Kynurenine 3-monooxygenase Inhibitor Research Articles

Mechanism of Treadmill Exercise Combined with Rich Environmental Stimulation to Improve Depression in Post-stroke Depression Model Rats.

Post-stroke depression (PSD) is a common complication, occurring in approximately one-third of these patients. The neurological symptoms of PSD affect patients' daily life and subsequent recovery. Analyzing the pathogenesis of post-stroke depression from a psychological perspective, it was found that PSD patients often feel despair and anxiety, and it is crucial to explore non-pharmacological ways to improve post-stroke depressive symptoms. A combination of exercise and rich environmental stimulation (RES) has been found effective in improving post-stroke depressive symptoms. Therefore, this study aimed to explore the effects of exercise and rich environmental stimulation on PSD in rats and their potential underlying mechanisms and to provide a theoretical basis for managing PSD. The PSD rat model was constructed, and the depression-like behaviors of rats in each group were evaluated using the open field test (OFT), sucrose preference test (SPT), and forced swimming test (FST). Moreover, changes in the morphological behavior of rat hippocampus were observed using hematoxylin-eosin (HE) staining and Nissl staining. The expression levels of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in hippocampus tissues were assessed using enzyme linked immunosorbent assay (ELISA), and the levels of tryptophan-related proteins were determined employing western blot analysis. Additionally, a kynurenine-3-monooxygenase (KMO) inhibitor was administered to the combined stimulation group, and the levels of tryptophan (TRP), 5-HT, kynurenine (KYN), 3-hydroxy-kynurenine (3-HK), and quinolinic acid (QA) were evaluated using liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). Treadmill exercise combined with rich environmental stimulation significantly reduced the immobility time in the FST (p < 0.01), increased the exploratory behavior in the OFT (p < 0.05), and increased the sucrose water consumption in the SPT (p < 0.01), indicating that the depression-like behavior was improved. Treadmill exercise combined with rich environmental stimulation also improved the shape of the damaged hippocampus and increased the number of neurons in the hippocampus. Additionally, treadmill exercise combined with rich environmental stimulation significantly increased the levels of 5-HT and NE in hippocampus tissues (p < 0.01) and decreased KMO protein level (p < 0.01). In the KMO inhibitor group, the neural function was efficiently restored, the levels of 3-HK, QA, and KMO in the hippocampus were substantially reduced (p < 0.01), and the expression level of 5-HT was increased (p < 0.01). Exercise stimulation combined with enriched environmental stimuli alleviates post-stroke depression in rats, and the underlying mechanisms may be related to TRP/KYN/3-HK/QA excitotoxicity pathways and increased 5-hydroxytryptamine levels.

Identification of potent inhibitors of kynurenine-3-monooxygenase from natural products: In silico and in vitro approaches

Existing inhibitors of kynurenine-3-monooxygenase (KMO) have side effects and poorly cross the blood-brain barrier. Therefore, the discovery of new molecules targeting KMO isnecessary.This study aims to develop a novel therapeutic drug targeting KMO using computational methods and experimental validation of natural compounds.The results of our study show that the top four compounds, namely, 3′-Hydroxy-alpha-naphthoflavone exhibited the best docking scores with KMO (−10.0 kcal/mol), followed by 3′-Hydroxy-ss-naphthoflavone (−9.9 kcal/mol), genkwanin (−9.2 kcal/mol) and apigenin(-9.1 kcal/mol) respectively. Molecular dynamics was used to assess the stability of the primary target, KMO, and inhibitor complexes. We found stable interactions of 3′-Hydroxy-ss-naphthoflavone and apigenin with KMO up to 100 ns. Further, kinetic measurements showed that 3′-Hydroxy-alpha-naphthoflavone and 3′-Hydroxy-ss-naphthoflavone induce competitive inhibition with a good IC50 activity (15.85 ± 0.98 μM and 18.71 ± 0.78, respectively), while Genkwanin and Apigenin exhibit non-competitive inhibition mechanism (21.61 ± 0.97 μM and 24.14 ± 1.00 μM, respectively).Drug-likeness features and ADME analysis features also showed that the top four compounds could be used as potential candidates to replace the synthetic KMO inhibitor drugs with known side effects and poor brain-blood barrier penetration.

