Kynurenine 3-monooxygenase Research Articles

Viability of diffuse large B-cell lymphoma cells is regulated by kynurenine 3-monooxygenase activity.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous lymphoid malignancy that is the most common type of lymphoma in Japan. Previous studies have demonstrated that patients with DLBCL have a poor prognosis due to increased levels of indoleamine 2,3-dioxygnase and kynurenine (KYN). However, the roles of metabolites acting downstream of KYN and associated enzymes are not fully understood. The present study investigated the role of kynurenine 3-monooxygenase (KMO), which catalyzes the conversion of KYN to 3-hydroxykynurenine (3-HK), using serum samples from patients with DLBCL and human DLBCL cell lines with different KMO expression [STR-428 cells with high levels of KMO expression (KMOhigh) and KML-1 cells with low levels of KMO expression (KMOlow)]. Serum samples from 28 patients with DLBCL and 34 healthy volunteers were used to investigate the association between prognosis and KMO activity or 3-HK levels. Furthermore, to investigate the roles of KMO and its related metabolites, STR-428 and KML-1 cell lines, and the lymph nodes of patients with DLBCL were analyzed by reverse transcription-quantitative PCR for KMO, KYNU, 3-hydroxyanthranilate-3,4-dioxygenase and quinolinate phosphoribosyltransferase, by western blotting, and immunohistochemical or immunofluorescence staining for KMO, and by cell viability and NAD+/NADH assays. KYN pathway metabolites in serum samples were measured by HPLC. Serum 3-HK levels were regulated independently of serum KYN levels, and increased serum 3-HK levels and KMO activity were found to be associated with worse disease progression. Notably, the addition of KMO inhibitors and 3-HK negatively and positively regulated the viability of DLBCL cells, respectively. Furthermore, NAD+ levels in KMOhigh STR-428 cells were significantly higher than those in KMOlow KML-1 cells. These results suggested that 3-HK generated by KMO activity may be involved in the regulation of DLBCL cell viability via NAD+ synthesis.

Tryptophan Metabolism and Cancer Progression

Abstract: Intracellular tryptophan (Trp) is catabolized to a large repertoire of metabolites via two major pathways: indoleamine and tryptophan 2, 3-dioxygenases (IDO/TDO) and Trp hydroxylase (TPH) pathways. The catabolites possess diverse biological activities and carry out various physiological functions. Several catabolites such as kynurenine (Kyn) and serotonin promote while melatonin and 5-methoxytryptophan (5-MTP) suppress cancer growth and metastasis. Cancer cell-derived Kyn enhances cancer growth and evasion of immunosurveillance by interacting with cancer cell and immune cell membrane aryl hydrocarbon receptors (AHR), respectively. Serotonin exerts its tumor-promoting activities through type 1 and type 2 serotonin receptors. 5-MTP and melatonin suppress cancer growth and metastasis by common mechanisms, i.e., inhibition of p300 histone acetyltransferase (HAT) and NF-κB activation, and suppression of cyclooxygenase-2 and cytokine transcription. Both metabolites block p38 MAPK signaling pathway. Human cancer tissues express increased levels of IDO, TDO and kynurenine monooxygenase (KMO) which are correlated with reduced patient survival. In summary, cancer Trp metabolism regulates cancer growth and metastasis by complex mechanisms. 5-MTP and melatonin provide valuable lead to develop new drugs for chemo-prevention and adjuvant therapy of cancer.

