Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase

The involvement of tryptophan 2,3-dioxygenase (TDO) in cancer biology has recently been described, with the enzyme playing an immunomodulatory role, suppressing antitumour immune responses and promoting tumour cell survival and proliferation. This finding reinforces the need for specific inhibitors of TDO that may potentially be developed for therapeutic use. In this work we have screened ∼2800 compounds from the library of the National Cancer Institute USA and identified seven potent inhibitors of TDO with inhibition constants in the nanomolar or low micromolar range. All seven have antitumour properties, killing various cancer cell lines. For comparison, the inhibition potencies of these compounds were tested against IDO and their inhibition constants are reported. Interestingly, this work reveals that NSC 36398 (dihydroquercetin, taxifolin), with an in vitro inhibition constant of ∼16 μM, is the first TDO-selective inhibitor reported.     

Synthesis of 4- and 5-arylthiazolinethiones as inhibitors of indoleamine 2,3-dioxygenase

Docking studies of 4-phenylthiazolinethione on human IDO1 suggest complexation of the heme iron by the exocyclic sulfur atom further reinforced by hydrophobic interactions of the phenyl ring within pocket A of the enzyme. On this basis, chemical modifications were proposed to increase inhibition activity. Synthetic routes had to be adapted and optimized to yield the desired substituted 4- and 5-arylthiazolinethiones. Their biological evaluation shows that 5-aryl regioisomers are systematically less potent than the corresponding 4-aryl analogs. Substitution on the phenyl ring does not significantly increase inhibition potency, except for 4-Br and 4-Cl derivatives.     

Development of a series of novel o-phenylenediamine-based indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

A novel series of o-phenylenediamine-based inhibitors of indoleamine 2,3-dioxygenase (IDO) has been identified. IDO is a heme-containing enzyme, overexpressed in the tumor microenvironment of many cancers, which can contribute to the suppression of the host immune system. Synthetic modifications to a previously described diarylether series resulted in an additional degree of molecular diversity which was exploited to afford compounds that demonstrated significant potency in the HeLa human cervical cancer IDO1 assay..     

Identification and optimization of a novel series of indoleamine 2,3-dioxygenase inhibitors

The discovery of a series of structurally-novel biaryl urea IDO inhibitors is described. Optimization of a micromolar hit through iterative cycles of synthesis and screening in an assay measuring IDO-mediated intracellular conversion of tryptophan to kynurenine led to potent inhibitors with favorable selectivity and metabolic stability profiles.     

Molecular evolution of bacterial indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD⁺). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD⁺, like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K_m, V_max and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD⁺) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking the kynU gene suggests its IDO has a function similar to eukaryotic enzymes.     

Design,synthesis and biological evaluation of novel aryl-acrylic derivatives as novel indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors

Two series of novel aryl-acrylic derivatives were designed, synthesized, and screened in enzymatic and cellular inhibitory activities. All compounds showed moderate to significant potency. The SAR analyses indicated that the semicarbazone linker is better than the 1,2,3-triazole linker. Among semicarbazone compounds that R₁ bearing di-chain amino groups exhibited superior activities to those with morpholino group. Furthermore, compounds with electron-withdrawing groups at the 2-position or 4-position on the terminal phenyl ring were more active. Among these, compounds 7g, 7i, 7m and 7n exhibited the inhibitory potency in the low micromolar range and displayed negligible level of cytotoxicity against normal HeLa cells. In addition, the study suggested that the aryl-acrylic is an interesting novel scaffold for IDO1 inhibition for further development.     

4,6-Substituted-1H-Indazoles as potent IDO1/TDO dual inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are constitutively overexpressed in many types of cancer cells and exert important immunosuppressive functions. In this article, a series of 4,6-substituted-1H-indazole derivatives were synthesized and evaluated the inhibitory activities against IDO1 and TDO, as well as their structure-activity relationships (SARs). Among these, compound 35 displayed the most IDO1 inhibitory potency with an IC₅₀ value of 0.74?μM in an enzymatic assay and 1.37?μM in HeLa cells. Quantitative analysis of the Western blot results indicated that 35 significantly decreased the INFγ-induced IDO1 expression in a concentration-dependent manner. In addition, 35 showed promising TDO inhibition with an IC₅₀ value of 2.93?μM in the enzymatic assay and 7.54?μM in A172 cells. Moreover, compound 35 exhibited in vivo antitumor activity in the CT26 xenograft model. These findings suggest that 1H-indazole derivative 35 is a potent IDO1/TDO dual inhibitor, and has the potential to be developed for IDO1/TDO-related cancer treatment.     

