Indoleamine 2,3-dioxygenase 1 Enzyme Research Articles

IMMU-27. NON-TUMOR CELL IDO1 NON-ENZYMATICALLY DECREASES RT + PD-1 MAB TREATMENT EFFICACY IN OLDER ADULTS WITH GLIOBLASTOMA

Abstract Checkmate 498 is a large, randomized, phase 3 clinical trial that demonstrated an improved hazard ratio for younger but not older adults with MGMT unmethylated GBM treated with RT + PD-1 mAb. In an effort to better understand the biological determinant, indoleamine 2,3 dioxygenase 1 (IDO1), and its relationship with reducing RT + PD-1 mAb treatment efficacy in older adults with GBM, we created and/or compared 20-26 month-old C57BL/6 background mice that were IDO1-T2A-GFP [wild-type (WT); negative control (n=16)], IDO1 enzyme null [with normal IDO1 expression potential but incapable of tryptophan metabolism (n=16)], IDO1KO (n=4), Ido1fl/fl (n=14), Lysm-Cre+→Ido1fl/fl [IDO1-deficient in monocytes, macrophages, and microglia (n=20)], CD11c-Cre+→Ido1fl/fl [IDO1-deficient in dendritic cells (n=23)], Tie2-Cre+→Ido1fl/fl [IDO1-deficient in endothelial cells (n=14)]. Mice were intracranially (ic.) engrafted with 5×103 syngeneic GL261 followed by treatment with radiation (RT) + PD-1 mAb beginning at 14-days post ic. IDO1 WT and IDO1 enzyme null mice had a survival rate of 12.5% and 18.75% at 90-day post ic., respectively, which was significantly reduced as compared to IDO1KO mice with a 75% survival rate (p< 0.05). Tie2-Cre+→Ido1fl/fl mice, CD11c-Cre+→Ido1fl/fl, and Lysm-Cre+→Ido1fl/fl mice had a 100%, 96%, and 70% survival rate at 90-day post ic., respectively, as compared to Ido1fl/fl mice with a 42% survival rate (p< 0.05). Significantly reduced kynurenine (KYN) levels were found in IDO1 enzyme null and IDO1KO mice as compared to WT mice (p< 0.001). Unexpectedly, kynurenine levels were also reduced in Tie2-Cre+→Ido1fl/fl but not in CD11c-Cre+→Ido1fl/fl, Lysm-Cre+→Ido1fl/fl or Ido1fl/fl mice (p< 0.05). Tryptophan (TRP) levels did not change between mouse groups. These data support the hypothesis that non-tumor cell IDO1 reduces immunotherapeutic efficacy in older adults with GBM through a TRP-KYN pathway-independent mechanism.

Imidazo[2,1-b]thiazole based indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor: Structure based design, synthesis, bio-evaluation and docking studies

Indoleamine-2,3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme known to catalyse the initial and rate limiting step of kynurenine pathway of l-tryptophan metabolism. IDO1 enzyme over expression plays a crucial role in progression of cancer, malaria, multiple sclerosis and other life-threatening diseases. Several efforts over the last two decades have been invested by the researchers for the discovery of different IDO1 inhibitors and the plasticity of the IDO1 enzyme ligand binding pocket provide ample opportunities to develop new heterocyclic scaffolds targeting this enzyme. In the present work, based on the X-ray crystal structure of human IDO1 coordinated with few ligands, we designed and synthesized new fused heterocyclic compounds and evaluated their potential human IDO1 inhibitory activity (compound 30 and 41 showed IC50 values of 23 and 13 µM, respectively). The identified HITs were observed to be non-toxic to HEK293 cells at 100 µM concentration. The observed activity of the synthesized compounds was correlated with the specific interactions of their structures at the enzyme pocket using docking studies. A detailed analysis of docking results of the synthesized analogues as well as selected known IDO1 inhibitors revealed that most of the inhibitors have some reasonable docking scores in at least two crystal structures and have similar orientation as that of co-crystal ligands.