Pharmacokinetics and metabolic disposition of a potent and selective kynurenine monooxygenase inhibitor, CHDI-340246, in laboratory animals

The disposition of a novel kynurenine monooxygenase inhibitor, CHDI-340246, was investigated in vitro and in animals. In vitro, there was minimal metabolic turnover of CHDI-340246 in all species. The protein binding was higher in human plasma (99.7%) relative to other species. In all species, blood clearance was low (<20% of liver blood flow) and volume of distribution was small (<0.5 L/kg). The terminal half-life was longer in monkeys (9 hr) than in mice, rats, or dogs (1–2 hr). CHDI-340246 was orally bioavailable (>60%) in all species. In rats, [14C]CHDI-340246 showed wide distribution of radioactivity in all tissues except brain and testes. In rats, the parent drug was the major circulating moiety with minor amounts of a sulphate conjugate of an O-dealkylated metabolite. The elimination occurred via the urinary route and to a lesser extent by biliary route, but mostly as metabolites. In cynomolgus monkeys, the parent drug predominated in plasma with only trace amounts of metabolites detected. Acyl glucuronide conjugate of CHDI-340246 was not detected in plasma of rats or monkeys. Overall, the ADME profile of CHDI-340246 was favourable in rats and monkeys for potential evaluation of KMO inhibition in humans.

Assessment of the safety, pharmacokinetics and pharmacodynamics of GSK3335065, an inhibitor of kynurenine monooxygenase, in a randomised placebo-controlled first-in-human study in healthy volunteers.

GSK3335065 is an inhibitor of kynurenine monooxygenase (KMO) being developed for the treatment of acute pancreatitis. Healthy male volunteers were administered ascending doses of GSK3335065 or matched placebo as a single intravenous bolus injection to assess safety, tolerability, pharmacokinetics and pharmacodynamics. GSK3335065 displayed an apparent volume of distribution between 20.6L and 44.6L, a clearance between 0.462 L/h and 0.805 L/hr and a terminal half-life between 31.3 and 34.5hr. In the single subject who received 1.3mg GSK3335065, changes in tryptophan pathway metabolites were observed consistent with the changes seen in preclinical species suggesting that KMO enzyme activity was partially inhibited. However, a broad complex ventricular tachycardia was observed in this subject, which was judged to be a Serious Adverse Event (SAE) and resulted in early termination of the study. While development of GSK3335065 was subsequently discontinued, significant confounding factors hinder a clear interpretation that the tachycardia was directly related to administration of the compound.

Kynurenine Monooxygenase Expression and Activity in Human Astrocytomas.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The enzyme indoleamine-2,3-dioxygenase (IDO), which participates in the rate-limiting step of tryptophan catabolism through the kynurenine pathway (KP), is associated with poor prognosis in patients with GBM. The metabolites produced after tryptophan oxidation have immunomodulatory properties that can support the immunosuppressor environment. In this study, mRNA expression, protein expression, and activity of the enzyme kynurenine monooxygenase (KMO) were analyzed in GBM cell lines (A172, LN-18, U87, U373) and patient-derived astrocytoma samples. KMO mRNA expression was assessed by real-time RT-qPCR, KMO protein expression was evaluated by flow cytometry and immunofluorescence, and KMO activity was determined by quantifying 3-hydroxykynurenine by HPLC. Heterogenous patterns of both KMO expression and activity were observed among the GBM cell lines, with the A172 cell line showing the highest KMO expression and activity. Higher KMO mRNA expression was observed in glioma samples than in patients diagnosed with only a neurological disease; high KMO mRNA expression was also observed when using samples from patients with GBM in the TCGA program. The KMO protein expression was localized in GFAP+ cells in tumor tissue. These results suggest that KMO is a relevant target to be explored in glioma since it might play a role in supporting tumor metabolism and immune suppression.

CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say

Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

Protective Role of Kynurenine 3-Monooxygenase in Allograft Rejection and Tubular Injury in Kidney Transplantation.