Synthesis and biological evaluation of novel tryptoline derivatives as indoleamine 2,3-dioxygenase (IDO) inhibitors

Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer’s disease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and evaluated for their inhibitory activity against IDO. Substituted tryptoline derivatives (11a, 11c, 11e, 12b and 12c) were demonstrated to be more potent than known inhibitor MTH-Trp. Suzuki–Miyaura cross-coupling reaction of 11a–d with phenylboronic acid proceeded in high yields. In most cases, C5 and C6 substitutions on the corresponding indole ring were well tolerated. The tryptoline derivative 11c is a promising chemical lead for the discovery of novel IDO inhibitors.     

Substrate stereo‐specificity in tryptophan dioxygenase and indoleamine 2,3‐dioxygenase

The first and rate‐limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N‐formylkynurenine is catalyzed by two heme‐containing proteins, Indoleamine 2,3‐dioxygenase (IDO), and Tryptophan 2,3‐dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small‐molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work, we have used a combination of classical molecular dynamics (MD) and hybrid quantum‐classical (QM/MM) methodologies to establish the structural basis that determine the differences in (a) the interactions of TDO and IDO with small ligands (CO/O₂) and (b) the substrate stereo‐specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo‐specificity of TDO is achieved by the perfect fit of L ‐Trp, but not D ‐Trp, which exhibits weaker interactions with the protein matrix. For hIDO, the presence of multiple stable binding conformations for L /D ‐Trp reveal the existence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO‐selective inhibitors. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Synthesis and biological activity of 1-methyl-tryptophan-tirapazamine hybrids as hypoxia-targeting indoleamine 2,3-dioxygenase inhibitors

We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygenase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were prepared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3 showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1 was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotoxicity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function as IDO inhibitors.     

Synthesis and biological evaluation of benzocyclobutane-C-glycosides as potent and orally active SGLT1/SGLT2 dual inhibitors

Synthesis and biological evaluation of benzocyclobutane-C-glycosides as potent and orally active SGLT1/SGLT2 dual inhibitors are described. Compound 19 showed high inhibitory potency at SGLT1 (IC₅₀?=?45?nM), and excellent potency at SGLT2 (IC₅₀?=?1?nM). It also displayed excellent PK profiles in mice, rats, dogs and monkeys (F?=?78–107%). In SD rats, compound 19 treatments significantly reduced blood glucose levels in a dose-dependent manner. In ZDF rats, compound 19 displayed anti-hyperglycemic effect up to 24?h. Therefore, compound 19 may serve as valuable pharmacological tool, and potential use as a treatment for metabolic syndrome.     

Design,synthesis and preliminary bioactivity studies of indomethacin derivatives as Bcl-2/Mcl-1 dual inhibitors

Bcl-2 family proteins, which divides into pro-apoptosis proteins and anti-apoptosis proteins, are involved in cell apoptosis progression. As numerous studies illustrated, targeting Bcl-2 family proteins is more and more attractive and practicable to cancer treatment. In this work, we designed and synthesized a series of indomethacin derivatives as new inhibitors for Bcl-2 family proteins. Our results of binding assay to Bcl-2 proteins, MTT assay and apoptotic assay indicated that some compounds had potent binding affinity to Bcl-2/Mcl-1 but not Bcl-X_L. Furthermore, compound 8j showed improved anti-proliferative activity than known Bcl-2 inhibitor WL-276.     

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.     