Abstract 1849: Superiority of dual IDO1/TDO2 inhibition versus IDO1 selective inhibition in reducing immunosuppressive KYN levels in tumors co-expressing IDO1 and TDO2

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) catalyze the oxidation of L-Tryptophan (TRP) leading to the formation of immunosuppressive kynurenine (KYN) pathway metabolites. According to TCGA, a substantial fraction of different tumor types expresses both IDO1 and TDO2. In 82 tumor samples (GBM, hepatocellular carcinoma, NSCLC, and ovarian cancer) analyzed for IDO1/TDO2 RNA expression (RNA sequencing) and TRP metabolite content (LC-MS/MS), we found TDO2 highly expressed in hepatocellular carcinoma and to lower levels in NSCLC and ovarian cancer. IDO1 was found to be expressed in NSCLC and ovarian cancer but at higher levels and its expression was correlating with KYN levels. Immunosuppressive properties of KYN and downstream metabolites are mediated through T cell apoptosis and regulatory T cell (Treg) induction. In our hands, KYN at concentrations ≥ 50uM was shown to be immunosuppressive, notably inducing T cell apoptosis. According to our and published data, such high KYN concentrations (≥ 50uM) can be found in a subset of human tumors. As outlined, both IDO1 and TDO2 can contribute to high KYN levels in tumors. Hence, dual IDO1/TDO2 inhibition should be superior in decreasing tumor KYN levels and levering immunosuppression as compared to IDO1 selective inhibition. In this regard, we have generated potent dual IDO1/TDO2 inhibitors inhibiting IDO1 and TDO2 enzyme activity in cellular assays in vitro. To prove the concept of superiority in vivo, we established a dual IDO1/TDO2 expressing human tumor model (LOVO+IFNg) in the mouse and showed that the oral administration of a dual IDO1/TDO2 inhibitor as compared to an IDO1 selective inhibitor showed enhanced KYN/TRP modulation in the tumor. Lead compounds with favorable drug-like properties are currently investigated for pre-clinical development. Citation Format: Carina Lotz-Jenne, Sylvaine Cren, Christoph Joesch, Sabine Ackerknecht, Jennifer Brandes, Claire Moebs, Dominik Hartl, Felix Hartrampf, Philippe Guerry, Julien Pothier, Alexia Chavanton-Arpel. Superiority of dual IDO1/TDO2 inhibition versus IDO1 selective inhibition in reducing immunosuppressive KYN levels in tumors co-expressing IDO1 and TDO2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1849.

Indoleamine 2,3 dioxygenase 1 immobilization on magnetic nanoparticles for screening inhibitors from coffee

In this study, a ligand fishing method was developed to screen potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors from coffee extracts by immobilization of IDO1 enzyme on amino-modified magnetic nanoparticles combined with UHPLC-Q-TOF-MS/MS analysis. Parameters including enzyme concentration, immobilization time, the pH of glutaraldehyde and the amount of magnetic nanoparticles were optimized. The results indicated that immobilized IDO1 could be reused 5 times and was stable during storage for 7 days. Several IDO1 ligands were captured by incubating immobilized IDO1 with coffee extract, of which 10 showed an obvious difference comparing to non-conjugated bare nanoparticles. In vitro inhibitory activity was further performed by CE analysis, in which ferulic acid and chlorogenic acid had better IDO1 inhibitory activity, with IC50 value of 113.7 μM and 307.5 μM. These results demonstrate that this method provides an effective platform for identifying and screening IDO1 inhibitors from natural products.

Targeting the STAT5A/IDO1 axis overcomes radioresistance and reverses the immunosuppressive tumor microenvironment in NSCLC.

As a metabolic mediator of antitumor immunity, indoleamine‑2,3‑dioxygenase 1 (IDO1) is upregulated in various types of cancer; however, the regulatory mechanism and clinical significance of IDO1 in non‑small cell lung cancer (NSCLC) radiotherapy (RT) remain unclear. The present study investigated the role of IDO1 in the NSCLC microenvironment. MTT assay, immunofluorescence, apoptosis analysis, cell cycle analysis, and C57BL/6 and BALB/c nude mouse tumor models were utilized to evaluate the roles of the STAT5A/IDO1/kynurenine axis in radioresistance and in the immune microenvironment of NSCLC. Protein expression levels were evaluated by western blotting, immunofluorescence and immunohistochemistry. Flow cytometry was performed to assess the status of CD8+ T lymphocytes, regulatory T cells (Tregs) and immune‑related inflammatory factors in C57BL/6 mice. Notably, IDO1 and STAT5A were positively associated with the immune microenvironment. RT treatment significantly promoted the expression levels of IDO1. IDO1 knockdown markedly enhanced the radiosensitivity of lung tumor cells and the anti‑apoptotic properties of T lymphocytes. It was demonstrated that STAT5A knockdown suppressed T‑cell apoptosis by inhibiting IDO1 enzyme function. Finally, invivo experiments showed that STAT5A knockdown combined with RT was associated with greater numbers of CD8+ T cells and fewer Tregs. Results from the present study indicated that targeting the STAT5A/IDO1 axis may reshape the immune microenvironment and promote the efficacy of RT in NSCLC treatment. The present study may provide a theoretical foundation for more efficient use of immunotherapy regimens in NSCLC treatment.

