Screeing Of Indoleamine 2,3 Dioxygenase 1 Inhibitors By Capillary Electrophoresis

Indoleamine 2,3-dioxygenase 1（IDO1）plays a key role in the process of tumor immune escape,so it is considered to be a promising target of tumor immunotherapy.In our study,off-line and on-line capillary electrophoresis was established to screen IDO1 inhibitors from natural product extracts.The optimal separation conditions were obtained as follows:background electrolyte was 32 m M sodium tetraborate（p H 9.22）and separation voltage was21 k V.The effectiveness of the off-line capillary electrophoresis method was confirmed by measuring the enzyme kinetic parameters and the inhibition modes of INCB024360 and Vitamin K₃.The combination of partial filling online capillary electrophoresis with rapid polarity switching technology to screen IDO1 inhibitors enables the entire reaction and separation process to take only 5 min.There was no difference between online capillary electrophoresis results and offline results.The results showed that among the 18 natural products extracts,the extracts of Carthamus tinctorius,Schisandra fruit,Raisin,Coffee,Hawthorn and Angelica had inhibition effect on IDO1.All these reflect the practicability of this research method in the screening of inhibitors.The coffee extract was then isolated and analyzed by UHPLC-Q-TOF/MS to identify 10main specific active components.Molecular docking results showed that nine natural compounds had binding interactions with IDO1,which may be potential IDO1 inhibitors.In this thesis,IDO1 was immobilized on amino-functionalized magnetic nanoparticles with glutaraldehyde as crosslinking agent.The immobilization conditions were optimized by investigating the initial p H values of glutaraldehyde,immobilized time,enzyme concentration and magnetic nanoparticles concentration.The results showed that the immobilized enzyme could not only be used for 5 times at 37℃without losing enzyme activity,but also could be stably stored at 4℃for 14 days.It has the advantages of good storage stability and repeatability.Therefore,magnetic nanoparticles have been proved to be an effective carrier for immobilizing IDO1.Finally,the immobilized enzyme method was applied to screen 10 main active ingredients in coffee extract,among which ferulic acid had the highest inhibitory activity of IDO1.Therefore,a combination of CE and UHPLC-Q-TOF/MS provides a good choice for high-throughput screening of IDO1 enzyme inhibitors from complex systems such as natural products.