Novel Lactone Compound:Design、Synthesis And Promotes The Apoptosis Of SMMC-7721 By Targeting SPOP

Spop protein is a member of the SCF (Skpl and cull and F-box) E3 ligase family, involved in the regulation of many physiological processes, the main is modified by ubiquitin regulates a variety of protein stability and signaling pathway of transduction. SPOP protein is overexpressed in many cancer cell lines, and it has been shown to inhibit tumor proliferation in tumor cells and in mice. Therefore, taking SPOP as a new target of anti-tumor drugs has become a hot spot in the present study. Although people have discovered or designed and synthesized inhibitor with a role, but to spop for targets of high selectivity, high lead structures and pharmacological mechanism research still has seldom been reported. Therefore, the research group based on the crystal structure of the ubiquitin ligase SPOP and potential activity center, the initial establishment of the molecular docking model, virtual screening of the skeleton structure of some high activity, and taking the opportunity of skeleton based design synthesized a series of small molecular compounds. The screening of antitumor activity of the small molecule compounds and the mechanism of the pharmacological activity of this series of compounds were also studied. The main research contents of this subject are as follows:1. Design and synthesis of the target compoundsBased on ubiquitin ligase SPOP protein crystal structure and potential activity center, the establishment of the protein inhibitor of molecular docking model, through virtual screening, selected the skeleton structure of some highly active compounds, and through the relative theory and method of chemical synthesis of this compound.2. Detection the effective of compounds on the proliferation of tumor cell linesThe synthesis of small molecular compounds to design anticancer activity screening:in people human gastric cancer cell SGC-7901、 human gastric cancer cell MGC-803、human breast cancer cells Bcap-37 and hepatoma cell SMMC-7721 were MTT assay. Results showed that compounds in this series of compounds 8 on SMMC-7721 tumor cell proliferation inhibition effect is the most obvious. At the same time, compound 8 influenced on SMMC-7721 tumor cell cycle and apoptosis could be observed by flow cytometry. The results showed that SMMC-7721 cells by compound 8 treatment had a significant increase in the number of apoptotic cells after 48 h, suggesting that compound 8 is to achieve anti-tumor effect by promoting apoptosis.3. Detection the effective of compound 8 on SPOPThe expression of SPOP protein was detected by blot Western assay. The results showed that the expression of SPOP protein decreased after 48 h treatment. Suggest that compound 8 has a strong inhibitory effect on SPOP protein.4. Detection the effective of compounds 8 on NF-κB/P65 signaling pathway and testing the expression of P65, κB-α, IL-6 and TNF-αThe Q-PCR and Western blot experiments found that the compounds 8 had a downward effect on the protein expression of key molecule P65 and ixB-α protein in the NF-κB cell signaling pathway expression, mRNA and protein expression of its downstream signaling molecule IL-6 and TNF-α, this result indicated that the promotion effect on the apoptosis of SMMC-7721 cells of compounds 8 may be related to NF-κB pathway.5. Detection the involvement of SPOP in NF-κB signal pathway in SMMC-7721.PDTC (inhibitor of the NF-κB pathway) and SKPin C1 (SPOP inhibitor) were used to study the involvement of SPOP in NF-κB signal pathway in SMMC-7721.According to the results of ELISA, Western blotting and Real-time quantitative RT-PCR showed that SPOP could affect NF-κB signal pathway and downstream signal molecules, like TNF-α, IL-6.In summary, the research group on the molecular docking model selected target to spop high activity of skeleton structure of myricitrin based were designed and synthesized. The a series of new lactone derivatives, and through the method of pharmacology screened a potential small molecular inhibitor -- compound 8 and on its possible pharmacological mechanism of further study. A series of experimental results show that the target to ubiquitin ligase compounds 8 promote the apoptosis of SMMC-7721 cells, inhibition of cell proliferation. At the same time, it is found that the realization of this function may be related to the down-regulation of the NF-κB classical signal pathway related to cell proliferation and activation.