Identification and Characterization of a Novel Indoleamine 2,3-Dioxygenase 1 Protein Degrader for Glioblastoma.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunosuppressive enzyme that inhibits the antitumor immune response through both tryptophan metabolism and non-enzymatic functions. To date, most IDO1-targeted approaches have focused on inhibiting tryptophan metabolism. However, this class of drugs has failed to improve the overall survival of patients with cancer. Here, we developed and characterized proteolysis targeting chimeras (PROTACs) that degrade the IDO1 protein. IDO1-PROTACs were tested for their effects on IDO1 enzyme and non-enzyme activities. After screening a library of IDO1-PROTAC derivatives, a compound was identified that potently degraded the IDO1 protein through cereblon-mediated proteasomal degradation. The IDO1-PROTAC: (i) inhibited IDO1 enzyme activity and IDO1-mediated NF-κB phosphorylation in cultured human glioblastoma (GBM) cells, (ii) degraded the IDO1 protein within intracranial brain tumors in vivo, and (iii) mediated a survival benefit in mice with well-established brain tumors. This study identified and characterized a new IDO1 protein degrader with therapeutic potential for patients with glioblastoma.

Evaluation of Novel Inhibitors of Tryptophan Dioxygenases for Enzyme and Species Selectivity Using Engineered Tumour Cell Lines Expressing Either Murine or Human IDO1 or TDO2.

Indoleamine 2, 3-dioxygenase 1 (IDO1) is commonly expressed by cancers as a mechanism for evading the immune system. Preclinical and clinical studies have indicated the potential of combining IDO1 inhibitors with immune therapies for the treatment of cancer, strengthening an interest in the discovery of novel dioxygenase inhibitors for reversing tumour-mediated immune suppression. To facilitate the discovery, development and investigation of novel small molecule inhibitors of IDO1 and its hepatic isozyme tryptophan dioxygenase (TDO2), murine tumour cells were engineered to selectively express either murine or human IDO1 and TDO2 for use as tools to dissect both the species specificity and isoenzyme selectivity of newly discovered inhibitors. Lewis lung carcinoma (LLTC) lines were engineered to express either murine or human IDO1 for use to test species selectivity of the novel inhibitors; in addition, GL261 glioma lines were engineered to express either human IDO1 or human TDO2 and used to test the isoenzyme selectivity of individual inhibitors in cell-based assays. The 20 most potent inhibitors against recombinant human IDO1 enzyme, discovered from a commissioned screening of 40,000 compounds in the Australian WEHI compound library, returned comparable IC50 values against murine or human IDO1 in cell-based assays using the LLTC-mIDO1 and LLTC-hIDO1 line, respectively. To test the in vivo activity of the hits, transfected lines were inoculated into syngeneic C57Bl/6 mice. Individual LLTC-hIDO1 tumours showed variable expression of human IDO1 in contrast to GL261-hIDO1 tumours which were homogenous in their IDO1 expression and were subsequently used for in vivo studies. W-0019482, the most potent IDO1 inhibitor identified from cell-based assays, reduced plasma and intratumoural ratios of kynurenine to tryptophan (K:T) and delayed the growth of subcutaneous GL261-hIDO1 tumours in mice. Synthetic modification of W-0019482 generated analogues with dual IDO1/TDO2 inhibitory activity, as well as inhibitors that were selective for either TDO2 or IDO1. These results demonstrate the versatility of W-0019482 as a lead in generating all three subclasses of tryptophan dioxygenase inhibitors which can be applied for investigating the individual roles and interactions between IDO1 and TDO2 in driving cancer-mediated immune suppression.