Renal tubular epithelial cells (TECs) are the primary targets of ischemia–reperfusion injury (IRI) and rejection by the recipient’s immune response in kidney transplantation (KTx). However, the molecular mechanism of rejection and IRI remains to be identified. Our previous study demonstrated that kynurenine 3-monooxygenase (KMO) and kynureninase were reduced in ischemia–reperfusion procedure and further decreased in rejection allografts among mismatched pig KTx. Herein, we reveal that TEC injury in acutely rejection allografts is associated with alterations of Bcl2 family proteins, reduction of tight junction protein 1 (TJP1), and TEC-specific KMO. Three cytokines, IFNγ, TNFα, and IL1β, reported in our previous investigation were identified as triggers of TEC injury by altering the expression of Bcl2, BID, and TJP1. Allograft rejection and TEC injury were always associated with a dramatic reduction of KMO. 3HK and 3HAA, as direct and downstream products of KMO, effectively protected TEC from injury via increasing expression of Bcl-xL and TJP1. Both 3HK and 3HAA further prevented allograft rejection by inhibiting T cell proliferation and up-regulating aryl hydrocarbon receptor expression. Pig KTx with the administration of DNA nanoparticles (DNP) that induce expression of indoleamine 2,3-dioxygenase (IDO) and KMO to increase 3HK/3HAA showed an improvement of allograft rejection as well as murine skin transplant in IDO knockout mice with the injection of 3HK indicated a dramatic reduction of allograft rejection. Taken together, our data provide strong evidence that reduction of KMO in the graft is a key mediator of allograft rejection and loss. KMO can effectively improve allograft outcome by attenuating allograft rejection and maintaining graft barrier function.

Identification of Metabolic Enzymes ENO1 and KMO As Novel Immunotherapeutic Targets in Myeloma

Introduction. Our previous studies showed an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs are present in increased numbers in MM BM, promote tumor growth and survival, protect tumor from therapy-induced cytotoxicity; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323) . Aberrant pDC function is evidenced from their interactions with MM tumor and T/NK effector cells in the BM milieu (Ray et al, Leukemia 2015, 29:1441-1444) . Direct interruption of pDC-MM and pDC-T cells interactions and their sequelae utilizing novel targeted agents is necessary to further enhance both anti-tumor immunity and cytotoxicity. Based on DNA microarray analysis, we identified several novel targets on MM cells that are upregulated after coculture with pDCs, and focused on metabolic pathway enzymes that are upregulated both in pDC and MM cells. We identified alpha-Enolase (ENO1) and Kynurenine 3-monooxygenase (KMO) as the two most promising therapeutic targets, based on their roles in normal and cancer metabolism. ENO1 is a glycolytic enzyme, and its surface localization is a unique feature of the tumor cells. Moreover, ENO1 promotes tumor metastasis. KMO is one of the two rate-limiting enzymes involved in eukaryotic tryptophan catabolic kynurenine pathway; and importantly, its overexpression has been reported to inhibit functional immune responses in cancer. Here, we utilized our pDC/MM or pDC/T cells co-culture model to show that targeting ENO1 or KMO generates MM-specific cytotoxic T lymphocyte activity.MethodsDNA MicroArray Analysis: MM.1S cells were cocultured with pDCs for 72h, separated using CD138 Microbeads, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM.1S cells cultured in the presence vs absence of pDCs were compared, and heat map was generated (>1.5-fold change in transcript was considered significant, CI > 95%). pDC-induced overexpression of target proteins : MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in target protein expression. CD138+ tumor cells were gated out, and KMO-FITC or ENO1-AlexaFluor488-stained cells were analyzed. C TL activity assays : MM patient BM CD8+ T cells were cocultured with autologous pDCs at 1:10 (pDC:T) ratio in the presence or absence of KMO inhibitor Ro 61-8048 (10 nM) or ENO1 inhibitor ENOblock (0.1-0.2 µM) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T:MM), followed by quantification of viable MM cells by FACS. Statistical parameters were calculated using GraphPad Prism. Pharmacological inhibitors were obtained from Selleck Chemicals.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of both ENO1 and KMO transcripts in MM cells (1.772 and 2.153-fold, respectively, vs MM.1S alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high KMO and surface ENO1 expression. Coculture of MM cells with pDCs further increased ENO1 surface expression levels in MM cells (5-6-fold increase; n=3; p = 0.003), as well as the ENO1-positive MM cell population (3-4-fold versus MM alone; mean ± SD; n=3; p = 0.005). 3) Like ENO1, pDCs also increased both KMO expression on MM cells and KMO-positive MM cells (MFI: 2-3-fold in the presence vs absence of pDCs; p = 0.006). 4) Functional studies showed that treatment of pDC-T cell cocultures with ENO1 antagonists triggers a robust MM-specific CD8+ CTL activity against autologous CD138-positive MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2-fold vs control; p = 0.008). Finally, 6) KMO inhibitor Ro 61-8048 activates patient BM-CD8+T cells in autologous pDCs/T cell cocultures, and triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0-fold vs control; p = 0.009).Conclusions Our study shows that pDC-MM interactions upregulate tumor promoting and immunosuppressive metabolic enzymes in MM. Blockade of ENO1 or KMO enzymes tiggers MM-specific CD8-positive CTL activity. These data therefore identify novel immune-based therapeutic strategies targeting ENO1 and KMO to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

