Screening Of IDO Inhibitors For Tumor Immunotherapy

Part1Screening of drug IDO inhibitors for tumor immunotherapyBackground:In the development of cancer,cancer cells could employ a number of mechanisms to escape clearance by immune system.Indoleamine 2,3-dioxygenase(IDO)is a kind of enzyme existing in immune cells,which catalyzed the metabolism of tryptophan through the kynurenine pathway.Inflammation induced by cancer development could result in overexpression of IDO in protuberance cells and tumor cells in the tumor microenvironment,which caused sustained consumption of tryptophan,inhibition of tryptophan-sensitive T cells functional activity,and finally decrease in local immune activity of tumor tissue.Inhibition of IDO activity and its expression could effectively activate the immune system to kill the tumor cells.Therefore,IDO has become a new target in cancer immunotherapy.Currently,four IDO inhibitors,Epacadostat,Indoximod,GDC-0919 and BMS-986205,are in the clinical research stage as potential new anti-cancer molecule immunotherapy drugs.At the same time,new IDO inhibitors such as PF-06840003 and RG70099 are also being investigated.Purpose:The screening models for IDO inhibitors at the molecular level and at the cellular level were established,and screening for IDO inhibitors was conducted.Methods:1.The prokaryotic expression system was used to express human recombinant IDO1 protein and the His-Tag tag was used to isolate and purify the protein.TheIDO1 protease activity was verified by method 2 and used for the screening of IDO inhibitor at the protein level.2.The colorimetric method was used to detect the concentration of kynurenine in the enzymatic reaction product of IDO1 in the enzymatic reaction system,which reflected the enzyme activity of IDO1 itself.The activity of IDO1 was inhibited to varying degrees by different concentrations of the test compound.The effect curve can determine the IC50 of different compounds.3.The IDO can be highly expressed in He La cells that do not express IDO by stimulation of IFN-reaction product of IDO1 in the enzymatic reaction system,which reflected the enzymel culture solution can reflect the enzyme activity of IDO1 itself.The activity of IDO1 was inhibited to varying degrees under the effect of different concentrations of the test compound.The IC50 of different compounds can be obtained from the drug efficacy curve.Result:1.The human recombinant IDO1 protein with His-Tag tag having enzymatic activity was successfully expressed and purified by the E.coli prokaryotic expression system.2.Using the IDO1 protein with enzymatic activity obtained by isolation and purification,the construction of an enzyme-level IDO1 inhibitor screening model was completed,and 15 compounds were successfully screened,from which TT-00181 had good IDO1 inhibitory activity.3.The construction of a cell-level IDO1 inhibitor screening model was completed.The above 15 compounds were screened on a cell-level screening model and it was found that TT-00181 has a good IDO1 inhibitory activity.Conclusion:IDO is involved in the immune escape of tumors through its own enzymaticactivity.Previous studies have shown that inhibiting the activity of IDO enzymes in tumor patients can relieve the tumor tissue immunosuppression in the tumor microenvironment to some extent.Therefore,screening and obtaining low-toxicity and high-efficiency IDO inhibitors is an important direction of tumor immunotherapy.This paper established an experimental model for screening IDO inhibitors at the molecular and cellular levels.Screening dozens of compounds and screening them to obtain active compounds TT-00181,can effectively inhibit IDO activity.It is proved that this screening model has the advantages of being simple and intuitive,which can be used for the screening of IDO inhibitors,and provides a new method for the development of new tumor molecular immunotherapy drugs.Part2Effect of ES2 on multidrug resistance of colon cancer and its mechanismBackground:In the course of cancer treatment,the occurrence of tumor multidrug resistance is an important reason for the failure of chemotherapy drugs to treat cancer patients.An important mechanism for multidrug resistance of tumors is the tumor cell P-glycoprotein family.The drug development against P-gp is also one of