Mechanism of miR-760 Reversing Lung Cancer Immune Escape by Downregulating IDO1 and Eliminating Regulatory T Cells Based on Mathematical Biology

In cancer biology, mathematical models have become indispensable. They are useful for gaining a mechanistic grasp of cancer's dynamic processes. In cancer research, mathematical modelling approaches are becoming more common. The complexity of cancer is well suited to quantitative approaches as it provides challenges and opportunities for new developments (Altrock et al., 2015). Background. MicroRNA-760 (miR-760), as an early discovered tumor suppressor gene, is poorly expressed in lung cancer (LC). Indoleamine 2,3-dioxygenase 1 (IDO1), as an important regulator of T cell function, is active in immune tolerance. We discovered that miR-760 has a targeted relationship with IDO1, but the regulatory mechanism between miR-760 and IDO1 is still unclear. Method. The miR-760 and IDO1 levels in NSCLC were tested via real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting (WB). Cell growth was tested by CCK8, and NSCLC cell migration and invasion were analyzed through Transwell analysis. The binding conditions and target gene of miR-451 in NSCLC cells were determined via double luciferase reporter gene. The CD8+ T and CD4+ T cell ratio in CD45+cells was assessed by flow cytometry. Results. qRT-PCR revealed that miR-760 was low-expressed and IDO2 was highly expressed in LC. miR-760 mimics suppressed cell growth, invasiveness, and migration. We also observed that miR-760 could downregulate the IDO1 protein level. Significantly, we revealed that miR-760 could inhibit CD8+ T cell apoptosis by controlling IDO1 enzyme function. Conclusion. Our findings show that miR-760 inhibits CD8+ T cell responses in LC through regulating IDO1, laying the groundwork for the development of novel vaccination therapies for the treatment of LC.

Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation.

The indoleamine 2,3-dioxygenase 1 (IDO1) metabolic circuitry, comprising the first tryptophan (Trp) catabolite L-kynurenine (Kyn) and the aryl hydrocarbon receptor (AHR), has emerged as a mechanism of cancer immune evasion. Here, we investigated the functional role of the IDO1/Kyn/AHR axis in chronic lymphocytic leukemia (CLL). Our data show that CLL cells expressed an active form of the IDO1 enzyme and microenvironmental stimuli can positively modulate its expression. Interferon (IFN)-γ induces IDO1 expression through the Jak/STAT1 pathway and mediates Kyn production concomitantly with Trp consumption in CLL-conditioned media, while INCB018424 (ruxolitinib), a JAK1/2 inhibitor, impaired both effects. To characterize the involvement of IDO1 in leukemic cell maintenance, we overexpressed IDO1 by vector transfection measuring enhanced resistance to spontaneous apoptosis. IDO1 pro-survival influence was confirmed by treating CLL cells with Kyn, which mediated the increase of induced myeloid leukemia cell differentiation protein (MCL1). Conversely, AHR silencing or its blockade via CH-223191 improved the apoptosis of leukemic clones and mitigated MCL1 expression. Moreover, Kyn-treated CLL cells are less affected by the pro-apoptotic effect of ABT-199 (venetoclax), while CH-223191 showed synergistic/additive cytotoxicity with this drug. Lastly, targeting directly MCL1 in CLL cells with AMG-176, we abrogate the pro-survival effect of Kyn. In conclusion, our data identify IDO1/Kyn/AHR signaling as a new therapeutic target for CLL, describing for the first time its role in CLL pathobiology.

Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

The Role of Serum Level of Indoleamine 2,3–dioxygenase 1 (IDO1) in Patients with Prostate Cancer

Prostate cancer or carcinoma of prostate is the development of cancer in the prostate a gland in the malereproductive system. This is a cross sectional, hospital based study. This study was carried out at theOncology Center in Kirkuk City- Iraq from the 1st of February 2020 to the end of June 2020. A verbalconsent was taken from each Men included in this study whether considered as a case or control. Sixty_ Seven men with prostatic cancer were screened to participate in the present study. Men with prostaticcancer attended to Oncology Center were enrolled in this study. The diagnosis of prostate cancer was madeby the patient’s file contain information about his state after take agreement from each Men included inthis study, that is mean that the diagnosis of prostate cancer was according to discussion of the oncologist. Men ages were between 52 to 89 years, and they were from the center and periphery of Kirkuk City.Forty-Seven Men with prostatic cancer were considered as a study group, three Men did not meeting theinclusion criteria and 17 Men refuse to participate in this study due to the personal reasons. Forty apparentlyhealthy Men without prostatic cancer and with negative family history. Each Men with benign tumor, benignprostatic hyperplasia or prostatitis diseases were excluded from this study. By using a sterile disposablesyringe, 5 ml of venous blood sample was drawn from the vein of each Men at morning and was kept intodisposable gel tube and allowed to clot for(2hour) at room temperature, then each sample was centrifugedat 6000 rpm for 10 minutes to obtain serum. The serum was aspirated by mechanical micropipette andtransferred into clean Eppendorf tubes which labeled with number, then put this Eppendorf tubes in urinecups for safety and addition more information like name, date, number and stored at - 20 0C until the timeof estimation. Serum of the patients and controls had assay for: Human IDO1 (Indoleamine 2,3-dioxygenase1) and Human acid phosphatase/prostate by ELISA. The study demonstrated that, the rate of prostate canceroccurrence was increased with age and majority of patients were belonged to the age group (70-79 years).The study also showed non-significant difference between patients and control regarding their BMI mean(27.36±5.16 vs. 27.24±3.34kg\m²) respectively. The study showed that, serum level of IDO1 enzyme waselevated significantly (P<0.01) in prostate cancer patients (11.74±0.28 pg/ml) as compared with healthycontrol group (4.78±0.15 pg/ml). The study showed that, the highest mean of PAP enzyme was recorded inprostate cancer patients (0.111±0.075 pg/ml) as compared with healthy control group (0.069±0.024 pg/ml).The difference was highly significant (P<0.01), Table 3. The study showed positive correlation between PAPenzyme and stage of prostate cancer (r: 0.34). The study showed positive correlation between IDO1 leveland stage of prostate cancer (r: 0.62). The study showed negative correlation between IDO1 level and PAPlevel in prostate cancer patients (r: -0.22).

Jejucarbazoles A-C, carbazole glycosides with indoleamine 2,3-dioxygenase 1 inhibitory activity from Streptomyces sp. KCB15JA151.

A bioassay-guided investigation led to the isolation of three new carbazole glycosides, jejucarbazoles A–C (1–3), from Streptomyces sp. KCB15JA151. Their planar structures were elucidated by detailed NMR and MS spectroscopic analysis with a literature study. Their relative and absolute configurations were established by ROESY correlations, coupling constants, LC-MS analysis of thiocarbamoyl-thiazolidine carboxylate derivatives, and ECD calculation. Compounds 1–3 showed indoleamine 2,3-dioxygenase 1 (IDO1) inhibitory activity with IC50 values of 18.38, 9.17, and 8.81 μM. The molecular docking analysis suggested that all compounds act as heme-displacing inhibitors against IDO1 enzyme.

Diverse chemical space of indoleamine-2,3-dioxygenase 1 (Ido1) inhibitors.

Indoleamine-2,3-dioxygenase 1 (IDO1) catalyses the first and rate limiting step of kynurenine pathway accounting for the major contributor of L-Tryptophan degradation. The Kynurenine metabolites are identified as essential cofactors, antagonists, neurotoxins, immunomodulators, antioxidants as well as carcinogens. The catalytic active site of IDO1 enzyme consists of hydrophobic Pocket-A positioned in the distal heme site and remains connected to a second hydrophobic Pocket-B towards the entrance of the active site. IDO1 enzyme also relates directly to the modulation of the innate and adaptive immune system. Various studies proved that the over expression of IDO1 enzyme play a predominant role in the escape of immunity during cancer progression. Recently, there has been considerable interest in evaluating the potential of IDO1 inhibitors to mobilize the body's immune system against solid tumours. In the last two decades, enormous attempts to advance new IDO1 inhibitors are on-going both in pharmaceutical industries and in academia which resulted in the discovery of a diverse range of selective and potent IDO1 inhibitors. The IDO1 inhibitors have therapeutic utility in various diseases and in the near future, it may have utility in the treatment of COVID-19. Despite various reviews on IDO1 inhibitors in last five years, none of the reviews provide a complete overview of diverse chemical space including naturally occurring and synthetic IDO1 inhibitors with detailed structure activity relationship studies. The present work provides a complete overview on the IDO1 inhibitors known in the literature so far along with the Structure-Activity Relationship (SAR) in each class of compounds.