Synthesis and biological evaluation of pyrimidine derivatives as novel human Pin1 inhibitors

Pin1 (Protein interacting with NIMA1) is a cis–trans isomerase and promotes the amide bond rotation of phosphoSer/Thr-Pro motifs in its substrates. Inhibition of Pin1 might be a novel strategy for developing anticancer agents. Herein, a series of pyrimidine derivatives were synthesized and their Pin1 inhibitory activities were evaluated. Among them, four compounds (2a, 2f, 2h and 2l) displayed potent inhibitory activities against Pin1 with IC₅₀ values lower than 3?µM. This series of pyrimidine-based inhibitors presented time-dependent inhibition against Pin1. The structure–activity relationships on the 2-, 4- and 5-positions of the pyrimidine ring were analyzed in details, which would facilitate further exploration of new Pin1 inhibitors.     

盐霉素类似物的分离及抑制吲哚胺2,3-双加氧酶活性研究

为研究盐霉素发酵粗产物中的新型盐霉素类活性化合物。实验通过制备色谱从盐霉素发酵粗产物中分离得到两个盐霉素类似物,并利用波谱手段表征了这两个化合物的结构,化合物A为40-甲基盐霉素,化合物B为35-甲基盐霉素,均为新化合物。生物活性研究表明,化合物B具有一定的吲哚胺2,3-双加氧酶(IDO)抑制活性。     

Design,synthesis and biological evaluation of 2,5-dimethylfuran-3-carboxylic acid derivatives as potential IDO1 inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a vital role in tumor immune escape and has emerged as a promising target for cancer immunotherapy. In this study, a novel series of 2,5-dimethylfuran-3-carboxylic acid derivatives were designed, synthesized and evaluated for inhibitory activities against IDO1, and their structure-activity relationship was investigated. Among these, compound 19a exhibited excellent IDO1 inhibitory activity (HeLa cellular IC₅₀?=?4.0?nM, THP-1 cellular IC₅₀?=?4.6?nM). Further molecular docking studies revealed that the compound 19a formed a coordinate bond with the heme iron through the carboxylic acid moiety. These results indicate that compound 19a is a potential IDO1 inhibitor for further investigation.     

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of d-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of d-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary l-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from d-tryptophan and l-tryptophan in mice.     

A multicomponent approach in the discovery of indoleamine 2,3-dioxygenase 1 inhibitors: Synthesis,biological investigation and docking studies

Indoleamine 2,3-dioxygenase plays a crucial role in immune tolerance and has emerged as an attractive target for cancer immunotherapy. In this study, the Passerini and Ugi multicomponent reactions have been employed to assemble a small library of imidazothiazoles that target IDO1. While the p-bromophenyl and the imidazothiazole moieties have been kept fixed, a full SAR study has been performed on the side-chain, leading to the discovery of nine compounds with sub-micromolar IC₅₀ values in the enzyme-based assay. Compound 7d, displaying a α-acyloxyamide substructure, is the most potent compound, with an IC₅₀ value of 0.20?µM, but a low activity in a cell-based assay. Compound 6o, containing a α-acylaminoamide moiety, shows an IC₅₀ value of 0.81?µM in the IDO1-based assay, a full biocompatibility at 10?µM, together with a modest inhibitory activity in A375 cells. Molecular docking studies show that both 7d and 6o display a unique binding mode in the IDO1 active site, with the side-chain protruding in an additional pocket C, where a crucial hydrogen bond is formed with Lys238. Overall, this work describes an isocyanide based-multicomponent approach as a straightforward and versatile tool to rapidly access IDO1 inhibitors, providing a new direction for their future design and development.     

Discovery and characterisation of hydrazines as inhibitors of the immune suppressive enzyme,indoleamine 2,3-dioxygenase 1 (IDO1)

Screening of a fragment library identified 2-hydrazinobenzothiazole as a potent inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme expressed by tumours that suppresses the immune system. Spectroscopic studies indicated that 2-hydrazinobenzothiazole interacted with the IDO1 haem and in silico docking predicted that the interaction was through hydrazine. Subsequent studies of hydrazine derivatives identified phenylhydrazine (IC₅₀ = 0.25 ± 0.07 μM) to be 32-fold more potent than 2-hydrazinobenzothiazole (IC₅₀ = 8.0 ± 2.3 μM) in inhibiting rhIDO1 and that it inhibited cellular IDO1 at concentrations that were noncytotoxic to cells. Here, phenylhydrazine is shown to inhibit IDO1 through binding to haem.