Discovery of highly potent heme-displacing IDO1 inhibitors based on a spirofused bicyclic scaffold.

Through structural modification of an oxalamide derived chemotype, a novel class of highly potent, orally bioavailable IDO1-specific inhibitors was identified. Representative compound 18 inhibited human IDO1 with IC50 values of 3.9 nM and 52 nM in a cellular and human whole blood assay, respectively. In vitro assessment of the ADME properties of 18 demonstrated very high metabolic stability. Pharmacokinetic profiling in mice showed a significantly reduced clearance compared to the oxalamides. In a mouse pharmacodynamic model 18 nearly completely suppressed lipopolysaccharide-induced kynurenine production. Hepatocyte data of 18 suggest the human clearance to be in a similar range to linrodostat (1).

Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production without inhibiting indoleamine 2,3-dioxygenase activity.

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC50 = 0.34 µM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment.

Quinolinic acid toxicity on oligodendroglial cells: relevance for multiple sclerosis and therapeutic strategies.

The excitotoxin quinolinic acid, a by-product of the kynurenine pathway, is known to be involved in several neurological diseases including multiple sclerosis (MS). Quinolinic acid levels are elevated in experimental autoimmune encephalomyelitis rodents, the widely used animal model of MS. Our group has also found pathophysiological concentrations of quinolinic acid in MS patients. This led us to investigate the effect of quinolinic acid on oligodendrocytes; the main cell type targeted by the autoimmune response in MS. We have examined the kynurenine pathway (KP) profile of two oligodendrocyte cell lines and show that these cells have a limited threshold to catabolize exogenous quinolinic acid. We further propose and demonstrate two strategies to limit quinolinic acid gliotoxicity: 1) by neutralizing quinolinic acid's effects with anti-quinolinic acid monoclonal antibodies and 2) directly inhibiting quinolinic acid production from activated monocytic cells using specific KP enzyme inhibitors. The outcome of this study provides a new insight into therapeutic strategies for limiting quinolinic acid-induced neurodegeneration, especially in neurological disorders that target oligodendrocytes, such as MS.

Identification of novel kynurenine production-inhibiting benzenesulfonamide derivatives in cancer cells.

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is known to be a key regulator of human immune responses including cancer immune tolerance. Therefore, abrogation of Kyn production from cancer cells by small molecules may be a promising approach to anticancer therapy. Indeed, several small molecule inhibitors of indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the catabolism of Trp to Kyn, exert antitumor effects in animal models. We screened our chemical libraries using a cell-based Kyn production assay to identify a new type of small molecules that regulate Kyn production, and for the first time identified a benzenesulfonamide derivative (compound 1) as a hit with the ability to inhibit Kyn production in interferon-γ (IFN-γ)-stimulated A431 and HeLa cells. Unlike the previously identified S-benzylisothiourea derivative, compound 2, compound 1 had little effect on the enzymatic activity of recombinant human IDO in vitro but suppressed the expression of IDO at the mRNA level in cells. Furthermore, compound 1 suppressed STAT1-dependent transcriptional activity and DNA binding, whereas no decrement in either the expression or phosphorylation level of STAT1 was observed. The inhibition of IDO expression by several benzenesulfonamide derivatives is associated with the suppression of STAT1. Thus, compound 1 and its analogs might be useful for analyzing the regulation of IDO activation, and STAT1-targeting could be an alternative to the IDO-directed approach for the regulation of Kyn levels by small molecules in the tumor microenvironment.

Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.

Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling.IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.

The indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive T regulatory cell generation.

Human plasmacytoid dendritic cells (PDCs) can drive naive, allogeneic CD4(+)CD25(-) T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). However, the intracellular mechanism or mechanisms underlying PDC-induced Treg generation are unknown. In this study, we show that human PDCs express high levels of IDO, an intracellular enzyme that catabolizes tryptophan degradation. Triggering of TLR 9 with CpG oligodeoxynucleotides activates PDCs to up-regulate surface expression of B7 ligands and HLA-DR Ag, but also significantly increases the expression of IDO and results in the generation of inducible Tregs from CD4(+)CD25(-) T cells with potent suppressor cell function. Blocking IDO activity with the pharmacologic inhibitor 1-methyl-D-tryptophan significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine, the immediate downstream metabolite of tryptophan, bypasses the 1-methyl-D-tryptophan effect and restores PDC-driven Treg generation. Our results demonstrate that the IDO pathway is essential for PDC-driven Treg generation from CD4(+)CD25(-) T cells and implicate the generation of kynurenine pathway metabolites as the critical mediator of this process.

Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer.

Cisplatin resistance is a major barrier in the effective treatment of lung cancer. Cisplatin-resistant (CR) lung cancer cells do not primarily use glucose but rather consume amino acids such as glutamine and tryptophan (Trp) for survival. CR cells activate the kynurenine (KYN) pathway (KP) to cope with excessive reactive oxygen species (ROS) and maintain homeostasis for growth and proliferation. Consequently, indoleamine 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR cells' survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in CR cells versus cisplatin-sensitive lung cancer. Importantly, significantly greater KYN/Trp ratio (P = 0.005) is detected in serum of patients who fail cisplatin when compared with naïve treatment. Knocking down IDO1 using shRNA or IDO1 inhibitors heightens ROS levels and results in a significant growth inhibitory effect only on CR cells and not on cisplatin-sensitive cells. Exposing CR cells to antioxidant (TIRON) results in suppression of IDO1 activity and confers resistance to IDO1 inhibition, indicating an interrelationship between ROS and IDO1. Because KYN plays a critical role in reprogramming naïve T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype, we observed higher expression of TGFß, FoxP3, and CD4+CD25+ in mice bearing CR tumors compared with tumors from cisplatin-sensitive counterparts. IMPLICATIONS: Findings suggest that the enzyme-inhibitory activity and antitumor efficacy of IDO1 inhibitors rely in part on ROS levels, arguing that IDO1 expression alone may be insufficient to determine the clinical benefits for this class of experimental cancer drugs. Importantly, IDO1 inhibitors may be more suitable to treat patients with lung cancer who failed cisplatin therapy than naïve treatment patients.

Pt(IV) hybrids containing a TDO inhibitor serve as potential anticancer immunomodulators.

Tryptophan 2,3-dioxygenase (TDO), an immunosuppressive enzyme, can involve in immune evasion and tumor tolerance. TDO inhibitors can boost the efficacy of chemotherapeutics by promoting immunity. Herein, a strategy to introduce a TDO inhibitor into Pt(IV) complexes for reversing tumor immune suppression was adopted. A mono-modified Pt(IV) complex, 3, displayed significant antitumor activity against human liver cancer cells. Flow cytometry study revealed that complex 3 could induce cell death via a mitochondrial-dependent apoptosis pathway and arrest the cell cycle at S phase. Furthermore, complex 3 was effective to enhance T-cell immune responses by inhibiting the TDO enzyme expression to block the kynurenine production and inactivating the downstream of aryl hydrocarbon receptor (AHR).

Targeting a metabolic pathway to fight the flu.

Our antiviral arsenal to fight influenza viruses is limited and we need novel anti-flu drugs. Recently, cellular drug targets came into focus and omics analysis were instrumental to suggest candidate factors. In this issue of The FEBS Journal, Kainov and colleagues used transcriptome data to investigate virus-induced changes in tryptophan metabolism that may serve as immunomodulatory approach against viruses.

Regulation of kynurenine biosynthesis during influenza virus infection.

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.

Characterization of the Kynurenine Pathway in CD8+ Human Primary Monocyte-Derived Dendritic Cells.

The kynurenine (KYN) pathway (KP) is a major degradative pathway of the amino acid, L-tryptophan (TRP), that ultimately leads to the anabolism of the essential pyridine nucleotide, nicotinamide adenine dinucleotide. TRP catabolism results in the production of several important metabolites, including the major immune tolerance-inducing metabolite KYN, and the neurotoxin and excitotoxin quinolinic acid. Dendritic cells (DCs) have been shown to mediate immunoregulatory roles that mediated by TRP catabolism. However, characterization of the KP in human DCs has so far only been partly delineated. It is critical to understand which KP enzymes are expressed and which KP metabolites are produced to be able to understand their regulatory effects on the immune response. In this study, we characterized the KP in human monocyte-derived DCs (MDDCs) in comparison with the human primary macrophages using RT-PCR, high-pressure gas chromatography, mass spectrometry, and immunocytochemistry. Our results show that the KP is entirely expressed in human MDDC. Following activation of the KP using interferon gamma, MDDCs can mediate apoptosis of T h cells in vitro. Understanding the molecular mechanisms regulating KP metabolism in MDDCs may provide renewed insight for the development of novel therapeutics aimed at modulating immunological effects and peripheral tolerance.

