Novel Compound Containing Dihydropyrazole Moiety Inhibits The Proliferation Of SMMC-7721 By Targeting H TERT

Human telomerase was composed of human telomerase reverse transcriptase(hTERT),human telomerase RNA(hTR),human telomerase associated protein(hTP1) and other components.Telomerase was widely researched since discovered from 1994, because of its critical role in responsible for maintaining chromosome integrity, cell immortalization and the key role of tumor development.Therefore, as a new target for screening antitumor drugs,telomerase has become a hot research focus.Although people have designed,synthesized and discovered some effective telomerase inhibitors,however, the crystal structure of telomerase was confirmed until 2008, the efficient and highly selective precursor structures against hTERT and there pharmacological mechanisms were still rarely reported. Therefore, based on the crystal structure and the potential active site,our research group established the model of molecular docking and screen out some highly active skeletal structures virtually, meanwhile,designed and synthesis a series of molecule compounds. Then we indicated the anticancer activity and pharmacological mechanism of this series of compounds.The main research contents of this subject are as follows: 1. Design and synthesis of the target compoundsBased on the crystal structure of TERT protein and potential telomerase activity center, we established the docking model of the compounds inhibitor initially, and screen out some highly active skeleton structure of the compounds virtually, and synthesized a series of this compounds through the methods of medicinal chemistry. 2. Detection the effective of compounds on the proliferation of tumor cell linesAnticancer activity of screening of small molecular compounds: MTT experiment on human hepatocellular carcinoma cell line SMMC-7721, human liver cancer cell line HepG2, human gastric carcinoma cell line MGC-803 and human breast cancer cell line MCF-7, found that compound 4a had the most obvious inhibitory effects on the proliferation of SMMC-7721 tumor cells. At the same time, through the flow cytometry cell cycle experiment: it was found that SMMC-7721 cells were treated with compound 4a after 48 h, the number of S cells increased significantly and the significant decrease in G0/G1 phase cells, it suggest that the compound 4a inhibit cell proliferation by blocking the cells in S phase. 3. Detection the effective of compound 4a on telomerase activity and hTERTDetection the telomerase activity of tumor cells by modified TRAP assay: the compound 4a also showed inhibitory effects on the telomerase activity obviously with IC50 value of 7.81 ± 1.32 μM. Meanwhile the Western blot assay demonstrated the expression of hTERT protein decreased significantly treated with the compound 4a cells after 48 h. The results suggest that the compound 4a has a strong inhibitory effect on telomerase activity and the expression of hTERT protein. 4. Detection the effective of compounds 4a on NF-κb/P65 signaling pathway and testing the expression of P65, IL-6 and TNF-αThe Q-PCR and Western blot experiments found that the compounds 4a had a downward effect on the protein expression of key molecule P65 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 inhibitory effect on the proliferation of SMMC-7721 cells of compounds 4a may be related to NF-κb/P65 pathway. 5. Detection the effective of the compound 4a on NF-κb/P65 signaling pathway and testing the expression of P65, IL-6 and TNF-α in cells overexpressing hTERTThe Q-PCR and Western blot experiments showed that cells overexpressing hTERT, which treated with compound 4a after 48 h,expressed lower levers of P65, IL-6 and TNF-α mRNA and protein than the untreated cells obviously. This result indicated that human hepatocellular carcinoma cell line SMMC-7721 with hTERT expression increased, the expression of P65 protein and the expression level of IL-6 and TNF-α mRNA and protein was up-regulated, while compound 4a could down-regulat the protein expression of P65 and the mRNA and protein expression of IL-6 and TNF-α. The results further suggest that by inhibiting hTERT, compound 4a had an effect on the downstream of NF-κb/P65 signaling pathway to inhibit the proliferation of SMMC-7721 hepatocellular carcinoma cells.In summary, basised on the high activity skeletal structure-N- sulfone- aryl dihydropyrazolo selected by molecular docking model, our group designed and synthesized a series of novel dihydropyrazole compounds targeted hTERT and screened out a potentially highly selective and efficient telomerase inhibitors- compounds 4a, and study its pharmacological mechanisms further. The results found that the compounds 4a inhibit telomerase activity by targeting hTERT, which will block SMMC-7721 hepatoma cells in S phase to inhibit cell proliferation. Furthermore this effect may be associated with NF-κb/P65 classic signaling pathway.