Design, Synthesis and Biological Evaluation of Novel 1,2,5-Oxadiazol-3- Carboximidamide Derivatives as Indoleamine 2, 3-Dioxygenase 1 (IDO1) Inhibitors.

Indoleamine-2,3-dioxygenase 1 (IDO1), which catalyzes the degradation of L-tryptophan (L-Trp) to N-formyl kynurenine (NFK) in the first and rate-limiting step of Kynurenine (KYN) pathway has been identified as a promising therapeutic target for cancer immunotherapy. The small molecule Epacadostat developed by Incyte Corp is the most advanced IDO1 inhibitor in clinical trials. In this study, various amidine derivatives were individually installed as the polar capping group onto the amino ethylene side chain to replace the sulfamoylamino moiety of Epacadostat to develop novel IDO1 inhibitors. A series of novel 1,2,5-oxadiazol-3-carboximidamide derivatives were designed, prepared, and evaluated for their inhibitory activities against human IDO1 enzyme and cellular IDO1. In vitro human IDO1 enzyme and cellular IDO1 assay results demonstrate that the inhibitory activities of compound 13a and 13b were comparable to Epacadostat, with the enzymatic IC50 values of 49.37nM and 52.12nM and cellular IC50 values of 12.34nM and 14.34nM, respectively. The anti-tumor efficacy of 13b is slightly better than Epacadosta in Lewis Lung Cancer (LLC) tumor-bearing mice model. 13b is a potent IDO1 inhibitor with therapeutic potential in tumor immunotherapy.

Abstract 5612: Targeting the non-enzymatic function of IDO1 in glioblastoma immunotherapy

Abstract Purpose: More effective immunotherapies against GBM are urgently needed considering current failures in several phase III clinical trials. Indoleamine 2, 3-dioxygenase 1 (IDO1) has been shown to mediate tumor-induced immunosuppression through its tryptophan catabolic activity. Surprisingly, pharmacological IDO1 enzyme inhibitors have not demonstrated any survival benefit in cancer patients either mono-therapeutically or in combination with other therapies. Previously we confirmed that tumor IDO1 mRNA expression is significantly upregulated in GBM patients compared to those from patients with lower grades gliomas. Furthermore, knockdown of IDO1 in mouse GBM cells significantly increased mouse survival and decreased tumor infiltrating regulatory T cells (Treg) but did not affect tumor local tryptophan catabolism. Consistently, inhibition of IDO1 enzymatic activity on GBM-bearing mice did not yield any survival benefit. Collectively, these findings prompt us to test the hypothesis whether GBM cell derived IDO1 can suppress immune response through a non-enzymatic pathway. Methods: IDO1-deficient mouse GBM cells were created and modified to stably express either wild-type (WT) or enzyme-null IDO1. Tumor infiltrating lymphocytes (TILs) and survival rate were analyzed from mice engrafted with these modified cells. Ex vivo co-culture of splenic monocytes with the modified GBM cells was also employed. To interrogate potential IDO1-regulated gene(s) in GBM, gene expression profile in human GBM cell lines under either IDO1 knockdown or overexpression conditions were investigated by microarray analysis and verified by real-time RT-PCR. Result: Overexpression of WT IDO1 in GBM cells led to significantly increased Treg accumulation in GBM and much poorer survival in comparison to IDO1-deficent tumor cells. Interestingly, ablation of IDO1 enzyme activity in the enzyme-null IDO1 overexpressing tumor cells showed similar Treg accumulation as in the WT IDO1-overexpressing tumor cells and poor survival rate. Moreover, overexpression of both WT and enzyme-null IDO1 in GBM cells induced higher level of mature macrophages (CD11c-CD11b+Ly6c+Ly6glow) than IDO1-deficient GBM cells. Finally, an IDO1 regulated gene encoding a secreted protein was identified in the microarray analysis, which also demonstrates high correlation with IDO1 in many different tumor types. Conclusions: Results from this study strongly imply an enzyme independent pathway associated with IDO1-driven immunosuppression in mouse GBM. Current investigation of the newly identified IDO1 regulated gene in the setting of human GBM will provide novel perspective in our understanding of the non-enzyme function of IDO1 in tumor immunity and enlighten the strategic design for future IDO1-based GBM immunotherapy. Citation Format: Lijie Zhai, Erik Ladomersky, Jun Qian, Brenda Nyugen, April Bell, Kristen Lauing, Derek Wainwright. Targeting the non-enzymatic function of IDO1 in glioblastoma immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5612.