Abstract TMP113: Kynurenine-3-monooxygenase Inhibition Confers Neuroprotection After Ischemic Stroke In Aged Mice

Background: The kynurenine pathway (KP) is the major route of tryptophan catabolism in mammals. KP branches into two major pathways - one (dependent on the kynurenine aminotransferases) implicated in neuroprotection, the other (dependent on the enzyme kynurenine-3-monooxygenase (KMO) in neurotoxicity. Under physiological conditions, the neuroprotective branch is more active, but under inflammatory conditions, such as seen in stroke, metabolism is shifted through the KP via KMO to produce toxic metabolites, including quinolinic acid. Recent studies have demonstrated that stroke leads to the activation of the KP. Here we tested whether inhibiting KMO activity after stroke could prevent and/or minimize the neuronal death and improve outcomes. Methods: WT C57Bl6 aged male mice were subjected to a 60-minute reversible middle cerebral artery occlusion (MCAO). Tissues collected from a sub-cohort of these mice was used for metabolomics. Other sub-cohort of mice was used for pharmacological studies. For this, mice received either an inhibitor of KMO (Ro 61-8048, 40mg/kg/day i.p) or vehicle immediately after stroke, and infarct was evaluated at day 3 post-stroke. Neurological deficit scores and open field analysis was performed to assess recovery. A separate cohort of aged WT and KMO knockout (KMO-/-) mice was also subjected to MCAO and assessed for infarct and recovery at 3 days after stroke to further evaluate the involvement of KMO on outcome. Results: Stroke caused significant reduction in the aged brain kynurenic acid/quinolinic acid (KYNA/QUIN) ratio, suggesting activation of the neurotoxic/KMO branch of the KP pathway. KMOi treatment administered immediately after the stroke reduced ischemic infarct size (p&lt;.05; n=8 mice/grp) and also improved spontaneous locomotor activity by 72h after stroke compared to vehicle treatment. We also found that genetic deletion of KMO reduces ischemic injury size (p&lt;.05; n=6-7mice/grp) and improves neurological deficit scores. Conclusions: Inhibition or deletion of KMO reduces neuronal death and improves motor function after stroke in aged male mice. Further studies are needed to ascertain if beneficial effects of KMOi on post-stroke recovery is not due to smaller injury, using aged animals of both sexes.

Gram-Scale Preparation of Cannflavin A from Hemp (Cannabis sativa L.) and Its Inhibitory Effect on Tryptophan Catabolism Enzyme Kynurenine-3-Monooxygenase.

Simple SummaryKynurenine-3-monooxygenase (KMO) is an enzyme in the neurotoxic branch of the kynurenine pathway (KP) and is a target of inhibitors with therapeutic potential against neuroinflammatory and neurodegenerative diseases. Phytochemicals from hemp (Cannabis sativa L.) including phytocannabinoids and flavonoids, can modulate enzymes involved in the KP metabolism, but their direct inhibition on KMO is unknown. In the current study, we purified a unique C. sativa flavonoid, namely, cannflavin A (CFA), from the hemp aerial material at a gram-scale. We evaluated the anti-KMO activity of CFA and a panel of phytocannabinoids. CFA was the most active compound with an IC50 of 29.4 μM, which was comparable to the positive control Ro 61-8048 (IC50 = 5.1 μM). We used a modeling method to demonstrate the interactions between CFA and the KMO protein and measured the binding affinity by a biophysical tool (surface plasmon resonance; SPR). We also conducted a competitive SPR assay to show that CFA and Ro 61-8048 may bind to the same location of the KMO protein. Our findings help the understanding of C. sativa compounds’ effects on KMO.Inhibitors targeting kynurenine-3-monooxygenase (KMO), an enzyme in the neurotoxic kynurenine pathway (KP), are potential therapeutics for KP metabolites-mediated neuroinflammatory and neurodegenerative disorders. Although phytochemicals from Cannabis (C. sativa L.) have been reported to show modulating effects on enzymes involved in the KP metabolism, the inhibitory effects of C. sativa compounds, including phytocannabinoids and non-phytocannabinoids (i.e., cannflavin A; CFA), on KMO remain unknown. Herein, CFA (purified from hemp aerial material at a gram-scale) and a series of phytocannabinoids were evaluated for their anti-KMO activity. CFA showed the most active inhibitory effect on KMO, which was comparable to the positive control Ro 61-8048 (IC50 = 29.4 vs. 5.1 μM, respectively). Furthermore, a molecular docking study depicted the molecular interactions between CFA and the KMO protein and a biophysical binding assay with surface plasmon resonance (SPR) technique revealed that CFA bound to the protein with a binding affinity of . A competitive SPR binding analysis suggested that CFA and Ro 61-8048 bind to the KMO protein in a competitive manner. Our findings show that C. sativa derived phytochemicals, including CFA, are potential KMO inhibitors, which provides insight into the development of therapeutics targeting the KP and its related pathological conditions.

