Identification Of Target Protein That Flavonoids Enhance Glucose Consumption By Photoaffinity Labeling Technique

A lot of flavonoids,which has low toxicity and side effects,possess hypoglycemic effect,and could be used as functional food or drug candidate.Current studies showed that the hypoglycemic hypoglycemic of flavonoids are mainly through their effects on regulation of several pathways,such as increasing insulin sensitivity,enhancing glucose uptake.However,the bounding protein is still not clear.With the development of proteomic,photo-affinity labeling(PAL)represents a useful biochemical strategy for target identification of protein targets and the interaction between drugs and the corresponding target proteins.In this thesis,17 kinds of flavonoids were screened by glucose consumption activity tests on HepG2 cells.The results showed that 5,6,7-trimethoxyflavone,quercetin,magnolol and dihydromyricetin can significantly promote glucose consumption,and only 5,6,7-trimethoxyflavone did not showed obvious activity of proliferation inhibiton against HepG2 cells.Therefore,5,6,7-trimethoxyflavone was selected as the molecular to study the target protein in promoting glucose consumption by flavonoids.To explore the potential target proteins of 5,6,7-trimethoxyflavone,we designed and synthesized a novel photoaffinity probe BE-9.We modified 5,6,7-trimethoxyflavone at C-4' of B ring with an azide photophore for covalent labeling and introduced a propargyl at C-5 of A ring as a bioorthogonal handle for the visualization of cross-linked protein complex using a fluorescence reporter.The structures of main products were characterized by mass spectroscopy and 1HNMR spectrum.We also designed and synthesized three reporter groups to explore the optimal chain length.The structures of the main products were characterized by mass spectroscopy.The synthesized probes could be used as powerful tools to isolate and identify the target proteins.We then evaluated the effect of the photoaffinity probes on glucose consumption and cell viability of HepG2 cells.The results showed that photoaffinity probe BE-9 retained its bioactivity in cellular glucose consumption.Meanwhile,the probe and 5,6,7-trimethoxyflavone exhibited no obvious cytotoxicity against HepG2 cells.The results suggest that the probe and 5,6,7-trimethoxyflavone selectively enhance cellular glucose consumption.The synthetic probes was used to characterize the interaction of flavonoids with its cellular targets.We compared the detection limit of Rhodamine B and biotin and found that the detection limit of Rhodamine B was higher than that of biotin.Photoaffinity labeling studies were also performed and a protein with a molecular weight 23 kDa were specifically tagged by probe BE-9.We further enriched the probe-labeled target proteins with streptavidin-coated beads,and the gel bands containing the enriched protein have been sent for proteome analysis.