One Key and Multiple Locks: Substrate Binding in Structures of Tryptophan Dioxygenases and Hydroxylases

Since its discovery at the beginning of the past century, the essential nutrient l -Tryptophan (l -Trp) and its catabolic pathways have acquired an increasing interest in an ever wider scientific community for their pivotal roles in underlying many important physiological functions and associated pathological conditions. As a consequence, enzymes catalyzing rate limiting steps along l -Trp catabolic pathways - including IDO1, TDO, TPH1 and TPH2 - have turned to be interesting drug targets for the design and development of novel therapeutic agents for different disorders such as carcinoid syndrome, cancer and autoimmune diseases. This article provides a fresh comparative overview on the most recent advancements that crystallographic studies, biophysical and computational works have brought on structural aspects and molecular recognition patterns of these enzymes toward l -Trp. Finally, a conformational analysis of l -Trp is also discussed as part of the molecular recognition process governing the binding of a substrate to its cognate enzymes.     

Characterization of Apo-Form Selective Inhibition of Indoleamine 2,3-Dioxygenase**

Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As it is an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo-form-binding IDO1 inhibitor (GSK5628) to the heme-coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo-binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1), whereas epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis-à-vis specificity and pharmacokinetic parameters.     

New Insights from Crystallographic Data: Diversity of Structural Motifs and Molecular Recognition Properties between Groups of IDO1 Structures

A large number of crystallographic structures of IDO1 in different ligand-bound and -unbound states have been disclosed over the last decade. Yet, only a few of them have been exploited for structure-based drug design (SBDD) campaigns. In this study, we analyzed the structural motifs and molecular-recognition properties of three groups of IDO1 structures: 1) structures containing the heme group and inhibitors in the catalytic site; 2) heme-free structures of IDO1; 3) substrate-bound structures of IDO1. The results suggest that unrelated conformations of the enzyme have been solved with different ligand-induced changes of secondary motifs that localize even in regions remote from the catalytic site. Moreover, the study identified an uncharted region of molecular-recognition space covered by IDO1 binding sites that could guide the selection of diverse structures for additional SBDD studies aimed at the identification of novel lead compounds with differentiated chemical scaffolds.     

The Kynurenine Pathway—New Linkage between Innate and Adaptive Immunity in Autoimmune Endocrinopathies

The kynurenine pathway (KP) is highly regulated in the immune system, where it promotes immunosuppression in response to infection or inflammation. Indoleamine 2,3-dioxygenase 1 (IDO1), the main enzyme of KP, has a broad spectrum of activity on immune cells regulation, controlling the balance between stimulation and suppression of the immune system at sites of local inflammation, relevant to a wide range of autoimmune and inflammatory diseases. Various autoimmune diseases, among them endocrinopathies, have been identified to date, but despite significant progress in their diagnosis and treatment, they are still associated with significant complications, morbidity, and mortality. The precise cellular and molecular mechanisms leading to the onset and development of autoimmune disease remain poorly clarified so far. In breaking of tolerance, the cells of the innate immunity provide a decisive microenvironment that regulates immune cells’ differentiation, leading to activation of adaptive immunity. The current review provided a comprehensive presentation of the known role of IDO1 and KP activation in the regulation of the innate and adaptive arms of the immune system. Significant attention has been paid to the immunoregulatory role of IDO1 in the most prevalent, organ-specific autoimmune endocrinopathies—type 1 diabetes mellitus (T1DM) and autoimmune thyroiditis.     

Increased Indoleamine 2,3-Dioxygenase Levels at the Onset of Sjögren’s Syndrome in SATB1-Conditional Knockout Mice

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by dysfunction of salivary and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Autoantibodies, such as anti-SSA and anti-SSB antibodies, are hallmarks and important diagnostic factors for SS. In our previous study, we demonstrated that SS-like xerostomia was observed in SATB1 conditional knockout (SATB1cKO) mice, in which the floxed SATB1 gene was specifically deleted in hematopoietic cells as early as 4 weeks of age. In these mice, autoantibodies were not detected until 8 weeks of age in SATB1cKO mice, although exocrine gland function reached its lowest at this age. Therefore, other markers may be necessary for the diagnosis of SS in the early phase. Here, we found that mRNA expression of the interferon γ (IFN-γ) gene and the IFN-responsive indoleamine 2,3-dioxygenase (IDO) gene is upregulated in the salivary glands of SATB1cKO mice after 3 and 4 weeks of age, respectively. We detected l-kynurenine (l-KYN), an intermediate of l-tryptophan (l-Trp) metabolism mediated by IDO, in the serum of SATB1cKO mice after 4 weeks of age. In addition, the upregulation of IDO expression was significantly suppressed by the administration of IFN-γ neutralizing antibodies in SATB1cKO mice. These results suggest that the induction of IFN-dependent IDO expression is an initial event that occurs immediately after the onset of SS in SATB1cKO mice. These results also imply that serum l-KYN could be used as a marker for SS diagnosis in the early phases of the disease before autoantibodies are detectable.     