Kynurenine pathway (KP) inhibitors: Novel agents for the management of depression.

Depression is a highly prevalent and severely disabling condition globally. Despite being a major cause of disability worldwide, little progress has been made in the last three decades in developing rational and novel pharmacological treatment options for the management of depression. Recently there has been growing interest in the role of kynurenine pathway in pathophysiology of depression. In this paper, the potential role of kynurenine pathway inhibitors in the management of depression particularly in secondary and reactive depression and the development of novel antidepressant drugs targeting kynurenine pathway are discussed.

Kynurenine pathway inhibition reduces neurotoxicity of HIV-1-infected macrophages.

The AIDS dementia complex (ADC) is a consequence of excessive immune activation driven at least in part by systemic HIV infection and probably brain infection. Quinolinic acid (QUIN) is a neurotoxic tryptophan metabolite produced by macrophages in response to stimulation with cytokines or infection with HIV-1. Consequently it has been implicated in ADC pathogenesis. However, macrophages infected with HIV-1 synthesize numerous neurotoxic substances. Therefore we conducted experiments using human fetal brain tissue to determine the relative importance of QUIN as a neurotoxin in ADC. Human macrophages were infected with HIV-1 in vitro using a viral isolate from a demented patient. 6-Chloro-D-tryptophan, an inhibitor of QUIN biosynthesis, was added to half the macrophage cultures to block formation of QUIN. Supernatants containing QUIN (SQpos) or in which QUIN biosynthesis had been inhibited (SQneg) were then added to human fetal brain aggregate cultures. Toxicity was evaluated using lactate dehydrogenase efflux, trypan blue exclusion, immunohistochemistry, image analysis, and electron microscopy. Each technique showed a reduction of toxicity in SQneg-treated cultures. These studies confirm the significance of QUIN as a neurotoxin in ADC and suggest that neuroprotective strategies may have a place in the treatment of this disease.

In vivo effect of chloramphenicol and thiamphenicol on some enzymes of normal mouse liver.

Chloramphenicol a potent inhibitor of bacterial and some mammalian cell protein synthesis, was administered i.p. to a group of mice for 6 consecutive days. Another group of animals was treated similarly with thiamphenicol and a third group served as control. The effects of the two antibiotics on the activity of some liver enzymes; the two pyridoxal 5-phosphate dependent enzymes, kynurenine hydrolase and kynurenine amino-transferase; pyridoxal phosphokinase; beta-glucuronidase and acid ribonuclease were determined. Chloramphenicol and thiamphenicol decreased significantly the activities of kynurenine hydrolase, beta-glucuronidase and acid ribonuclease and both drugs increased the activity of pyridoxal phosphokinase significantly. Their effect on kynurenine amino-transferase was different, chloramphenicol decreased while thiamphenicol increased the enzyme activity. Results are discussed and possible explanations suggested.

Kynurenic acid antagonists and kynurenine pathway inhibitors.

The kynurenine pathway accounts for the metabolism of around 80% of non-protein tryptophan metabolism. It includes both an agonist (quinolinic acid) at NMDA receptors and an antagonist (kynurenic acid). Since their discovery, there has been a major development of kynurenic acid analogues as neuroprotectants for the treatment of stroke and neurodegenerative disease. Several prodrugs of kynurenic acid or its analogues that can be hydrolysed within the CNS are also available. More recently, the pathway itself has proved to be a valuable drug target, affected by agents which reduce the synthesis of quinolinic acid and increase the formation of kynurenic acid. The change in the balance of these, away from the excitotoxin and towards the neuroprotectant, has anticonvulsant and neuroprotective properties.

Pharmacological manipulation of brain kynurenine metabolism.

