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 6–8 depolymerize microtubules. See S10 Fig for compound 6–12 induced phenotypes.