Human Indoleamine Research Articles

Inhibitory effect of human indoleamine 2,3-dioxygenase 1 (hIDO1) by kazinols of 1,3-diphenylpropane derivatives

This study focused on identifying and characterizing 1,3-diphenylpropane derivatives from flavonoids that inhibit human indoleamine 2,3-dioxygenase 1 (hIDO1) enzymes, which play a role in immune regulation and are associated with various diseases. A series of isolated metabolites (1–7) demonstrated modest to high inhibition of hIDO1, with binding degree values ranging from 26.31 to 72.17%. In particular, during a target-based screening of natural products using hIDO1, kazinol J (6, a 1,3-diphenylpropane derivative) was found to potently inhibit hIDO1, with a binding degree of 72.17% at 1 ppm. Kazinol J (6) showed concentration-dependent and mixed inhibition kinetics and achieved slow and time-dependent inhibition of hIDO1. Additionally, docking simulations were performed to evaluate the inhibitory potential and binding interactions of the compounds with hIDO1. These findings suggest that these 1,3-diphenylpropane derivatives can serve as therapeutic agents for conditions involving hIDO1 dysregulation, such as cancer, autoimmune disorders, and infectious diseases.

Exploring a repurposed candidate with dual hIDO1/hTDO2 inhibitory potential for anticancer efficacy identified through pharmacophore-based virtual screening and in vitro evaluation

Discovering effective anti-cancer agents poses a formidable challenge given the limited efficacy of current therapeutic modalities against various cancer types due to intrinsic resistance mechanisms. Cancer immunochemotherapy is an alternative strategy for breast cancer treatment and overcoming cancer resistance. Human Indoleamine 2,3-dioxygenase (hIDO1) and human Tryptophan 2,3-dioxygenase 2 (hTDO2) play pivotal roles in tryptophan metabolism, leading to the generation of kynurenine and other bioactive metabolites. This process facilitates the de novo synthesis of Nicotinamide Dinucleotide (NAD), promoting cancer resistance. This study identified a new dual hIDO1/hTDO2 inhibitor using a drug repurposing strategy of FDA-approved drugs. Herein, we delineate the development of a ligand-based pharmacophore model based on a training set of 12 compounds with reported hIDO1/hTDO2 inhibitory activity. We conducted a pharmacophore search followed by high-throughput virtual screening of 2568 FDA-approved drugs against both enzymes, resulting in ten hits, four of them with high potential of dual inhibitory activity. For further in silico and in vitro biological investigation, the anti-hypercholesterolemic drug Pitavastatin deemed the drug of choice in this study. Molecular dynamics (MD) simulations demonstrated that Pitavastatin forms stable complexes with both hIDO1 and hTDO2 receptors, providing a structural basis for its potential therapeutic efficacy. At nanomolar (nM) concentration, it exhibited remarkable in vitro enzyme inhibitory activity against both examined enzymes. Additionally, Pitavastatin demonstrated potent cytotoxic activity against BT-549, MCF-7, and HepG2 cell lines (IC50 = 16.82, 9.52, and 1.84 µM, respectively). Its anticancer activity was primarily due to the induction of G1/S phase arrest as discovered through cell cycle analysis of HepG2 cancer cells. Ultimately, treating HepG2 cancer cells with Pitavastatin affected significant activation of caspase-3 accompanied by down-regulation of cellular apoptotic biomarkers such as IDO, TDO, STAT3, P21, P27, IL-6, and AhR.

Immediate administration of hTERT-MSCs-IDO1-EVs reduces hypoalbuminemia after spinal cord injury.

Spinal cord injury (SCI) presents challenging and unpredictable neurological recovery. During inflammatory conditions, the amount of serum albumin and nutrition consumption decreases. Currently, it is proposed to measure serum albumin and glucose content in human or animal subjects to predict the recovery rate and the efficiency of treatments following SCI. In this study, the effect of extra-cellular vesicles (EVs) from immortalized human adipose tissue-derived mesenchymal stem cells (hTERT-MSCs) equipped with the ectopic expression of the human indoleamine 2,3-dioxygenase-1 (IDO1) gene on serum albumin and glucose levels was investigated. After pre-clearing steps of 72-hr conditioned media, small EVs (sEVs) were isolated based on the ultra-filtration method. They were encapsulated with a chitosan-based hydrogel. Five experimental groups (female rats, N = 30, ~ 230 g) were considered, including SCI, sham, hydrogel, control green fluorescent protein (GFP)-EVs and IDO1-EVs. The 60.00 µL of hydrogel or hydrogels containing 100 µg sEVs from GFP or IDO1-EVs were locally injected immediately after SCI (laminectomy of the T10 vertebra and clip compression). After 8 weeks, non-fasting serum glucose and albumin levels were measured. The results indicated that the level of serum albumin in the animals received IDO1-EVs (3.52 ± 0.04) was increased in comparison with the SCI group (3.00 ± 0.94). Also, these animals indicated higher glucose levels in their serum (250.17 ± 69.61) in comparison with SCI ones (214 ± 45.34). Although these changes were not statistically significant, they could be considered as evidence for the beneficial effects of IDO1-EVs administration in the context of SCI to reduce hypoalbuminemia and improve energy consumption. More detailed experiments are required to confirm these results.