Identification of Human IDO1 Enzyme Activity by Using Genetically Encoded Nitrotyrosine.

Human indoleamine 2,3-dioxygenase 1 (IDO1) has become an increasingly valuable target for cancer immunotherapy because it promotes immune escape by tumor cells. To date, the function of post-translational modifications (PTMs) on IDO1 has not been fully elucidated. Among the many forms of PTMs, it has been identified that three tyrosine sites (Y15, Y345, and Y353) on IDO1 are nitrated and play important roles in catalytic function. Herein, by genetically encoding 3-nitro-l-tyrosine into the tyrosine nitration sites of IDO1, the homogeneous and native nitrated IDO1 have been obtained. It is found that the nitration of different tyrosine sites has different effects on the IDO1 structure and enzyme activity. Nitration at position Y15 has a negligible effect, but nitration at Y345 or Y353 decreases the enzyme activity, especially Y353. Furthermore, these results demonstrate that the regulation of the catalytic function caused by tyrosine nitration is related to perturbation of the protein structure and heme-binding disruption.

Discovery of 5-(pyridin-3-yl)-1H-indole-4,7-diones as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Early studies demonstrated that over expression of indoleamine 2,3-dioxygenase (IDO1) in tumor microenvironment results in tumor immune escape. Herein, in order to simplify the structure of two kinds of IDO1 inhibitors from marine alkaloid, Exiguamine A and Tsitsikammamines, we designed, synthesized a series of 1H-indole-4,7-dione derivatives and evaluated their inhibitory activity in IDO1 enzyme and in IFN-γ stimulated Hela cells in vitro. The structure-activity relationship demonstrated that 5-(pyridin-3-yl)-1H-indole-4,7-dione is a promising scaffold for IDO1 inhibitors and most compounds with this core showed moderate inhibition potency at micromole level. Our further enzyme kinetics experiments reveal that these new developed compounds might act as reversible competitive inhibitors of IDO1.

4,5-Disubstituted 1,2,3-triazoles: Effective Inhibition of Indoleamine 2,3-Dioxygenase 1 Enzyme Regulates T cell Activity and Mitigates Tumor Growth

The improvement of body’s own immune system is considered one of the safest approaches to fight against cancer and several other diseases. Excessive catabolism of the essential amino acid, L-tryptophan (L-Trp) assists the cancer cells to escape normal immune obliteration. The formation of disproportionate kynurenine and other downstream metabolites suppress the T cell functions. Blocking of this immunosuppressive mechanism is considered as a promising approach against cancer, neurological disorders, autoimmunity, and other immune-mediated diseases. Overexpression of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme is directly related to the induction of immunosuppressive mechanisms and represents an important therapeutic target. Several classes of small molecule-based IDO1 inhibitors have been already reported, but only few compounds are currently being evaluated in various stages of clinical trials as adjuvants or in combination with chemo- and radiotherapies. In the quest for novel structural class(s) of IDO1 inhibitors, we developed a series of 4,5-disubstituted 1,2,3-triazole derivatives. The optimization of 4,5-disubstituted 1,2,3-triazole scaffold and comprehensive biochemical and biophysical studies led to the identification of compounds, 3i, 4i, and 4k as potent and selective inhibitors of IDO1 enzyme with IC50 values at a low nanomolar level. These potent compounds also showed strong IDO1 inhibitory activities in MDA-MB-231 cells with no/negligible level of cytotoxicity. The T cell activity studies revealed that controlled regulation of IDO1 enzyme activity in the presence of these potent compounds could induce immune response against breast cancer cells. The compounds also showed excellent in vivo antitumor efficacy (of tumor growth inhibition = 79–96%) in the female Swiss albino mice. As a consequence, this study describes the first example of 4,5-disubstituted 1,2,3-triazole based IDO1 inhibitors with potential applications for immunotherapeutic studies.

