Structural Basis Of Nitrogen Heterocyclic Podophyllotoxin Derivatives Targeting Tubulin

Tubulin is an important component of the spindle,which plays a critical role in the process of cell mitosis.Compared with normal cells,tumor cells have the property of infinite proliferation.Therefore,tubulin has become one of the important targets of antitumor drugs.Podophyllotoxin is the main pharmacodynamic component of podophyllotoxin in traditional Chinese medicine.Pharmacological mechanism studies have shown that podophyllotoxin binds to the colchicine site of tubulin and exerts antitumor effects.Previous studies have discovered podophyllotoxin lead compounds with good antitumor activity by targeting a tubulin,however,the molecular mechanism of its enhanced anti-tumor activity is not yet clear.By analyzing the crystal structure of podophyllotoxin derivatives and tubulin complex to reveal the structural basis of its interaction with tubulin,which is helpful to provide a theoretical basis for the design of other drug molecules targeting tubulin.In this paper,nitrogen-containing heterocyclic podophyllotoxin derivatives with nanomolar antitumor activity were taken as the research object to carry out the structural basic research on their targeted inhibition of tubulin.Tubulin complexes T2R-TTL-QAP and T2RTTL-IMP containing quinoline podophyllotoxin(QAP)and pyrimidine podophyllotoxin(IMP)were obtained,respectively.By means of protein crystal culture and X-ray diffraction,the T2RTTL-QAP complex crystals with a resolution of 2.7 A and T2R-TTL-IMP complex crystals with a resolution of 2.89 A were obtained.Then,by analyzing the crystal structure of the complex,it was found that the podophyllotoxin derivative QAP and IMP containing nitrogen heterocyclic ring were bound to the colchicine site between the αβ tubulin heterodimer,the podophyllotoxin parent nucleus was deeply buried in the β-tubulin,and the quinoline substituted at the C-ring 4 site was located at the junction of the αβ heterodimer,and reached into the α-tubulin.By comparing the apo structure of the T2R-TTL protein complex without any compound,it was found that the introduction of QAP and IMP deflected the T7 ring of β-tubulin and the T5 ring of α-tubulin.To analyze the structural differences between podophyllotoxin derivatives containing different nitrogen heterocycles with tubulin,the crystal structures of T2R-TTL-QAP and T2R-TTL-IMP were compared with those of triazoline podophyllotoxin(THP)obtained earlier.Among them,QAP connected by carbon-nitrogen bond has the weakest interaction with tubulin,resulting in the least conformational change;IMP and THP connected by carbon-sulfur bond have stronger interaction with tubulin,resulting in greater conformational change,and THP is greater than IMP.Finally,the results of the investigation on tubulin affinity,intracellular and extracellular tubulin polymerization and cell cycle arrest showed that consistent with the differences in the structural effects of the three compounds,the affinity of podophyllotoxin derivatives to tubulin,the effect of inhibiting microtubule polymerization,and the effect of causing cell cycle arrest,THP is greater than IMP is greater than QAP.This paper focuses on the structural basis of targeted inhibition of tubulin by nitrogencontaining heterocyclic podophyllotoxin derivatives.It was revealed that podophyllotoxin derivatives inhibited the conformation change of microtubules from crooked to straight by changing the conformation of tubulin βT7 and αT5 rings,thus inhibiting the polymerization of microtubules,causing the disorder of spindle behavior during mitosis,cell arrest in G2/M phase,and finally inducing cell apoptosis.According to the structural and functional differences generated by the introduction of different nitrogen heterocycles,we analyzed that the introduction of C-S bonding substituents which can strongly interact with a tubulin at the Cring 4 position of podophyllotoxin may be an important direction for the subsequent optimization of podophyllotoxin targeting a tubulin.