Podophyllotoxin derivatives-tubulin complex reveals a potential binding site of tubulin polymerization inhibitors in α-tubulin adjacent to colchicine site

The colchicine site of beta-tubulin has been proven to be essential binding sites of microtubule polymerization inhibitors. Recent studies implied that GTP pocket of alpha-tubulin adjacent to colchicine sites is a potential binding site for developing tubulin polymerization inhibitors. However, the structural basis for which type of structural fragments was more beneficial for enhancing the affinity of alpha-tubulin is still unclear. Here, podophyllotoxin derivatives-tubulin complex crystals indicated that heterocyclic with the highly electronegative and small steric hindrance was conducive to change configuration and enhance the affinity of the residues in GTP pocket of alpha-tubulin. Triazole with lone-pairs electrons and small steric hindrance exhibited the strongest affinity for enhancing affinity of podophyllotoxin derivatives by forming two hydrogen bonds with alpha T5 Ser178. Pyrimidine with the secondary strong affinity could bind Asn101 to make the alpha H7 configuration deflection, which reduces the stability of tubulin result in its depolymerization. Conversely, 4 beta-quinoline-podophyllotoxin with the weakest affinity did not interact with alpha-tubulin. The molecular dynamics simulation and protein thermal shift results showed that 4 beta-triazole-podophyllotoxin-tubulin was the most stable mainly due to two hydrogen bonds and the higher van der Waals force. This work provided a structural basis of the potential binding sites for extending the alpha/beta-tubulin dual-binding sites inhibitors design strategy.