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.     

Indoleamine 2,3 Dioxygenase 1—The Potential Link between the Innate Immunity and the Ischemia-Reperfusion-Induced Acute Kidney Injury?

Ischemia-reperfusion injury (IRI) is of the most common causes of acute kidney injury (AKI); nevertheless, the mechanisms responsible for both early kidney injury and the reparative phase are not fully recognised. The inflammatory response following ischemia is characterised by the crosstalk between cells belonging to the innate immune system—dendritic cells (DCs), macrophages, neutrophils, natural killer (NK) cells, and renal tubular epithelial cells (RTECs). A tough inflammatory response can damage the renal tissue; it may also have a protective effect leading to the repair after IRI. Indoleamine 2,3 dioxygenase 1 (IDO1), the principal enzyme of the kynurenine pathway (KP), has a broad spectrum of immunological activity from stimulation to immunosuppressive activity in inflamed areas. IDO1 expression occurs in cells of the innate immunity and RTECs during IRI, resulting in local tryptophan (TRP) depletion and generation of kynurenines, and both of these mechanisms contribute to the immunosuppressive effect. Nonetheless, it is unknown if the above mechanism can play a harmful or preventive role in IRI-induced AKI. Despite the scarcity of literature in this field, the current review attempts to present a possible role of IDO1 activation in the regulation of the innate immune system in IRI-induced AKI.     

Spiro-Oxindole Skeleton Compounds Are Efficient Inhibitors for Indoleamine 2,3-Dioxygenase 1: An Attractive Target for Tumor Immunotherapy

Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive heme enzyme for its significant function in cancer immunotherapy. Potent IDO1 inhibitors have been discovered for decades, whereas no clinical drugs are used for cancer treatment up to now. With the goal of developing medically valuable IDO inhibitors, we performed a systematic study of SAR405838 analogs with a spiro-oxindole skeleton in this study. Based on the expression and purification of human IDO1, the inhibitory activity of spiro-oxindole skeleton compounds to IDO1 was evaluated by IC₅₀ and K_i values. The results demonstrated that inhibitor 3 exhibited the highest IDO1 inhibitory activity with IC₅₀ at 7.9 μM among all inhibitors, which is ~six-fold of the positive control (4−PI). Moreover, inhibitor 3 was found to have the most effective inhibition of IDO1 in MCF-7 cancer cells without toxic effects. Molecular docking analysis revealed that the hydrophobic interaction stabilized the binding of inhibitor 3 to the IDO1 active site and made an explanation for the uncompetitive mode of inhibitors. Therefore, this study provides valuable insights into the screen of more potent IDO1 inhibitors for cancer immunotherapy.     

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.     

Improving the Potency of Cancer Immunotherapy by Dual Targeting of IDO1 and DNA

Herein we report the first exploration of a dual‐targeting drug design strategy to improve the efficacy of small‐molecule cancer immunotherapy. New hybrids of indoleamine 2,3‐dioxygenase 1 (IDO1) inhibitors and DNA alkylating nitrogen mustards that respectively target IDO1 and DNA were rationally designed. As the first‐in‐class examples of such molecules, they were found to exhibit significantly enhanced anticancer activity in vitro and in vivo with low toxicity. This proof‐of‐concept study has established a critical step toward the development of a novel and effective immunotherapy for the treatment of cancers.     

Discovery of a Novel Scaffold as an Indoleamine 2,3‐Dioxygenase 1 (IDO1) Inhibitor Based on the Pyrrolopiperazinone Alkaloid,Longamide B

Indoleamine 2,3‐dioxygenase 1 (IDO1) has emerged as a key target for cancer therapy, as IDO1 plays a critical role in the capacity of tumor cells to evade the immune system. The pyrrolopiperazinone alkaloid longamide B and its derivatives were identified as novel IDO1 inhibitors based on docking studies and small library synthesis. The thioamide derivative showed higher IDO1 inhibitory activity than longamide B, and displayed an activity similar to that of a representative IDO1 inhibitor, 1‐methyl‐tryptophan. These results suggest that the pyrrolopiperazinone scaffold of longamide B could be used in the development of IDO1 inhibitors.     