Kynurenine-3-monooxygenase (KMO): From its biological functions to therapeutic effect in diseases progression.

Kynurenine-3-monooxygenase (KMO) is a mitochondrial enzyme involved in the eukaryotic kynurenine pathway (KP), which is the major catabolic route of tryptophan. KMO can convert the substrate kynurenine into the neurotoxin3-hydroxykynurenine and quinolinic acid, which promote the production of toxic metabolites and formation of free radical in the blood, while decrease the neuroprotective metabolite kynurenic acid. As a result of branch point, KMO is predicted as an attractive drug target for several diseases, especially neurodegenerative diseases, psychosis, and cancer. This review mainly pays attention to KMO structure and the research of mechanisms and functions, with a particular emphasis on the roles of KMO in the pathogenesis of various conditions. Furthermore, we also summarized important KMO inhibitors to supporting their effects on these diseases, indicating the prospect to find novel KMO inhibitors for diseases therapy.

Blast-related traumatic brain injury is mediated by the kynurenine pathway.

The overactivation of the kynurenine pathway, the major metabolic pathway of tryptophan, induced by inflammation and oxidative stress, might bring about excessive neurotoxic metabolites. This study aimed to investigate whether kynurenine pathway is overactivated in blast-related traumatic brain injury (bTBI) and whether inhibitors of kynureninase and kynurenine-3-monooxygenase (KMO), important enzymes in kynurenine pathway, could alleviate bTBI in rats. A shock tube was used to establish the bTBI animal models. Pathological changes in the hippocampus were observed using Nissl, propidium iodide and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. Immunohistochemistry was used to evaluate the expression levels of kynureninase and KMO. After the establishment of bTBI rat models, they were treated with KMO inhibitor (Ro 61-8048) and kynureninase inhibitor (benserazide hydrochloride), and the animals' behavioral performance was assessed using an elevated plus maze (EPM). After blast exposure, the number of neurons decreased, whereas the expression of kynureninase and KMO increased in the CA1 area of the rat hippocampus. In vitro, KMO inhibitor (Ro) and kynureninase inhibitor (benserazide hydrochloride) intervention could reduce the proportion of TUNEL-positive neurons in the hippocampus. In vivo, after Ro treatment, the behavior of the bTBI rats was significantly improved, and more neurons survived in the hippocampus CA1 region; however, following benserazide hydrochloride treatment, the behavior of bTBI rats was not significantly improved, and neuron survival could not be improved in the hippocampal CA1 region. The expression levels of KMO and kynureninase were increased in the hippocampus of the bTBI rats, suggesting that these factors might mediate the bTBI damage. Furthermore, the KMO inhibitor showed a significant protective effect on bTBI.

The Kynurenine Pathway and Kynurenine 3-Monooxygenase Inhibitors.

Under normal physiological conditions, the kynurenine pathway (KP) plays a critical role in generating cellular energy and catabolizing tryptophan. Under inflammatory conditions, however, there is an upregulation of the KP enzymes, particularly kynurenine 3-monooxygenase (KMO). KMO has garnered much attention due to its production of toxic metabolites that have been implicated in many diseases and disorders. With many of these illnesses having an inadequate or modest treatment, there exists a need to develop KMO inhibitors that reduce the production of these toxic metabolites. Though prior efforts to find an appropriate KMO inhibitor were unpromising, the development of a KMO crystal structure has provided the opportunity for a rational structure-based design in the development of inhibitors. Therefore, the purpose of this review is to describe the kynurenine pathway, the kynurenine 3-monooxygenase enzyme, and KMO inhibitors and their potential candidacy for clinical use.

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.

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.

