Structural Insights Into The Molecular Mechanism Of Microtubule Destabilization By N-heterocyclic Ring Substituted Podophyllum Derivative 1S With Antitumor Activity

As an important class of specific anticancer drugs,tubulin inhibitors could induce tumor cell apoptosis by damaging tubulin structure.Many new drugs that target microtubule are in clinical trials and proven to be effective.The design and synthesis of effective microtubule-targeted agents is research hotspot of development of novel anti-tumor drug.Most of the microtubule-targeted agents have good antitumor activity in vitro.However,their clinical use has been limited by unintended side effects.Therefore,it is very significance to study the interaction between microtubule-targeted agents and tubulin by using X-ray diffraction,then based on the structures design and synthesize new potent agents.Our group synthesized a series of the N-heterocyclic aromatic ring substituted podophyllum compounds,which exhibited a marked anti-tumor activity,especially compound 1S.Compared with the typical microtubule-targeted agents colchicine,the anticancer activity in the tumor cell lines of HeLa,BGC-823 and A549 was significantly enhanced,the rate of cell cycle arrest and apoptosis in these tumor cell lines was significantly increased.What is more,the toxic-side effect on normal human cell line HK-2 was decreased,the damage of microtubule in the tumor cell lines of HeLa was stronger.By taking the N-heterocyclic aromatic ring substituted podophyllum compound 1S as typical tubulin inhibitor,the project is to study the novel structural insights into the understanding of the interaction between 1S and tubulin,and explaining the molecular mechanism.Here,we present the structure,at 2.6 ? resolution,of tubulin in complex with 1S.It shows the binding site of 1S is the intermediate domain of the ? subunit.1S occupies a pocket that is known as the colchicine site.Comparison of the tubulin structure in the bound and unbound states shows that the side chains of the T7 loop of ?-tubulin flip outward and T5 loop of ?-tubulin changes its conformation.Because ?-subunit T7 loop participates in a reversible way in the resistance to straightening by flipping in and out,and the ?-subunit T5 loop participates in structural changes of the ??-tubulin heterodimer during the microtubule assembly/disassembly cycle by the change of its conformation,these findings explain how 1S inhibit the tubulin polymerization and break microtubule stability.In addition,superimposition of the tubulin–1S onto the tubulin–PTOX structure revealed an structural basis that N-containing aromatic heterocycle triazole substituted at the cycloparaffin(C-ring)4-position(C-4)of PTOX was a good modification direction.Taken together,our results offer fundamental structural insights into the mechanisms of 1S's activity and provide a structural basis to guide the design of new potent podophyllum derivatives for the treatment of cancer.