3-Hydroxykynurenine Regulates Lipopolysaccharide-Stimulated IL-6 Production and Protects against Endotoxic Shock in Mice.

Despite advances in our understanding of endotoxic shock, novel therapeutic interventions that can reduce the burden of sepsis remain elusive. Current treatment options are limited, and it is only through refinements in the ways that we deliver supportive care that mortality has fallen over the years. In this study, the role of kynurenine 3-monooxygenase (KMO) in immune regulation was examined in LPS-induced endotoxemia using KMO-/- and KMO+/+ mice treated with the KMO inhibitor Ro61-8048. We showed that LPS-induced or cecal ligation and puncture-induced mortality and hepatic IL-6 production increased in the absence of KMO, possibly involving increased activating transcription factor 4 (ATF4) signaling in hepatic macrophages. Moreover, treatment of septic mice with 3-hydroxykynurenine reduced mortality rates and inflammatory responses regardless of the presence or absence of KMO. According to our results, the administration of 3-hydroxykynurenine as part of the treatment approach for sepsis or as an adjuvant therapy might reduce the overproduction of IL-6, which is responsible for severe endotoxemia, and ultimately improve the survival rates of patients with sepsis.

Novel promising serum biomarkers for canine mammary tumors

Canine mammary tumors (CMT) are the second most common form of neoplasia accounting for 40% of tumors in dogs. Today, as we know cancer is a disease of multiple genes and molecules. Research across the last three decades has enhanced our understanding of these molecules which is making possible new strategies of predicting, diagnosing and clinical treatment of this malady. Biomarkers are the modern day “neoplastic signatures” which enhances the specificity of tumor diagnosis allowing the researchers to differentiate tumors at the molecular level. This review enlists a concise summary of novel serum biomarkers counting matrix metalloproteinases, lysyl oxidase, heat shock proteins, mammaglobin, kynurenine 3-monooxygenase, WT1, nectin 4, and ERBB2. These new-fangled markers of canine malignancy could might help in early and accurate diagnosis resulting in more effective treatment and better prognosis. Nevertheless, more detailed research of these biomarkers along with their role in other cancers should be further validated in future.

Downregulation of Kynurenine 3-Monooxygenase Inhibits cancer stemness-related genes in both human and canine breast tumor cell lines

Downregulation of Kynurenine 3-Monooxygenase Inhibits cancer stemness-related genes in both human and canine breast tumor cell lines

Pharmacophore-Based Virtual Screening of Novel Competitive Inhibitors of the Neurodegenerative Disease Target Kynurenine-3-Monooxygenase.

