Discovery and biological evaluation of a new type of dual inhibitors of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase from ethnomedicinal plant Dactylicapnos scandens.

The root of Dactylicapnos scandens (D.Don.) Hutch (Papaveraceae), one of the most famous ethno-medicinal plants from the Bai communities in P. R. China, is used to treat various inflammations and tumours. Bioassay-guided phytochemical research on D. scandens followed by semi-synthesis led to a series of undescribed tetrahydroisoquinoline alkaloids with dual inhibitory activities against indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO). The previously undescribed dark-green alkaloid dactycapnine A exhibited the best dual inhibitor effects among the identified compounds. Structure-activity relationship analysis revealed the importance of the base skeleton with a hyperconjugation system. The performed semi-synthesis further yielded bioactive dimeric and trimeric compounds with hyperconjugated systems. Performed STD NMR experiments disclosed direct interactions between dactycapnine A and IDO1/TDO. Inhibition kinetics indicated dactycapnine A as a mixed-type dual inhibitor. These findings provided a possible explanation for the anticancer properties of the ethno-medicinal plant species D. scandens.

Investigation of chalcogen bioisosteric replacement in a series of heterocyclic inhibitors of tryptophan 2,3-dioxygenase.

Selenium is an underexplored element that can be used for bioisosteric replacement of lower molecular weight chalcogens such as oxygen and sulfur. More studies regarding the impact of selenium substitution in different chemical scaffolds are needed to fully grasp this element's potential. Herein, we decided to evaluate the impact of selenium incorporation in a series of tryptophan 2,3-dioxygenase (TDO2) inhibitors, a target of interest in cancer immunotherapy. First, we synthesized the different chalcogen isosteres through Suzuki-Miyaura type coupling. Next, we evaluated the isosteres' affinity and selectivity for TDO2, as well as their lipophilicity, microsomal stability and cellular toxicity on TDO2-expressing cell lines. Overall, chalcogen isosteric replacements did not disturb the on-target activity but allowed for a modulation of the compounds' lipophilicity, toxicity and stability profiles. The present work contributes to our understanding of oxygen/sulfur/selenium isostery towards increasing structural options in medicinal chemistry for the development of novel and distinctive drug candidates.

Synthesis and in vivo antitumor evaluation of an orally active potent phosphonamidate derivative targeting IDO1/IDO2/TDO.

Targeting Trp-Kyn pathways has been identified as an attractive approach for the cancer immunotherapies. In this study, a novel phosphonamidate containing compound was designed, synthesized and evaluated for its inhibitory activity against key dioxygenases in Trp-Kyn pathway, including IDO1, IDO2 and TDO. This compound showed potent IDO1 inhibitory activity with an IC50 value of 94 nM in an enzymatic assay and 12.6 nM in HeLa cells. In addition, this compound showed promising IDO2 inhibition and TDO inhibition with IC50 values of 310 nM and 2.6 µM, respectively, in enzyme assay. Based on the promising enzyme inhibitory activity toward IDO/TDO, compound F04 was evaluated of its antitumor effects in two tumor models. Further evaluation of mechanism demonstrated compound F04 with the remarkable capacity of reducing kynurenine level in plasma/TME and restoring anti-tumor immune response. F04 could be further developed as a potential immunotherapeutic agent combined with immune checkpoint inhibitors or chemotherapeutic drugs for cancer treatment.

Identification of Substituted Naphthotriazolediones as Novel Tryptophan 2,3-Dioxygenase (TDO) Inhibitors through Structure-Based Virtual Screening.

A structure-based virtual screening strategy, comprising homology modeling, ligand-support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

St. John's Wort increases brain serotonin synthesis by inhibiting hepatic tryptophan 2, 3 dioxygenase activity and its gene expression in stressed rats.

