Figure 5.

WTC-01 treatment interferes with microtubule assembly in NPC cells.In vitro (A) and ex vivo (B) analysis of WTC-01 inhibition of tubulin polymerization. (A) In vitro tubulin polymerization was performed in the absence or present of 20 nM colchicine or WTC-01 and level of polymerized tubulin was measured. (B) Cancer cells were treated either with paclitaxel (40 nM), colchicine (800 nM) or various concentrations of WTC-01 for 5.5 h and the amount of polymerized tubulin was measured by Western blots. (C) The colchicine competition-binding SPAs (upper panel) were conducted and the inhibition constants (K_i; lower panel) were calculated using the Cheng-Prusoff equation. WTC-01 strongly competes with [³H] colchicine for binding to the same pocket in tubulin (D). Overlay of the docking pose of WTC-01 (yellow) with the bound orientation of colchicine (blue) in the crystal structure of tubulin. Computer simulation results suggest WTC-01 is able to dock into the colchicine-binding site of tubulin. Some tubulin amino acid residues interacting with WTC-01 are shown as stick structures. The dashed lines indicate hydrogen-bonding interactions.