The pathogenesis of several neurodegenerative diseases such as Alzheimer’s or Huntington’s disease has been associated with metabolic dysfunctions caused by imbalances in the brain and cerebral spinal fluid levels of neuroactive metabolites. Kynurenine monooxygenase (KMO) is considered an ideal therapeutic target for the regulation of neuroactive tryptophan metabolites. Despite significant efforts, the known KMO inhibitors lack blood–brain barrier (BBB) permeability and upon the mimicking of the substrate binding mode, are subject to produce reactive oxygen species as a side reaction. The computational drug design is further complicated by the absence of complete crystal structure information for human KMO (hKMO). In the current work, we performed virtual screening of readily available compounds using several protein–ligand complex pharmacophores. Each of the pharmacophores accounts for one of three distinct reported KMO protein-inhibitor binding conformations. As a result, six novel KMO inhibitors were discovered based on an in vitro fluorescence assay. Compounds VS1 and VS6 were predicted to be BBB permeable and avoid the hydrogen peroxide production dilemma, making them valuable, novel hit compounds for further drug property optimization and advancement in the drug design pipeline.

Discovery of N-(6-(5-fluoro-2-(piperidin-1-yl)phenyl)pyridazin-3-yl)-1-(tetrahydro-2H-pyran-4-yl)methanesulfonamide as a brain-permeable and metabolically stable kynurenine monooxygenase inhibitor

Kynurenine monooxygenase (KMO) is expected to be a good drug target to treat Huntington’s disease (HD). This study presents the structure-activity relationship of pyridazine derivatives to find novel KMO inhibitors. The most promising compound 14 resolved the problematic issues of lead compound 1, i.e., metabolic instability and reactive metabolite-derived side-effects. Compound 14 exhibited high brain permeability and a long-lasting pharmacokinetics profile in monkeys, and neuroprotective kynurenic acid was increased by a single administration of 14 in R6/2 mouse brain. These results demonstrated 14 may be a potential drug candidate to treat HD.

Paliperidone Reversion of Maternal Immune Activation-Induced Changes on Brain Serotonin and Kynurenine Pathways.

Emerging evidence indicates that early-life exposure to environmental factors may increase the risk for schizophrenia via inflammatory mechanisms. Inflammation can alter the metabolism of tryptophan through the oxidative kynurenine pathway to compounds with neurotoxic and neuroprotective activity and compromise serotonin (5-HT) synthesis. Here we investigate the role of serotonergic and kynurenine pathways in the maternal immune activation (MIA) animal model of schizophrenia. The potential reversion exerted by long-term antipsychotic treatment was also evaluated. MIA was induced by prenatal administration of polyinosinic:polycytidylic acid (poly (I:C)) in mice. Expression of different proteins and the content of different metabolites involved in the function of serotonergic and kynurenine pathways was assessed by RT-PCR, immunoblot and ELISA analyses in frontal cortex of the offspring after puberty. MIA decreased tissue 5-HT content and promoted changes in the expression of serotonin transporter, 5-HT2A and 5-HT2C receptors. Expression of indoleamine 2,3-dioxygenase 2 (IDO2) and kynurenine 3-monooxygenase (KMO) was increased by poly (I:C) whereas kynurenine aminotransferase II and its metabolite kynurenic acid were not altered. Long-term paliperidone was able to counteract MIA-induced changes in 5-HT and KMO, and to increase tryptophan availability and tryptophan hydroxylase-2 expression in poly (I:C) mice but not in controls. MIA-induced increase of the cytotoxic risk ratio of kynurenine metabolites (quinolinic/kynurenic acid) was also reversed by paliperidone. MIA induces specific long-term brain effects on serotonergic activity. Such effects seem to be related with alternative activation of the kynurenine metabolic pathway towards a cytotoxic status. Atypical antipsychotic paliperodine partially remediates abnormalities observed after MIA.

