The Screening Of Novel IDO1 Inhibitors And The Evaluation Of Anti-tumor Activity

Immune escape plays an important hallmark of cancer.Under normal physiological conditions,our own immune system has the immune surveillance,which can prevent the pathogens from invading our bodies.When our body exposed to the malignant cells,the immune system can recognize and clean those malignant cells specifically with the help of different kinds of immune mechanism.However,tumor cells may evade various attacks under the surveillance of immune system,the main reason of which is that the tumor cells can evolve a variety of immune escape mechanism.Tumor immune escape is mainly related to the modification of tumor cells and the changes of tumor immune microenvironment.Tumor cells can enhance the ability to avoid immune surveillance and attack,and interact with many kinds of cells as well as the factors,such as immune cells and immune factors,to form a complicated tumor immune microenvironment.Meanwhile,the composition and function of immune cells and immune factors will change due to the presence of tumor cells.Immune cells may be immune suppressed,and prompt tumor cells to escape from the surveillance and the killing of the immune system.Indoleamine 2,3-dioxygenase 1（IDO1）is an oxidoreductase containing 403amino acids and heme,which is a monomeric protein containing an active pocket.The IDO1 gene is located on the chromosome 8 of human with the length of approximately 15 kb.IDO1 is expressed in low levels in vivo under the normal physiological conditions.Moreover,it is the first rate-limiting enzyme of kynurenine pathway（KP）.Recent studies have found that IDO1 is highly expressed in a variety of tumor issues,and the expression of IDO1 is correlated to the poor prognosis of various cancers positively.IDO1 can catalyze the metabolism of L-tryptophan and produce cytotoxic metabolites such as kynurenine and quinolinic acid.Herein,IDO1has been shown to be involved in the immune escape of tumors,and closely related to many kinds of major human diseases as well,such as Alzheimer’s disease,cataracts,Parkinson’s disease and so on.In addition,there is another T cells in our bodies with the effect of immune suppression,namely CD4⁺CD25⁺Treg cells.This kind of cells is involved in tumor immune escape and closely related to the development of tumor and the prognosis of cancer patients,which is one of the main factors affecting the effectiveness of immunotherapy.Further studies have shown that as a classical immune-regulatory factor,when the expression of IDO1 is elevated,it will activate the resting Treg cells in the tumor microenvironment and induce CD4⁺T cells to up-regulate Foxp3.Meanwhile,the expression of Foxp3 through Treg cells can up-regulate the transcription level of mRNA of IDO1,so the expression of IDO1 will be increased as well.IDO1 plays an important metabolic immunomodulatory role in the functions of suppressing T cells immunity and anti-tumor immunity,organs transplantation immune tolerance,tumor immune escape and other immune regulation.Therefore,IDO1 can be regarded as an important drug target.The discovery of IDO1 inhibitors is of great significance for the prevention and treatment of tumors.In this paper,we used E.coli.BL21（DE3）to construct a prokaryotic expression vector containing pET-28a（+）-hIDO1 with His-tag,and isolated and purified the IDO1 recombinant protein by prokaryotic expression system and imidazole-Ni column purification system.The activity of IDO1 protein was evaluated by the specific IDO1 substrate L-tryptophan.After researching and optimizing the reaction conditions,the positive compound Epacadostat and 1-MT were used to evaluate the reaction system,and by comparing with the reports,we indicated that the high-throughput screening modle for small molecule IDO1 inhibitors was successfully established.Next,we used different methods to evaluate different series of compounds for the IDO1 inhibitory activity.We found that the compounds containing triazole structure had better IDO1 inhibitory activity,among which the compound 4d has shown the best activity with the IC₅₀ at 1.536μM.Later we established the co-cultured modle of immune cells and cancer cells in vitro,and studied the mechanism of compound 4d on the functions of IDO1.The results showed that compound 4d could down-regulate the expression not only of the protein level but also of the mRNA level,block the function of IDO1,and reduce the expression of Foxp3 and the differentiation of Treg cells,so that it may be as the potential inhibitor of IDO1 for the further study.The discovery of compound 4d can serve as a lead compound of the novel inhibitors of IDO1,basing the foundation for the discovery and optimization of such kind of novel IDO1 inhibitors.Meanwhile,the binding of IDO1 may recover the proliferation and activity of T cells,so that it may get a better immune-therapy in clinic.