Selective inhibition of indoleamine and tryptophan 2,3-dioxygenases: Comparative study on kynurenine pathway in cell lines via LC-MS/MS-based targeted metabolomics

In the last decade, the kynurenine pathway, which is the primary metabolic route for tryptophan (TRP) catabolism, has sparked great interest in the pharmaceutical sciences due to its role in immune regulation and cancer immunoediting. In this context, the development of cell-based assays might represent a tool to: i) characterize the cell secretome according to cell types; ii) gain more insight into the role of kynurenines in different disease scenarios; iii) screen hIDO1 (human indoleamine 2,3-dioxygenase) inhibitors and evaluate their effect on downstream TRP-catabolizing enzymes. This paper reports a validated Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify TRP, L-kynurenine (KYN), xanthurenic acid (XA), 3-hydroxykynurenine (3OHKYN), kynurenic acid (KA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid (AA), 5-hydroxytryptamine (serotonin, 5HT) and tryptamine (TRYP) in Dulbecco’s Modified Eagle and Eagle’s Minimum Essential Media (DMEM and EMEM, respectively). The quantitative method was validated according to FDA, ICH and EMA guidelines, later applied: i) to assess the impact of selective inhibition of hIDO1 or hTDO (human tryptophan 2,3-dioxygenase) on the kynurenine pathway in A375 (melanoma), MDA-MB-231 (breast cancer), and U87 (glioblastoma) cell lines using multivariate analysis (MVA); ii) to determine the IC50 values of both well-known (i.e., epacadostat, linrodostat) and the novel hIDO1 inhibitor (i.e., BL5) in the aforementioned cell lines. The proposed LC-MS/MS method is reliable and robust. Furthermore, it is highly versatile and suitable for applications in the preclinical drug research and in vitro assays.

ProBiS-Dock: A Hybrid Multitemplate Homology Flexible Docking Algorithm Enabled by Protein Binding Site Comparison.

The protein data bank (PDB) is a rich source of protein ligand structures, but ligands are not explicitly used in current docking algorithms. We have developed ProBiS-Dock, a docking algorithm complementary to the ProBiS-Dock Database (J. Chem. Inf. Model. 2021, 61, 4097-4107) that treats small molecules and proteins as fully flexible entities and allows conformational changes in both after ligand binding. A new scoring function is described that consists of a binding site-specific scoring function (ProBiS-Score) and a general statistical scoring function. ProBiS-Dock enables rapid docking of small molecules to proteins and has been successfully validated in silico against standard benchmarks. It enables rapid search for new active ligands by leveraging existing knowledge in the PDB. The potential of the software for drug development has been confirmed in vitro by the discovery of new inhibitors of human indoleamine 2,3-dioxygenase 1, an enzyme that is an attractive target for cancer therapy and catalyzes the first rate-determining step of l-tryptophan metabolism via the kynurenine pathway. The software is freely available to academic users at http://insilab.org/probisdock.

X-ray Structure-Guided Discovery of a Potent, Orally Bioavailable, Dual Human Indoleamine/Tryptophan 2,3-Dioxygenase (hIDO/hTDO) Inhibitor That Shows Activity in a Mouse Model of Parkinson's Disease.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan 2,3-dioxygenase (hTDO) have been closely linked to the pathogenesis of Parkinson's disease (PD); nevertheless, development of dual hIDO1 and hTDO inhibitors to evaluate their potential efficacy against PD is still lacking. Here, we report biochemical, biophysical, and computational analyses revealing that 1H-indazole-4-amines inhibit both hIDO1 and hTDO by a mechanism involving direct coordination with the heme ferrous and ferric states. Crystal structure-guided optimization led to 23, which manifested IC50 values of 0.64 and 0.04 μM to hIDO1 and hTDO, respectively, and had good pharmacokinetic properties and brain penetration in mice. 23 showed efficacy against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse motor coordination deficits, comparable to Madopar, an anti-PD medicine. Further studies revealed that different from Madopar, 23 likely has specific anti-PD mechanisms involving lowering IDO1 expression, alleviating dopaminergic neurodegeneration, reducing inflammatory cytokines and quinolinic acid in mouse brain, and increasing kynurenic acid in mouse blood.

