| BackgroundMalignant tumor is the leading cause of morbidity and mortality in the world, with almost 8.2 million deaths in 2012. Lung cancer is of the highest mortality, it accounts for 20%, and nearly 80% of lung cancer cases are non-small cell lung cancer patients, while 75% of the patients were diagnosed during the middle and advanced stages,5-year overall survival rate is very low. Currently, the therapeutic efficacy of non-small cell lung cancer is not very satisfied, and easy to invade and metastasize is the major reason of deaths clinically. It is necessary to enhance the efficiency of prevention and management.The study found that the interaction between mesenchymal cells and tumor cells was induced by the large number of chemokines in the tumor microenvironment, the immune inflammatory response promoted the proliferation, invasion, and adhesion of tumor. In recent years, many researches revealed that CCL5 is related to the invasion and migration of malignant tumor. A549 is a non-small cell lung cancer cell line, the combination of CCR1, CCR3 or CCR5 on its surface and CCL5 could accelerate the tumor cells metastasize to other sites. We screened lead compounds and made computer simulation through computer aided drug design (CADD) of virtual screening, dynamics simulations (MD) and the combination of free energy (MMGB/PBSA), and studied relationship between the structure of receptor antagonist of CCR1/3/5 and their inhibition of invasion or migration of non-small cell lung cancer by tested the biological activity of the synthetic lead compounds. It makes sense to design a more effective receptor antagonist of CCR1, CCR3 and CCR5.ObjectiveAccording to the crystal structure of the CCR5, homologous modeling and optimize the protein structure of CCR1/3, then use the virtual screening techniques such as molecular docking and ADMET, finally get six lead compounds. CCK8 and transwell assay are used to have the proliferation-toxicity detection, tumor invasion and migration evaluation, further study the relationship between the receptor antagonist and activity.Methods1. Building the pharmacophores which are focused on screened lead compounds and modified compounds, and virtually screen the pharmacophores and CCR5 as well as the CCR1/3 modeled homologously. Finally, we screen three compounds;2. Study the combination relationship between screened lead compounds and modified compounds, then analyse the free energy with the amino acid residues through molecular dynamics;3. Identified and analyzed the structures and purity of synthetic compounds;4. Using the method of CCK8 to test the proliferation and toxicity of lead compounds and modified compounds;5. Using transwell assay to test the compounds ability about antagonism of tumor invasion and migration.Results1. Build the pharmacophores of lead compounds;2. Get stable CCR1/3 structure after the structure of homologous modeling is optimized;3. Successful completion of the receptor for lead compounds, compounds and dynamics simulation, and the combination of free energy calculation;4. Get synthesis of high purity lead compounds and modified compound;5. CCK8 method prove that lead compounds and modified compounds have no cell cytotoxicity;6. Transwell experiments proved that the compounds with the role of tumor invasion and migration.Conclusion1. The pharmacophore filter combined with dynamic simulation, computer aided drug design method not only save the time to find lead compounds, but also study the micro aspect about the antagonists and the combination of the relationship between receptor.2. Using substitution reaction of carboxyl and carboxyl derivatives of ammonia solution reaction method to synthesis high purity compounds.3. Lead compound was studied by means of biological active compounds and transformation, and combined with dynamic experimental results analysis, which found that the two is roughly same. According to the dynamic experimental results we can design more effective CCR1 and CCR5 and CCR3 receptor antagonist. |
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