Tolerogenic IDO1+CD83− Langerhans Cells in Sentinel Lymph Nodes of Patients with Melanoma

Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin⁺ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1⁻CD83⁺ LCs; real immature IDO1⁻CD83⁻ LCs; tolerogenic mature IDO1⁺CD83⁺ LCs; tolerogenic immature IDO1⁺CD83⁻ LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma.     

Kynurenine/Tryptophan Ratio as a Potential Blood-Based Biomarker in Non-Small Cell Lung Cancer

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) degrade tryptophan (Trp) into kynurenine (Kyn) at the initial step of an enzymatic pathway affecting T cell proliferation. IDO1 is highly expressed in various cancer types and associated with poor prognosis. Nevertheless, the serum Kyn/Trp concentration ratio has been suggested as a marker of cancer-associated immune suppression. We measured Kyn and Trp in blood samples of a wide cohort of non-small-cell lung cancer (NSCLC) patients, before they underwent surgery, and analyzed possible correlations of the Kyn/Trp ratio with either IDO1 expression or clinical–pathological parameters. Low Kyn/Trp significantly correlated with low IDO1 expression and never-smoker patients; while high Kyn/Trp was significantly associated with older (≥68 years) patients, advanced tumor stage, and squamous cell carcinoma (Sqcc), rather than the adenocarcinoma (Adc) histotype. Moreover, high Kyn/Trp was associated, among the Adc group, with higher tumor stages (II and III), and, among the Sqcc group, with a high density of tumor-infiltrating lymphocytes. A trend correlating the high Kyn/Trp ratio with the probability of recurrences from NSCLC was also found. In conclusion, high serum Kyn/Trp ratio, associated with clinical and histopathological parameters, may serve as a serum biomarker to optimize risk stratification and therapy of NSCLC patients.     

A Series of 2-((1-Phenyl-1H-imidazol-5-yl)methyl)-1H-indoles as Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising therapeutic target in cancer immunotherapy and neurological disease. Thus, searching for highly active inhibitors for use in human cancers is now a focus of widespread research and development efforts. In this study, we report the structure-based design of 2-(5-imidazolyl)indole derivatives, a series of novel IDO1 inhibitors which have been designed and synthesized based on our previous study using N1-substituted 5-indoleimidazoles. Among these, we have identified one with a strong IDO1 inhibitory activity (IC₅₀=0.16 μM, EC₅₀=0.3 μM). Structural-activity relationship (SAR) and computational docking simulations suggest that a hydroxyl group favorably interacts with a proximal Ser167 residue in Pocket A, improving IDO1 inhibitory potency. The brain penetrance of potent compounds was estimated by calculation of the Blood Brain Barrier (BBB) Score and Brain Exposure Efficiency (BEE) Score. Many compounds had favorable scores and the two most promising compounds were advanced to a pharmacokinetic study which demonstrated that both compounds were brain penetrant. We have thus discovered a flexible scaffold for brain penetrant IDO1 inhibitors, exemplified by several potent, brain penetrant, agents. With this promising scaffold, we provide herein a basis for further development of brain penetrant IDO1 inhibitors.     

Critical Assessment of a Structure-Based Screening Campaign for IDO1 Inhibitors: Tips and Pitfalls

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.     

Galectin 1—A Key Player between Tissue Repair and Fibrosis

Galectins are ten family members of carbohydrate-binding proteins with a high affinity for β galactose-containing oligosaccharides. Galectin-1 (Gal-1) is the first protein discovered in the family, expressed in many sites under normal and pathological conditions. In the first part of the review article, we described recent advances in the Gal-1 modulatory role on wound healing, by focusing on the different phases triggered by Gal-1, such as inflammation, proliferation, tissue repair and re-epithelialization. On the contrary, Gal-1 persistent over-expression enhances angiogenesis and extracellular matrix (ECM) production via PI3K/Akt pathway activation and leads to keloid tissue. Therefore, the targeted Gal-1 modulation should be considered a method of choice to treat wound healing and avoid keloid formation. In the second part of the review article, we discuss studies clarifying the role of Gal-1 in the pathogenesis of proliferative diabetic retinopathy, liver, renal, pancreatic and pulmonary fibrosis. This evidence suggests that Gal-1 may become a biomarker for the diagnosis and prognosis of tissue fibrosis and a promising molecular target for the development of new and original therapeutic tools to treat fibrosis in different chronic diseases.     