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.

Discovery of 5-(N-hydroxycarbamimidoyl) benzofuran derivatives as novel indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are rate-limiting enzymes in the kynurenine pathway (KP) of l-tryptophan (l-Trp) metabolism and are becoming key drug targets in the combination therapy of checkpoint inhibitors in immunoncology. To discover a selective and potent IDO1 inhibitor, a structure–activity relationship (SAR) study of N-hydroxybenzofuran-5-carboximidamide as a novel scaffold was investigated in a systematic manner. Among the synthesized compounds, the N-3-bromophenyl derivative 19 showed the most potent inhibition, with an IC50 value of 0.44 μM for the enzyme and 1.1 μM in HeLa cells. The molecular modeling of 19 with the X-ray crystal structure of IDO1 indicated that dipole-ionic interactions with heme iron, halogen bonding with Cys129 and the two hydrophobic interactions were important for the high potency of 19.

Temporary Intermediates of L-Trp Along the Reaction Pathway of Human Indoleamine 2,3-Dioxygenase 1 and Identification of an Exo Site.

Protein dynamics governs most of the fundamental processes in the human body. Particularly, the dynamics of loops located near an active site can be involved in the positioning of the substrate and the reaction mechanism. The understanding of the functioning of dynamic loops is therefore a challenge, and often requires the use of a multi-disciplinary approach mixing, for example, crystallographic experiments and molecular dynamics simulations. In the present work, the dynamic behavior of the JK-loop of the human indoleamine 2,3-dioxygenase 1 hemoprotein, a target for immunotherapy, is investigated. To overcome the lack of knowledge on this dynamism, the study reported here is based on 3 crystal structures presenting different conformations of the loop, completed with molecular dynamics trajectories and MM-GBSA analyses, in order to trace the reaction pathway of the enzyme. In addition, the crystal structures identify an exo site in the small unit of the enzyme, that is populated redundantly by the substrate or the product of the reaction. The role of this newer reported exo site still needs to be investigated.

Conformational Plasticity in Human Heme-Based Dioxygenases.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) are two important heme proteins that degrade the essential amino acid, l-tryptophan (Trp), along the kynurenine pathway. The two enzymes share a similar active site structure and an analogous catalytic mechanism, but they exhibit a variety of distinct functional properties. Here we used carbon monoxide (CO) as a structural probe to interrogate how the functionalities of the two enzymes are encoded in their structures. With X-ray crystallography, we detected an unexpected photochemical intermediate trapped in a crystal of the hIDO1-CO-Trp complex, where CO is photolyzed from the heme iron by X-rays at cryogenic temperatures (100 K). The CO photolysis triggers a large-scale migration of the substrate Trp, as well as the photolyzed CO, from the active site to a temporary binding site, Sa*. It is accompanied by a large conformational change to an active site loop, JK-LoopC, despite the severely restricted protein motion under the frozen conditions, which highlights the remarkable conformational plasticity of the hIDO1 protein. Comparative studies of a crystal of the hTDO-CO-Trp complex show that CO and Trp remain bound in the active site under comparable X-ray illumination, indicating a much more rigid protein architecture. The data offer important new insights into the structure and function relationships of the heme-based dioxygenases and provide new guidelines for structure-based design of inhibitors targeting them.

Evaluation and comparison of the commonly used bioassays of human indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO)

Inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are potential drugs for the treatment of tumor and neurological diseases. A variety of bioassays have been developed to evaluate IDO1/TDO (IDO1 and/or TDO) inhibitors, with uncertainty regarding how the differences in the assay methods or protocols may influence the assay outcomes. The enzymatic assays of IDO1/TDO are usually performed with NFK assay and Kyn adduct assay while the cellular assays of IDO1 are carried out with Hela assay and HEK293 assay. The present study focused on the comparison of the most common bioassays of IDO1/TDO. In addition, the effects of major factors of bioassays such as reaction time and culture medium on the assay outcomes were evaluated. The study will provide reference for the researchers to select IDO1/TDO inhibitors with bioassays, and promote the development of IDO1/TDO inhibitors.