In silico methods predict new blood-brain barrier permeable structure for the inhibition of kynurenine 3-monooxygenase

Kynurenine 3-monooxygenase (KMO) regulates the levels of bioactive substances in the kynurenine pathway of tryptophan catabolism and its activity is tied to so many diseases that finding an appropriate inhibitor for KMO has become an urgent task. This especially proved to be difficult for the central nervous system related diseases due to the requirement that the supposed inhibitor should be both blood brain barrier permeable and should not cause hydrogen peroxide as a harmful side product. In this in silico study, we present our step-wise approach, whose starting point is based on the important experimental observations. To tackle the problem, a library of 7561938 structures was obtained from Zinc15 database utilizing the tranche browser. From this library, a subset of 501777 structures was determined with the considerations of their functional groups that constrain their applicability. Then, the binding affinity ranking of this set of structures was determined via virtual screening. Starting from the structures whose affinities are the highest among this subset, the ADMET properties were checked through in silico methods and the binding properties of the selected inhibitor candidates were further investigated via molecular dynamics simulations and MM/GBSA calculations. According to the computational results of this study, ZINC_71915355 has passed all the evaluations and is a potentially BBB permeable structure that can inhibit KMO. Additionally, ZINC_19827377 was identified as a new potential KMO inhibitor which may be more suitable for peripheral administration. From the in silico study presented herein, ZINC_71915355 and ZINC_19827377 structures, which showed high binding affinity without harmful H2O2 production, along with the tailored properties can now serve as powerful candidates for KMO inhibition and these hits are worth of further experimental validation.

Kynurenine 3-Monooxygenase Inhibition during Acute Simian Immunodeficiency Virus Infection Lowers PD-1 Expression and Improves Post-Combination Antiretroviral Therapy CD4+ T Cell Counts and Body Weight.

The kynurenine pathway (KP) is a key regulator of many important physiological processes and plays a harmful role in cancer, many neurologic conditions, and chronic viral infections. In HIV infection, KP activity is consistently associated with reduced CD4 T cell counts and elevated levels of T cell activation and viral load; it also independently predicts mortality and morbidity from non-AIDS events. Kynurenine 3-monooxygenase (KMO) is a therapeutically important target in the KP. Using the nonhuman primate model of SIV infection in rhesus macaques, we investigated whether KMO inhibition could slow the course of disease progression. We used a KMO inhibitor, CHDI-340246, to perturb the KP during early acute infection and followed the animals for 1 y to assess clinical outcomes and immune phenotype and function during pre-combination antiretroviral therapy acute infection and combination antiretroviral therapy-treated chronic infection. Inhibition of KMO in acute SIV infection disrupted the KP and prevented SIV-induced increases in downstream metabolites, improving clinical outcome as measured by both increased CD4+ T cell counts and body weight. KMO inhibition increased naive T cell frequency and lowered PD-1 expression in naive and memory T cell subsets. Importantly, early PD-1 expression during acute SIV infection predicted clinical outcomes of body weight and CD4+ T cell counts. Our data indicate that KMO inhibition in early acute SIV infection provides clinical benefit and suggest a rationale for testing KMO inhibition as an adjunctive treatment in SIV/HIV infection to slow the progression of the disease and improve immune reconstitution.

A brain-permeable inhibitor of the neurodegenerative disease target kynurenine 3-monooxygenase prevents accumulation of neurotoxic metabolites

Dysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington’s disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable. Here, 19 new KMO inhibitors have been identified. One of these (1) is neuroprotective in a Drosophila HD model but is minimally brain penetrant in mice. The prodrug variant (1b) crosses the blood–brain barrier, releases 1 in the brain, thereby lowering levels of 3-hydroxykynurenine, a toxic KP metabolite linked to neurodegeneration. Prodrug 1b will advance development of targeted therapies against multiple neurodegenerative and neuroinflammatory diseases in which KP likely plays a role, including HD, Alzheimer’s disease, and Parkinson’s disease.

