Structural Insights Into The Inhibitor Of Microtubule Polymerization By N-heterocyclic Ring Substituted Podophyllum Derivatives With Antitumor Activity

Cancer has become an important disease threatening human health.Microtubules play a key role in cell division and are a highly successful target for anticancer therapy research and development.Microtubule polymerization inhibitors inhibit microtubule polymerization by targeting tubulin,disturb the normal process of microtubule depolymerization-polymerization dynamic equilibrium,disorder the mitotic process of cancer cells,lead to cell cycle arrest,and ultimately induce cell apoptosis to play its anti-tumor role.Therefore,from the perspective of structural biology,it is of great significance to analyze the crystal structure of drug-tubulin complex by X-ray diffraction technology,to analyze the interaction between tubulin and microtubule polymerization inhibitors at atomic level,and to design microtubule polymerization inhibitors with high anti-tumor activity and low cytotoxicity based on structural guidance.According to the principle of bioelectronics and structure-based drug design,a series of nitrogen-containing heterocycles podophyllum derivatives with high activity and low toxicity were screened by targeting tubulin and C-NH modification at the fourth position of the C ring of podophyllotoxin.Using HepG2 cell lines of human liver carcinoma,HeLa cell lines of human cervical carcinoma,A549 cell lines of human lung cancer,and MCF-7 cell lines of human breast cancer as test cells respectively,compounds 6-IA-PTOX,5-ID-PTOX and 6-QL-PTOX were found to have better anti-tumor activity,reaching nanomolar level,when compared with Nocodazole,a well-known inhibitor of microtubule polymerization.The cytotoxicity to HL-7702 of normal human liver cell lines was lower.From the perspective of structural biology,the interactions of 6-IA-PTOX,5-ID-PTOX and 6-QL-PTOX with tubulin were analyzed at the atomic level to explain their action mechanism.Microtubulin was extracted and purified from mammalian brain tissue（fresh porcine brain）.Two microtubule-associated proteins,RB3-SLD and TTL,were obtained by recombinant expression in vitro.Then the tubulin-podophyllotoxin derivatives（T2R-TTL-PTOXs）complex crystals were cultured by co-crystallization.By optimizing the culture conditions,under the conditions of 100 mM MES（pH 5.7）,30mM MgCl₂,30 mM CaCl₂,7%PEG 4000,6%glycerol,and adding 3.3%Jeffamine M-600（pH 7.0）,the complex single crystals conforming to X-ray diffraction were obtained,with 2.4?resolution.Through structural analysis,no structure of small molecules of three compounds was found in the well-known podophyllotoxin binding pocket,the tubulin colchicine binding site,but the cavity of the binding site was sufficient to accommodate the compound.It may be that the compound has acted and then pulled out of the crystal.By comparison the structure of tubulin and compound with the tubulin structure without compound,it was found that the overall structure did not change obviously.Only the conformation ofαT5 andβT7 loop was deflected,andαT5 loop andβT7 loop participat in the structural changes during tubulin depolymerization-polymerization.This also indicates that the compound was released from the crystal after it has acted.Comparing the structure of tubulin and compound with the tubulin in straight conformation,it was found that the T7 loop,the H7,H6 and H10 helix,and the S7-S10-S8-S9 sheet of theβ-tubulin all had a large degree of conformation outward,which hindered the tubulin transitions from curved to an straight,thereby blocking the polymerization of the microtubules.It was found that the mechanism of 6-IA-PTOX inhibiting tubulin polymerization is that the tubulin is in a curved state and cannot be polymerized,so that the spindle in the process of cell mitosis can not be formed,cell cycle arrest,cell proliferation can be inhibited,and cells are finally apoptosis.By comparing the crystal structures of different compounds with tubulin,the interaction between different functional groups and tubulin was analyzed,which provided a theoretical reference for the structural design of podophyllotoxin derivatives with higher antitumor activity.