We aimed to investigate the effects of herbal St. John's Wort (SJW) on transcriptional regulation of hepatic tryptophan 2, 3 - dioxygenase (TDO) enzyme activity and brain regional serotonin (5-HT) levels in rats exposed to forced swim test (FST). TDO mRNA expression was quantified using real-time reverse transcription polymerase chain (RT-PCR) reaction and brain regional indoleamines were determined by high performance liquid chromatography coupled to fluorescence detector. Behavioral analysis shows significant reduction in immobility time in SJW (500mg/kg/ml) administered rats. It was found that pretreatment of SJW to rats did not prevent stress-induced elevation in plasma corticosterone levels however it increases serotonin synthesis by virtue of inhibiting hepatic TDO enzyme activity and its gene expression, ascertaining the notion that there exists an inverse relationship between hepatic TDO enzyme activity and brain 5-HT. The drug also decreases serotonin turnover in all the brain areas (hypothalamus, hippocampus amygdala) in stressed rats endorsing its monoamine oxidase inhibition property. Inhibition of TDO enzyme activity and its gene expression by the drug provides new insights for the development of therapeutic interventions for stress related mental illnesses.

High-throughput fluorescence-based screening assays for tryptophan-catabolizing enzymes.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

Citalopram decreases tryptophan 2,3-dioxygenase activity and brain 5-HT turnover in swim stressed rats.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressant class today and exert their effects by increasing synaptic concentrations of serotonin (5-HT). The forced swim test (FST) is the most widely used animal test predictive of antidepressant action. Rationale of the present study was to investigate the acute effects of citalopram on hepatic tryptophan metabolism and disposition in rats exposed to FST. METHODS: We investigated the effects of acute citalopram (20 mg/kg, ip) administration on rat's behavioral responses in FST paradigm, hepatic tryptophan 2,3-dioxygenase (TDO) activity, serum corticosterone levels and brain regional 5-HT metabolism. RESULTS: Citalopram administered to swim-stressed rats showed a decrease in FST-induced increases in plasma corticosterone concentration and 5-HT turnover in hypothalamus, amygdala and hippocampus. The drug also decreases immobility and increases swimming during the FST. Citalopram administration to unstressed rats increases plasma corticosterone concentration but decreases 5-HT turnover in all three brain areas examined. CONCLUSIONS: Our findings support the hypothesis that acute citalopram administration increases tryptophan (by inhibiting TDO activity) availability for 5-HT synthesis and activates serotonergic neurotransmission in limbic brain areas in rats exposed to FST paradigm. The mechanism of action of citalopram in ameliorating social stress related depressive disorder in humans is discussed.

Antioxidants inhibit indoleamine 2,3-dioxygenase in IFN-gamma-activated human macrophages: posttranslational regulation by pyrrolidine dithiocarbamate.

Induction of the heme-containing indoleamine 2,3-dioxygenase (IDO) by IFN-gamma is implicated in anti-microbial and pro-inflammatory activities of human macrophages. Antioxidants can modulate the expression of immune and inflammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-kappaB. Here we show that IFN-gamma treatment of human monocyte-derived macrophages (hMDMs) increased the proportion of oxidized glutathione. PDTC attenuated this increase and inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other antioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein expression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cellular IDO activity without affecting protein expression, whereas addition of hemin or the heme precursor delta-aminolevulinic acid increased IDO activity. Also, incubation of IFN-gamma-activated hMDM with delta-[(14)C]-aminolevulinic acid resulted in the incorporation of label into immunoprecipitated IDO, a process inhibited by PDTC and SA. Furthermore, supplementation of lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activity to control levels, and hemin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requirement for de novo heme synthesis for IDO activity in IFN-gamma-activated hMDM. They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes place posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.

Plasma nicotinic acid level in chloroquine treated rats.

The effect of chloroquine phosphate on plasma nicotinic acid levels in adult male albino rats was investigated. Pyrogen free chloroquine phosphate in physiological saline was administered subcutaneously to rats in a dose of 15 mg/kg body weight daily for eleven succeeding days in the treatment group. A control group was given equal volume of physiological saline daily for eleven succeeding days. Nicotinic acid concentration in the plasma was determined [1] Plasma nicotinic acid level was found to be significantly reduced (P < 0.01) throughout the duration of treatment. No change was observed in the control group. The significant reduction of plasma nicotinic acid level observed in this study may not be unrelated to competitive inhibition of the enzyme tryptophan dioxygenase by chloroquine phosphate.