Kynurenine Metabolism in the D2 mdx Mouse: A Muscle‐to‐Brain Connection

Introduction The kynurenine (KYN) pathway has been implicated in depression and neurotoxicity. Derived from tryptophan, KYN can be further degraded along one of two distinct branches. The KYN-KYNA branch is regulated by the enzyme kynurenine aminotransferase (KAT) and is considered neuroprotective, as it degrades KYN into the non-blood brain barrier (BBB) transportable metabolite kynurenic acid (KYNA). The KYN-NAD branch is regulated by the enzyme kynurenine monooxygenase (KMO) and is considered neurotoxic as it degrades KYN into the BBB transportable metabolite 3-hydroxykynurenine (3-HK) and, further in the cascade, quinolinic acid (QUIN). Recent studies have shown the importance of muscle health on directing kynurenine metabolism towards the neuroprotective branch, highlighting a novel muscle-to-brain axis. Specifically, exercise induced increases in the transcription factor PCG-1⍺ amplifies the content of KAT enzymes that convert KYNA from KYN. Duchenne muscular dystrophy (DMD) is an X-linked severe muscle disorder caused by a loss of dystrophin leading to muscle fragility, wasting, and weakness. In light of recent evidence revealing the cognitive and depressive behaviours in DMD patients and in the preclinical mdx mouse, we sought to determine whether KYN metabolism as well as PGC-1α and KAT content would be altered in the mdx model. Methods 8-10 week old male mdx and wild-type (DBA/2J) mice were purchased from Jackson laboratories. Behavioural changes (ie., grooming activity, food and water intake) were measured using a Promethion metabolic cage system along with fear and anxiety-like behaviour during a novel object recognition test (NORT). Mice were euthanized and serum KYN and KYNA were measured using commercially available ELISA kits. Extensor digitorum longus muscle was collected, homogenized, and Western blotting was performed for PGC-1⍺, KAT1, and KAT3. Results Metabolic cage results showed that fine activity (-5%), water intake (-25%), and food intake (-60%) were lower across light and dark stages in mdx mice compared to WT mice (main effect of genotype, p<0.0001 for all measures). The mdx mice also spent more time in the corners of the NORT arenas compared to their WT counterparts (+270s, p<0.0001). Though the change in serum KYN was insignificant across mdx and WT mice, the concentration of serum KYNA was lower in the mdx mice (-57%, p = 0.01), therefore causing a lower KYN:KYNA ratio in mdx mice compared with WT (-56%, p = 0.01). Western blotting demonstrated a reduction in PGC-1⍺ (-65%, p = 0.002) and KAT1 (-35%, p = 0.02) content in mdx mice compared to WT mice, whereas the KAT3 content was elevated in mdx mice (1.5-fold, p = 0.05). Conclusion Our results of lowered serum KYN:KYNA concentrations from mdx mice (compared to WT) correspond well with changes in affective and anxiety-related behaviours. The observed reduction in muscle PGC-1⍺ and KAT1 content likely contributes to these changes in KYN:KYNA ratio. Though KAT3 was upregulated in mdx muscle compared to WT, this could represent a failed compensatory response.

Significance of Kynurenine 3-Monooxygenase Expression in Colorectal Cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths. Because of the lack of reliable prognostic and predictive biomarkers for CRC, most patients are often diagnosed at a late stage. The tryptophan–kynurenine pathway plays a crucial role in promoting cancer progression. Kynurenine is considered an oncometabolite in colon cancer, and its downstream metabolites are also associated with CRC. Kynurenine 3-monooxygenase (KMO), a pivotal enzyme that catalyzes kynurenine metabolism, is essential for several cellular processes. In the current study, we explored the role of KMO in CRC. Immunohistochemical results showed that KMO was upregulated in CRC tissues relative to paired healthy tissue and polyps. Moreover, CRC patients with higher KMO expression were associated with higher metastasis and poorer survival rates. Knockdown of KMO decreased the expression of cancer stem cell markers, as well as the sphere-forming, migration, and invasion abilities of CRC cells. Additionally, blockade of the enzymatic activity of KMO using an inhibitor suppressed sphere formation and cell motility in CRC cells. These findings suggest the clinical relevance of KMO in CRC tumorigenesis and aggressiveness.