Human Platelet Releasate (hPR) - A xeno-free supplement for the expansion of stem cells

Background & Aim Background Recent regulatory guidelines for cell therapy applications recommend the use of xeno-free materials to mitigate the risk of immune reaction to animal-derived additives i.e. fetal bovine serum (FBS). On this note, human platelet lysate (hPL) has been accepted as a xeno-free alternative for stem cell expansion. With several methods available to manufacture hPL, the lack of standardization amongst manufacturers has led to inconsistencies in expansion rate, phenotype, and immunomodulatory properties of stem cells; altering the intrinsic therapeutic potential of cells. Further, some platelet lysates induce “conditioning of cells”, where the cell expansion occurs with a bias towards a lineage of differentiation, as directed by the composition and concentration of growth factors, cytokines and chemokines present in the hPL. We have developed a cGMP process to manufacture human platelet releasate (hPR), a potential alternative to hPL and FBS. hPR is a xeno-free, non-conditioning media supplement derived from expired-human platelets. Methods, Results & Conclusion Methods We have developed a proprietary closed-system manufacturing process designed for optimal extraction of growth factors, cytokines and chemokines (Fig.1). Every lot is tested for sterility, endotoxin, mycoplasma, total protein content, and growth factors. The performance characteristics was evaluated by expanding bone marrow derived MSCs (BM-MSCs) in basal growth medium (DMEM) supplemented with hPR and benchmarked with equivalent strengths of three commercially available non-heparin-requiring human platelet lysates, AB serum, and FBS. Cell proliferation and population doubling time were measured using Luna II automated cell counter. Tri-lineage differentiation potential of these cells was assessed with adipogenesis, chondrogenesis and osteogenesis assay. The phenotypic, and biochemical markers of BM-MSCs were quantified using flow cytometry and multiplex ELISA quantibody assay (Fig.2). The potency of expanded cells was assessed by i) measuring human indoleamine 2, 3-dioxygenase (IDO) using kynurenine assay and luminex assay (IDO human ProcartaPlex), ii) mixed lymphocyte reaction assay (a lab-developed co-culture assay), and regulatory T-cell conversion assay using flow cytometry. Results We have developed a cGMP-compliant process to manufacture consistent lots of hPR. Our results indicate that the potency, and proliferation rate of MSCs is higher in basal medium supplemented with hPR compared to FBS or hPL (Fig.3).

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.

A highly efficient modality to block the degradation of tryptophan for cancer immunotherapy: locked nucleic acid-modified antisense oligonucleotides to inhibit human indoleamine 2,3-dioxygenase 1/tryptophan 2,3-dioxygenase expression.

Tumors can utilize a diverse repertoire of immunosuppressive mechanisms to evade attack by the immune system. Despite promising success with blockade of immune checkpoints like PD-1 the majority of patients does not respond to current immunotherapies. The degradation of tryptophan into immunosuppressive kynurenine is an important immunosuppressive pathway. Recent attempts to target the key enzymes of this pathway-IDO1 and TDO2-have so far failed to show therapeutic benefit in the clinic, potentially caused by insufficient target engagement. We, therefore, sought to add an alternative, highly efficient approach to block the degradation of tryptophan by inhibiting the expression of IDO1 and TDO2 using locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs). We show that LNA-modified ASOs can profoundly inhibit the expression of IDO1 and TDO2 in cancer cells in vitro without using a transfection reagent with IC50 values in the sub-micromolar range. We furthermore measured kynurenine production by ASO-treated cancer cells in vitro and observed potently reduced kynurenine levels. Accordingly, inhibiting IDO1 expression in cancer cells in an in vitro system leads to increased proliferation of activated T cells in coculture. We furthermore show that combined treatment of cancer cells in vitro with IDO1-specific ASOs and small molecule inhibitors can reduce the production of kynurenine by cancer cells in a synergistic manner. In conclusion, we propose that a combination of LNA-modified ASOs and small molecule inhibitors should be considered as a strategy for efficient blockade of the degradation of tryptophan into kynurenine in cancer immunotherapy.

UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1

Human indoleamine 2,3-dioxygenase 1 (IDO) is a heme enzyme that catalyzes the first reaction of the main metabolic pathway of L-tryptophan (Trp) to produce N-formylkynurenin. The reaction involves cleavage of the C2=C3 bond in the Trp indole ring and insertion of two atomic oxygens from the iron-bound O2 into the indole 2 and 3 position. For establishment of the chemical mechanism of this unique enzymatic reaction, it is necessary to determine the conformation and electronic state of the substrate Trp bound to IDO. In this study, we measured the ultraviolet resonance Raman spectra of IDO in the presence of Trp to detect the vibrational modes of the substrate Trp. We compared the ultraviolet resonace Raman spectra of Trp in a ternary complex (Trp-bound cyanide enzyme) and a binary complex (Trp-bound reduced enzyme) of IDO with that of free Trp in solution and found that binding to IDO influences the conformation of Trp, resulting in similar changes in the two complexes, especially around the C3-Cβ bond. However, the presence of the diatomic ligand at the heme sixth coordination site in the ternary complex significantly alters the mobility and electronic structure of Trp, most likely resulting in the C2=C3 bond cleavage in the enzymatic reaction.

Mapping the Binding Trajectory of a Suicide Inhibitor in Human Indoleamine 2,3 Dioxygenase 1

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is a small heme-containing enzyme that is an important cancer immune therapeutic target. Several hIDO1 inhibitors have entered clinical trials, among which BMS-986205 (BMS) stands out as the only suicide inhibitor. Here, we crystalized the hIDO1-BMS complex and identified three snapshots along the inhibitor binding trajectory. BMS first binds in a surface cleft near the active site in an extended conformation. The initial binding leads to partial protein unfolding that triggers heme release and subsequent large-scale movement of the inhibitor to the vacant heme binding site, where it adopts a high-energy kinked conformation. Finally the protein reaches its energy-minimum state with the inhibitor relaxed to a bent conformation. The data offers new insights into structure-based design of hIDO1-selective inhibitors.

Mapping the Binding Trajectory of a Suicide Inhibitor in Human Indoleamine 2,3-Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an important heme-containing enzyme that is a key drug target for cancer immunotherapy. Several hIDO1 inhibitors have entered clinical trials, among which BMS-986205 (BMS) stands out as the only suicide inhibitor. Despite its "best-in-class" activity, the action mechanism of BMS remains elusive. Here, we report three crystal structures of hIDO1-BMS complexes that define the complete binding trajectory of the inhibitor. BMS first binds in a solvent exposed surface cleft near the active site in an extended conformation. The initial binding partially unfolds the active site, which triggers heme release, thereby exposing a new binding pocket. The inhibitor then undergoes a large scale movement to this new binding pocket, where it binds by adopting a high energy kinked conformation. Finally, the inhibitor relaxes to a bent conformation, via an additional large scale rearrangement, culminating in the energy minimum state. The structural data offer a molecular explanation for the remarkable efficacy and suicide inhibition activity of the inhibitor. They also suggest a novel strategy that can be applied for drug development targeting hIDO1 and related enzymes.

High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form.

Human indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme with important roles in many cellular processes and is a potential target for drug discovery against cancer and other diseases. Crystal structures of IDO1 in complex with various inhibitors have been reported. Many of these crystals belong to the same crystal form and most of the reported structures have resolutions in the range 3.2-2.3 Å. Here, three new crystal forms of human IDO1 obtained by introducing a surface mutation, K116A/K117A, distant from the active site are reported. One of these crystal forms diffracted to 1.5 Å resolution and can be readily used for soaking experiments to determine high-resolution structures of IDO1 in complex with the substrate tryptophan or inhibitors that coordinate the heme. In addition, this mutant was used to produce crystals of a complex with an inhibitor that targets the apo form of the enzyme under the same conditions; the structure of this complex was determined at 1.7 Å resolution. Overall, this mutant represents a robust platform for determining the structures of inhibitor and substrate complexes of IDO1 at high resolution.

Inhibition Mechanisms of Human Indoleamine 2,3 Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hIDO1, the indoleamine group of the substrate Trp is H-bonded to S167 through a bridging water, while that in hTDO is directly H-bonded to H76. Here we show that Trp binding to the Si site or the mutation of S167 to histidine in hIDO1 retards its turnover activity and that the inhibited activity can be rescued by an effector, 3-indole ethanol (IDE). Kinetic studies reveal that the inhibited activity introduced by Trp binding to the Si site is a result of retarded recombination of the ferryl moiety with Trp epoxide to form NFK and that IDE reverses the effect by preventing Trp from binding to the Si site. In contrast, the abolished activity induced by the S167H mutation is primarily a result of ∼5000-fold reduction in the O2 binding rate constant, possibly due to the blockage of a ligand delivery tunnel, and that IDE binding to the Si site reverses the effect by reopening the tunnel. The data offer new insights into structure-based design of hIDO1-selective inhibitors.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.