Synthesis,Biological Evaluation Of Benzyl Lipoic Acid Acyl Shikonin Twin Drugs

In recent years,with the increasing of environmental pollution,work pressure and food safety,the incidence of malignant tumors is higher and higher,and has become one of the most serious diseases that threat to human health,therefore,the development of new drugs or active molecules that can target tumor efficient become Particularly important.It is well known that microtubules play an important role in maintaining cell morphology,cell division,signal transduction and so on.Because of its particularity on the structure and function.So Tubulin has become one of the important targets for the development of anti-tumor drugs,meanwhile,metabolic changes in malignant tumors become a research focus and have received a generous concern.To induce cell apoptosis through change the energy metabolism of cancer cells also received much attention.PDK is the key enzyme of energy metabolism of cancer cells,we can disrupt the energy metabolic pathways of tumor cells and make tumor cells lake of energy through inhibit this enzyme.We according to the characteristics of tubulin and pyruvate dehydrogenase kinase to design twin drugs.we hope it can disrupt the energy supply and disturb the form of tubulin to induce the cell apoptosis.Shikonin is a highly effective natural antitumor active molecule,it has many targets.According to the researchs of our lab,shikonin can regulate the form of tubulin through act with the colchicine binding site to achieve the purpose of inhibiting tumor cell growth.CPI-613 is a commercially available drug for the treatment of acute myeloid leukemia,It has been reported in the literature that can inhibit the activity of pyruvate dehydrogenase kinase to inhibit tumor proliferation effectively.Based on the idea of twin drugs and the previou study,we have combined the shikonin molecule with CPI-613 to design the new type urinary benzoic acid bromide bitbromide(PMMB340).And then we do relate analysis to verify the two targets.The results indicate that it can act on tubulin and pyruvate dehydrogenase kinase respectively to reach anti-cancer effect,it's a double targets inhibitors with high efficientFirst,we use the Discovery Studio molecular docking simulation software to that PMMB340,shikonin and CPI-613 butt joint with tubulin and PDK proteins,we can see PMMB340 can combine two targets stably,it can combine tubulin with three hydrogen bonds and two ? bonds,the binding energy is 56.23,and it can combine PDK proteins with two ? bonds,the binding energy is 63.09.Which give Theroy support to following experiments.Compound PMMB340 were evaluated for its antiproliferation activities against five cancer cell lines Hela,MCF-7,A549,MDA-231 and H460 and a non-cancercell lines[L02(human normal liver cell)]by MTT assay.the results showed that compound PMMB340 has the strongest inhibitory activity on H460 cells(IC50=6.31±0.52 ?M)and was better than shikonin(IC50=7.36±0.67 ?M)and CPI-613(9.21±1.12 ?M),It exhibited low cytotoxicity to L02 cells(IC50=84.31±3.37 ?M).through tubulin polymerization experiment,we found that PMMB340 has similar mechanism with colchicine,which can promote the depolymerization of tubulin effectively,the cell cycle analysis indicate that it can cause accumulation of cells in the G2/M phase of the cell cycle in a time and does dependent manner.Subsequently,The PDH enzyme activity analysis,Cell apoptosis assay and mitochondrial membrane potential experiment confirmed that the compound could act on PDK to promote the activity of PDH.furthermore inhibited the glycolytic pathway and activate mitochondria-dependent apoptosis pathways,it lead to 76.6%cells apoptosis when cells were treated with 8?M PMMB340.The expression of relate protines of cell apoptosis PARP and caspase 3 is down regulate,the expression of Cyto C is up regulate.The result indicate that the PMMB340 can induce cell apoptosis through mitochondria-dependent apoptosis pathways.finally,the cell apoptosis assay and mitochondrial membrane potential experiment of normal cell line L02 showed that compound PMMB340 could reduce the toxicity to L02 cells compared with shikonin.all in all,the compound PMMB340 is a high effective and low toxicity molecule,which can play an anti-tumor effect by acting on two targets respectively.