IMMU-22. TARGETING NON-CANONICAL FUNCTION OF IDO1 IN GLIOBLASTOMA IMMUNOTHERAPY

Abstract Recent failure of immune checkpoint blockade, anti-PD-1 therapy in patients suffering recurrent glioblastoma (GBM) indicates that development of novel immunosuppressive mediator-targeted therapeutic strategy is critical for more effective immunotherapies against GBM. Indoleamine 2, 3-dioxygenase 1 (IDO1) has been shown to mediate tumor-induced immunosuppression through its tryptophan catabolic activity. Surprisingly, pharmacological IDO1 enzyme inhibitors have not demonstrated any survival benefit in cancer patients either mono-therapeutically or in combination with other therapies. Previously we confirmed that tumor IDO1 mRNA expression is significantly upregulated in GBM patients compared to those from patients with lower grades gliomas. Furthermore, knockdown of IDO1 in mouse GBM cells significantly increased mouse survival and decreased tumor infiltrating Tregs, but did not affect tumor local tryptophan catabolism. Consistently, inhibition of IDO1 enzymatic activity on GBM-bearing mice did not yield any survival benefit. These findings indicate that IDO1 may suppress immune response through a non-enzymatic pathway. To test this hypothesis, IDO1-deficient mouse GBM cells were created and modified to stably express either wild-type (WT) or enzyme-null IDO1. Syngeneic mice intracranially engrafted with both WT and enzyme-null IDO1 overexpressing GBM cells showed significantly increase of tumor infiltrating Tregs and decreased survival in comparison to mice engrafted with IDO1-deficient GBM cells. In contrast, no difference was observed between WT-IDO1 GBM and enzyme-null IDO1 GBM groups. Additionally, when co-cultured with splenic monocytes, both WT and enzyme-null IDO1 overexpressing GBM cells induced more mature macrophages than IDO1-deficient GBM cells. Taken together, our data strongly imply an enzyme independent pathway associated with IDO1-driven immunosuppression in mouse GBM. Validation of this novel mechanism on human GBM cells is ongoing. A soluble molecule has been identified as a potential mediator for the non-enzymatic function of IDO1. Results from this study will provide innovative strategies for IDO1-based immunotherapy against GBM.

Exploration of good and bad structural fingerprints for inhibition of indoleamine-2,3-dioxygenase enzyme in cancer immunotherapy using Monte Carlo optimization and Bayesian classification QSAR modeling

Indoleamine-2,3-dioxygenase 1 (IDO1) is an extrahepatic, heme-containing and tryptophan-catalyzing enzyme responsible for causing blockade of T-cell proliferation and differentiation by depleting tryptophan level in cancerous cells. Therefore, inhibition of IDO1 may be a useful strategy for immunotherapy against cancer. In this study, 448 structurally diverse IDO1 inhibitors with a wide range of activity has been taken into consideration for classification QSAR analysis through Monte Carlo Optimization by using different splits as well as different combinations of SMILES-based, graph-based and hybrid descriptors. The best model from Monte Carlo optimization was interpreted to find out the good and bad structural fingerprints for IDO1 and further justified by using Bayesian classification QSAR modeling. Among the three splits in Monte Carlo optimization, the statistics of the best model was obtained from Split 3: sensitivity = 0.87, specificity = 0.91, accuracy = 0.89 and MCC = 0.78. In Bayesian classification modeling, the ROC scores for training and test set were found to be 0.91 and 0.86, respectively. The combined modeling analysis revealed that the presence of aryl hydrazyl sulphonyl moiety, furazan ring, halogen substitution, nitro group and hetero atoms in aromatic system can be very useful in designing IDO1 inhibitors. All the good and bad structural fingerprints for IDO1 were identified and are justified by correlating these fragments to the inhibition of IDO1 enzyme. These structural fingerprints will guide the researchers in this field to design better inhibitors against IDO1 enzyme for cancer immunotherapy.Communicated by Ramaswamy H. Sarma