The amino acid tryptophan is a precursor for the neurotransmitter serotonin as well as for kynurenic and quinolinic acids. These latter molecules are antagonists and agonists, respectively, of the excitatory amino acid glutamate and arise through the kynurenine pathway of tryptophan metabolism. Significant differences exist in the sites and physiological control of serotonin versus kynurenine. While serotonin is formed within serotonin neurons (in the brain and intestine) and neuroendocrine cells of the intestine, kynurenine is formed by liver cells (as a precursor to nicotinic acid) and in macrophages, activated by inflammatory cytokines. Our studies are based on the hypothesis that inhibition of kynurenine metabolism (at the kynurenine hydroxylase [KH] step) allows the amino acid to be converted to kynurenic acid, a neuroprotective antagonist of excitatory amino acid receptors. Inhibition of KH also prevents formation of the neurotoxic species 3-hydroxykynurenine and quinolinic acid. To accomplish this end, inhibitors were identified and are described.

Antagonism of L-glycine to seizures induced by L-kynurenine, quinolinic acid and strychnine in mice.

L-glycine (1-12.5 micrograms, intracerebroventricularly, i.c.v.) completely prevented seizures induced by i.c.v. administration of L-kynurenine, and practically did not modify those induced by another convulsant quinolinic acid, a metabolite of tryptophan, and by strychnine. L-Glycine administered intraperitoneally (i.p.) (1000 mg/kg) decreased lethality after K-kynurenine and quinolinic acid; at doses of 3000 and 4000 mg/kg which are sedative and hypothermic it prolonged the latency of strychnine and L-kynurenine seizures. The convulsant action of pentylenetetrazol was not modified. Kynurenine seizures are suggested to be related to the action of kynurenine on glycine receptors in the central nervous system.

Nicotinamide, inosine and hypoxanthine, putative endogenous ligands of the benzodiazepine receptor, opposite to diazepam are much more effective against kynurenine-induced seizures than against pentylenetetrazol-induced seizures.

Nicotinamide (NAM, 1000 mg/kg), inosine (INS, 1000 mg/kg), hypoxanthine (HXT, 500 mg/kg), putative endogenous ligands of the benzodiazepine receptor, and nicotinic acid (NA, 500 mg/kg) diminished DL-kynurenine-(DL-K, 50 micrograms ICV) induced seizures in C57BL/6 adult male mice and only prolonged the latency of pentylenetetrazol (PTZ, 500 micrograms iCV) seizures. The same effect was previously observed when PTZ was administered IP. In albino male BALB/c and SHR (bred from Swiss) mice only NA was effective against DL-K. Diazepam in a dose of 0.5 mg/kg prevented PTZ-induced seizures in half of the animals but even in dose of 10 and 20 mg/kg it was ineffective against DL-K. When injected ICV NAM (1 and 10 micrograms), INS (10 micrograms) and HXT (10 micrograms) prevented seizures induced by DL-K and were ineffective against seizures induced by PTZ. It is suggested that if NAM, INS and HXT are of functional importance in the central nervous system, they can act as antagonists of endogenous brain kynurenine. NA and NAM are suggested to be functional feedback inhibitory regulators of the kynurenine pathway of metabolism of tryptophan.

Inhibitors of the kynurenine pathway.

Strokes (intracranial thomboses or haemorrhaging) cause death and disability, but effective treatments are lacking. The metabolism of tryptophan leads to the generation of quinolinic acid, an agonist potentially neurotoxic at glutamate receptors, and kynurenic acid, an antagonist at the same population of receptors. The commercial development of the kynurenine pathway has included the use of analogues of kynurenic acid as antagonists at glutamate receptors. A second has been to use prodrugs of kynurenic acid or its analogues. Alternatively, it is proving possible to interfere directly with the kynurenine pathway to block the synthesis of quinolinic acid and promote the formation of kynurenic acid. This change yields neuroprotectant and anticonvulsant compounds.

Serotonin-GABA treatment is hypothesized for self-injury in Lesch-Nyhan syndrome.

The self-injurious movements of Lesch-Nyhan patients are considered extremely severe and currently intractable. Lesch-Nyhan syndrome is a genetic disorder of purine metabolism resulting in several abnormalities, one of which is elevated levels of xanthine. The author reasons that elevated plasma xanthine sets off a chain of events that produce excessive endogenous convulsants and diminished endogenous anticonvulsants. Treatment is proposed for self-injury in Lesch-Nyhan that entails reducing production of two endogenous convulsants (kynurenine and quinolinic acid) and enhancing two endogenous anticonvulsants (serotonin and gamma aminobutyric acid).