Screening And Mechanism Of Action Of Nitrogen-containing Heterocyclic Compounds With Antitumor Activity

Nitrogen-containing heterocyclic compounds have the advantages of low toxicity,targeting,and excellent biological activities in anti-tumor,antibacterial,as well,most of the compounds are multi-target compounds.With the development of new targets and targeted drugs,it was discovered that tumor suppressor gene p53 plays an important role in cell cycle regulation,DNA repair,cell differentiation,and apoptosis;and tubulin also exerts a crucial role in the basic cellular processes such as mitotic spindle formation,tissue cell structure,intracellular transport,and cell signal transduction and secretion.Therefore,the p53 and tubulin have attracted much attention as anticancer targets.Taking the library of nitrogen-containing heterocyclic compounds as the research object.This paper preliminarily explored the possible mechanism of high activity nitrogen-containing heterocyclic compounds against the potential targets p53 and tubulin,to lay a foundation for the discovery of high efficiency and low toxicity of new nitrogen-containing heterocyclic p53 and tubulin inhibitors.The work of this paper is summarized as follows:The mechanism of action of 62 nitrogen-containing heterocyclic compounds synthesized by the research group was studied.First,the MTT assay was used to evaluate the antiproliferative activity of these nitrogen-containing heterocyclic compounds against A549(human non-small cell lung cancer cells),Hep G2(human liver cancer cells),PC-3(human prostate cancer cells),K562(human leukemia cells),and NRK-52E(normal rat renal tubular epithelial cells)in vitro.The results showed that triazolothiazide indole derivatives Q-12(IC₅₀=9.90±0.85?M),Q-37(IC₅₀=10.67±1.44?M),Q-43(IC₅₀=10.47±2.01?M)displayed better activity against PC-3cancer cells,As well,the cytotoxicity against NRK-52E was relatively small.We also further explored the antiproliferative mechanism of compounds Q-12,Q-43 from the aspects of cell migration and invasion,cycle,and apoptosis.First of all,the effect of wound healing and transwell assay on cell migration and invasion was studied.The results revealed that the compounds Q-12?Q-37?Q-43 could inhibit cell migration and invasion;Then,through Hoechst 33258 staining,Annexin V-FITC/PI double staining,and mitochondrial membrane potential test(MMP),the apoptosis of PC-3 cells induced by compounds Q-12,Q-37,Q-43 were detected.The results validated that both compounds Q-12,Q-37,Q-43 could cause the PC-3 cells to produce different degrees of apoptosis.Finally,the PC-3 cell cycle changes induced by compounds Q-12,Q-37,Q-43 were detected by PI staining combined with flow cytometry.The results revealed that Q-12,Q-43 could induce PC-3 cell cycle arrest in G2/GM stage,Q-37 induced cell cycle arrest in G0/G1 stage.To explore the possible mechanism of action of compounds on p53:First,The antiproliferative activity of Q-12,Q-43 against NCI-H1299(null p53)was evaluated by MTT assay in vitro.The results displayed that its IC₅₀value against NCIH1299was much higher than the other cancer cells;On that basis,the RT-PCR and Western blotting experiments were also used to investigate the effects of compounds Q-12,Q-43 on the m RNA and protein expression in PC-3 cells.The results indicated that the compounds Q-12,Q-43 induced PC-3 cells,p53 m RNA expression was slightly up-regulated,And MDM2 m RNA expression was slightly down-regulated;The results of the Western blotting analysis exhibited that Q-12,Q-43 could up-regulate the expression of p53 protein slightly,while the expression of MDM2,AKT,P-AKT was down-regulated slightly,it had nothing significant difference;Next,the MDM2proteins are selected for molecular docking to study the binding mode and affinity of compounds Q-12,Q-43 with MDM2.The results elucidated that the binding score of compounds Q-12 and Q-43 with MDM2 protein was low and the binding affinity was weak.Above all,it is inferred that the mechanism of the antiproliferative activity of these compounds has little correlation with tumor suppressor p53.To explore the possible mechanism of action of compounds on tubulin:First,Tubulin-Red tracker was explored to evaluate the effect of compounds Q-12,Q-43 on microtubule polymerization in PC-3 cells.The results showed that the formation of microfilaments was blocked to different degrees,and the concentration higher,the compounds could more effectively promote tubulin polymerization.Through transmission electron microscopy,it was found that the number of microtubule polymers increased with the concentration of drugs.At last,tubulin protein molecules were selected for molecular docking to predicted the possible mode of action and binding ability,it was found that the compounds Q-12,Q-43 could form a hydrogen bond with the tubulin,and displayed potent binding affinity.The above results show that both compounds Q-12,Q-43 showed the potent ability to inhibit cell proliferation,migration,and invasion,and regulate cycle and apoptosis.A preliminary mechanism study confirmed that the mechanism of antiproliferative activity had little correlation with the p53-MDM2 target and high correlation with tubulin.It was preliminarily determined that Q-12,Q-43 could be used as a novel lead compound for targeting tubulin.This study provided a theoretical and experimental basis for the further development and application of highly effective and low-toxic nitrogen-containing heterocyclic tubulin inhibitors.