the important directions for overcoming multidrug resistance of tumors.At present,the inhibitors of P-gp have been developed to the fourth generation,but due to the high toxic and side effects,no P-gp inhibitors have entered the clinical treatment stage.The abundant and diverse natural products have the features of diversity in chemical structure and low toxic and side effects.Therefore,finding low-toxic and effective drug-resistant reversal agents from natural products has become an important way to discover tumor multidrug resistance reversal agents.Colon cancer is one of the high-risk cancers in China.Patients with colon cancer often undergo resistance to various chemotherapeutic drugs after receiving chemotherapy drugs,which greatly hinders the continued treatment of colon cancer patients and affects the patients' postoperative rehabilitation.ES2 is a macrocyclic diterpene compound extracted from the seeds of Euphorbia sororia as a Chinese Uygur traditional medicine.Previous studies showed that ES2 showed good resistance to breast cancer and human oral squamous cell carcinoma cell lines.Drug resistance reversal effect.Purpose:The previous study of ES2 showed a good reversal activity of multidrug resistance in tumors.The purpose of this study was to observe the reversal effect of ES2 on the resistance of colon cancer and its mechanism,and to verify the reversal ofES2 on more tumor resistant cell lines.The role of multidrug resistance in tumors,expanding the spectrum of ES2 reversal of tumor MDR,provides more data support for the subsequent development of ES2.Methods:1.MMT assay was used to detect the resistance of ES2 to colon cancer and whether the parental cell line had cytotoxicity.2.The MTT method was used to detect the changes of IC50 when various chemotherapeutic drugs such as PTX,NVB,VCR and DDP were applied to colon cancer drug resistance and parental cell lines under different concentrations of ES2.3.Fluorescence microscopy and flow cytometry were used to detect the effects of different concentrations of ES2 on drug resistance of colon cancer and parental cell lines on the accumulation of rhodamine 123.4.Western Blot and q-PCR were used to detect the changes of ABCB1 m RNA expression and protein expression levels in colon cancer-resistant cells when ES2 was applied to colon cancer-resistant cell lines at different concentrations or at the same concentrations.Result:1.The cytotoxicity of ES2 on colon cancer-resistant cell lines(HCT8/T cells and HCT8/VCR cells)was extremely weak and was comparable to that of the colon cancer parental cell line HCT8(IC50 >30?M).At the same time,ES2 showed a strong ability to reverse the multidrug resistance of colon cancer resistant cell lines,and showed a certain concentration dependence.Compared with the first-generation drug resistance reversal agent verapamil,3 ce of colon cancer resistant cell lines,and showed a certain concentration dependence.rental cell line HCT8(IC cell lines at different concentrations or at the same concentratil.2.ES2 can effectively enhance the accumulation of rhodamine 123 in coloncancer-resistant cell lines(HCT8/T cells and HCT8/VCR cells),and the effect of 3?M ES2 is comparable to that of 10 ?M verapamil,and 10 ?M of ES2 is enhanced.Colon cancer drug-resistant cell lines had a much stronger effect on rhodamine 123 accumulation than verapamil at 10 ?M.3.ES2 does not affect the protein expression of ABCB1 in colon cancer-resistant cell lines,but does not affect the expression of ABCB1 m RNA.Conclusion:We have verified that ES2 has a good anti-tumor multidrug resistance effect on colon cancer-resistant cell lines.Comparison experiments with Verapamil,the first-generation drug resistance reversal agent,show that ES2 shows far more than dimensions.Lapamil has an anti-tumor multidrug resistance activity and acts like verapamil on the ABCB1 transmembrane transporter.ES2 can effectively inhibit the function of ABCB1,inhibit the chemotherapeutic drug from being excreted by ABCB1,and increase chemotherapeutic drugs.Accumulation in multidrug resistant tumor cells occurs.Our study demonstrated that ES2 can reverse the multidrug resistance of colon cancer cells,broaden the ES2 tumor-resistant multidrug resistant tumor types,and provide a basis for further research and development of ES2.