New 4‐Amino‐1,2,3‐Triazole Inhibitors of Indoleamine 2,3‐Dioxygenase Form a Long‐Lived Complex with the Enzyme and Display Exquisite Cellular Potency

Indoleamine‐2,3 dioxygenase 1 (IDO1) has emerged as a central regulator of immune responses in both normal and disease biology. Due to its established role in promoting tumour immune escape, IDO1 has become an attractive target for cancer treatment. A novel series of highly cell potent IDO1 inhibitors based on a 4‐amino‐1,2,3‐triazole core have been identified. Comprehensive kinetic, biochemical and structural studies demonstrate that compounds from this series have a noncompetitive kinetic mechanism of action with respect to the tryptophan substrate. In co‐complex crystal structures, the compounds bind in the tryptophan pocket and make a direct ligand interaction with the haem iron of the porphyrin cofactor. It is proposed that these data can be rationalised by an ordered‐binding mechanism, in which the inhibitor binds an apo form of the enzyme that is not competent to bind tryptophan. These inhibitors also form a very tight, long‐lived complex with the enzyme, which partially explains their exquisite cellular potency. This novel series represents an attractive starting point for the future development of potent IDO1‐targeted drugs.     

The [1,2,4]Triazolo[4,3-a]pyridine as a New Player in the Field of IDO1 Catalytic Holo-Inhibitors

Inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) are considered a promising strategy in cancer immunotherapy as they are able to boost the immune response and to work in synergy with other immunotherapeutic agents. Despite the fact that no IDO1 inhibitor has been approved so far, recent studies have shed light on the additional roles that IDO1 mediates beyond its catalytic activity, conferring new life to the field. Here we present a novel class of compounds originated from a structure-based virtual screening made on IDO1 active site. The starting hit compound is a novel chemotype based on a [1,2,4]triazolo[4,3-a]pyridine scaffold, so far underexploited among the heme binding moieties. Thanks to the rational and in silico-guided design of analogues, an improvement of the potency to sub-micromolar levels has been achieved, with excellent in vitro metabolic stability and exquisite selectivity with respect to other heme-containing enzymes.     

The Role of NRF2/KEAP1 Signaling Pathway in Cancer Metabolism

The nuclear factor-erythroid 2 p45-related factor 2 (NRF2, also called Nfe2l2) and its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (KEAP1), are major regulators of redox homeostasis controlling a multiple of genes for detoxification and cytoprotective enzymes. The NRF2/KEAP1 pathway is a fundamental signaling cascade responsible for the resistance of metabolic, oxidative stress, inflammation, and anticancer effects. Interestingly, a recent accumulation of evidence has indicated that NRF2 exhibits an aberrant activation in cancer. Evidence has shown that the NRF2/KEAP1 signaling pathway is associated with the proliferation of cancer cells and tumerigenesis through metabolic reprogramming. In this review, we provide an overview of the regulatory molecular mechanism of the NRF2/KEAP1 pathway against metabolic reprogramming in cancer, suggesting that the regulation of NRF2/KEAP1 axis might approach as a novel therapeutic strategy for cancers.     

Conformational features of human melanin-concentrating hormone: an NMR and computational analysis

The conformational features of human melanin-concentrating hormone (hMCH) [Asp1-Phe2-Asp3-Met4-Leu5-Arg6-cyclo(S[bond]S)(Cys7-Met8-Leu9-Gly10-Arg11-Val12-Tyr13-Arg14-Pro15-Cys16)-Trp17-Gln18-Val19], in water and in a CD(3)CN/H(2)O (1:1 v/v) mixture at 298 K, have been determined by NMR spectroscopy followed by simulated annealing and molecular dynamics analyses to identify conformer populations. Backbone clustering analysis of NMR-spectroscopy-derived structures in the 7-16 peptide region led to the identification of a single representative structure in each solvent. Both root mean square deviation clustering and secondary structure analysis of the final conformers in both solvents show substantial convergence of most conformers into a single fold in the 4-17 region, with a limited variability around Gly10 and Tyr13 on going from CD(3)CN/H(2)O to pure water. The main feature deduced from the analysis of secondary structures is the occurrence of an N-terminal alpha helix of variable length, which spans an overall residue range of 2-9. A comparative analysis in the two solvents highlights that these structures are substantially different from that reported in the literature for the cyclic MCH(5-14) subunit of salmon MCH, which was used to perform a molecular characterization of the MCH/receptor complex. Our conformational data call for a critical revision of the proposed MCH/receptor complex model.