Ablation of kynurenine 3-monooxygenase rescues plasma inflammatory cytokine levels in the R6/2 mouse model of Huntington\u2019s disease

Kynurenine 3-monooxygenase (KMO) regulates the levels of neuroactive metabolites in the kynurenine pathway (KP), dysregulation of which is associated with Huntington’s disease (HD) pathogenesis. KMO inhibition leads to increased levels of neuroprotective relative to neurotoxic metabolites, and has been found to ameliorate disease-relevant phenotypes in several HD models. Here, we crossed KMO knockout mice to R6/2 HD mice to examine the effect of KMO depletion in the brain and periphery. KP genes were dysregulated in peripheral tissues from R6/2 mice and KMO ablation normalised levels of a subset of these. KP metabolites were also assessed, and KMO depletion led to increased levels of neuroprotective kynurenic acid in brain and periphery, and dramatically reduced neurotoxic 3-hydroxykunurenine levels in striatum and cortex. Notably, the increased levels of pro-inflammatory cytokines TNFa, IL1β, IL4 and IL6 found in R6/2 plasma were normalised upon KMO deletion. Despite these improvements in KP dysregulation and peripheral inflammation, KMO ablation had no effect upon several behavioural phenotypes. Therefore, although genetic inhibition of KMO in R6/2 mice modulates several metabolic and inflammatory parameters, these do not translate to improvements in primary disease indicators—observations which will likely be relevant for other interventions targeted at peripheral inflammation in HD.

Kynurenine 3-monooxygenase deficiency induces depression-like behavior via enhanced antagonism of α7 nicotinic acetylcholine receptors by kynurenic acid

Tryptophan (TRP) is metabolized via the kynurenine (KYN) pathway, which is related to the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the metabolism of KYN to 3-hydroxykynurenine. In rodents, KMO deficiency induces a depression-like behavior and increases the levels of kynurenic acid (KA), a KYN metabolite formed by kynurenine aminotransferases (KATs). KA antagonizes α7 nicotinic acetylcholine receptor (α7nAChR). Here, we investigated the involvement of KA in depression-like behavior in KMO knockout (KO) mice. KYN, KA, and anthranilic acid but not TRP or 3-hydroxyanthranilic acid were elevated in the prefrontal cortex of KMO KO mice. The mRNA levels of KAT1 and α7nAChR but not KAT2−4, α4nAChR, or β2nAChR were elevated in the prefrontal cortex of KMO KO mice. Nicotine blocked increase in locomotor activity, decrease in social interaction time, and prolonged immobility in a forced swimming test, but it did not decrease sucrose preference in the KMO KO mice. Methyllycaconitine (an α7nAChR antagonist) antagonized the effect of nicotine on decreased social interaction time and prolonged immobility in the forced swimming test, but not increased locomotor activity. Galantamine (an α7nAChR allosteric agonist) blocked the increased locomotor activity and prolonged immobility in the forced swimming test, but not the decreased social interaction time in the KMO KO mice. In conclusion, elevation of KA levels contributes to depression-like behaviors in KMO KO mice by α7nAChR antagonism. The ameliorating effects of nicotine and galantamine on depression-like behaviors in KMO KO mice are associated with the activation of α7nAChR.

Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition

The structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.

Expanding the Toolkit for Genome Editing in a Disease Vector, Aedes aegypti: Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and the juvenile hormone receptor, Methoprene-tolerant (Met). PiggyBac transformation vectors were prepared to express sgRNAs targeting kmo and Met under the control of the U6 promoter. Transgenic Ae. aegypti expressing kmo-sgRNA or Met-sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in kmo-sgRNA and Met-sgRNA groups, respectively, showed mutations in their target genes.