Abstract 1711: Kynurenine 3-monooxygenase (KMO) acts as a novel oncoprotein in triple negative breast cancer

Abstract Purpose: Tryptophan-kynurenine pathway involves in inflammation, immune response and tumorigenesis, in which kynurenine 3-monooxygenase (KMO), an outer mitochondrial membrane protein, mediating kynurenine metabolism. Previous studies indicated KMO showed increased activity in breast cancer. Triple-negative breast cancer (TNBC) tumors exhibited elevated levels of tryptophan metabolites compared to estrogen receptor positive breast cancers. We aimed to study the role of KMO in human TNBC. Experimental design: The gene alterations and transcripts of enzymes in kynurenine metabolism were analyzed from The Cancer Genome Atlas (TCGA) database. Immunohistochemical staining for KMO was performed and a H-score was assigned to quantify protein expression. Epithelial-mesenchymal transition (EMT) phenotypes were examined by transwell assay and EMT markers expressions. Stemness properties were assessed by mammosphere assay and pluripotent genes expressions. The molecular events were analyzed by Western blot, quantitative real-time PCR and luciferase reporter assay. Tumor growth and metastasis were conducted in nude mice and NOD-SCID mice by subcutaneous and tail vein injection respectively. Results: TCGA databases showed KMO but not KYNU and KAT2 was amplified in breast cancer. Both the data from TCGA and our in-house IHC-based tissue-microarray exhibited increased KMO expression in TNBC compared to normal tissue. In vitro, overexpression of KMO in TNBC cells resulted in increased cell growth and colony formation. The abilities migration and invasion as well as EMT markers expressions of TNBC cells were elevated by KMO overexpression. In addition, KMO increased mammosphere formation, pluripotent genes expressions and promoter activities. However, inhibition of KMO enzymatic activity by KMO inhibitors did not affect cancer progression or mitochondrial respiration of TNBC cells. KMO upregulated β-catenin, the upstream regulator of pluripotent genes, CD44 and Nanog expressions. Mechanistically, data showed KMO expressed in both cytosol and nuclear fractions and was associated with β-catenin. KMO enhanced pluripotent genes expressions through β-catenin upregulation. Importantly, KMO knockdown suppressed tumor growth and expressions of β-catenin, CD44 and Nanog in TNBC tumors. Moreover, KMO knockout significantly decreased lung metastasis in vivo. Conclusion: Our data indicated KMO can play an oncogenic role in TNBC, acting as a novel regulator of pluripotent genes via β-catenin and promoted TNBC progression. Citation Format: Chun-Yu Liu, Tzu-Ting Huang, Ji-Lin Chen, Chia-Han Lee, Wan-Lun Wang, Ka-Yi Lau, Chun-Teng Huang, Pei-Yi Chu, Hsin-Chen Lee, Ling-Ming Tseng. Kynurenine 3-monooxygenase (KMO) acts as a novel oncoprotein in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1711.

Modification of kynurenine pathway via inhibition of kynurenine hydroxylase attenuates surgical brain injury complications in a male rat model.

Neurosurgical procedures result in surgically induced brain injury (SBI) that causes postoperative complications including brain edema and neuronal apoptosis in the surrounding brain tissue. SBI leads to the release of cytokines that indirectly cause the stimulation of kynurenine 3-monooxygenase (KMO) and the release of neurotoxic quinolinic acid (QUIN). This study tested a KMO inhibitor, RO 61-8048, to prevent postoperative brain edema and consequent neuronal apoptosis in an in vivo model of SBI. A rodent model of SBI was utilized which involves partial resection of the right frontal lobe. A total of 127 Sprague-Dawley male rats (weight 275-325g) were randomly divided into the following groups: Sham surgical group, SBI, SBI+DMSO, SBI+RO 61-8048 (10mg/kg), SBI+RO 61-8048 (40mg/kg), and SBI+RO 61-8048 (40mg/kg)+KAT II inhibitor PF-04859989 (5mg/kg). RO 61-8048 was administered by intraperitoneal injection after SBI. Postoperative assessment at different time points included brain water content (brain edema), neurological scoring, and western blot. SBI increased brain water content (ipsilateral frontal lobe), decreased neurological function, and increased apoptotic markers compared with sham animals. Treatment with RO 61-8048 (40mg/kg) reduced brain water content and improved long-term neurological function after SBI. RO 61-8048 increased the expression of kynurenic acid while reducing QUIN and apoptotic markers in the surrounding brain tissue after SBI. These neuroprotective effects were reversed by PF-04859989. This study suggests KMO inhibition via RO 61-8048 as a potential postoperative therapy following neurosurgical procedures.

Targeting Tryptophan Catabolic Kynurenine Pathway Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction. Plasmacytoid dendritic cells (pDCs) play an integral role in MM pathogenesis (Chauhan et al, Cancer Cell 2009, 16:309-323). pDCs interactions with tumor cells and T/NK effector cells in the MM-BM milieu induce immune suppression and MM cell proliferation (Chauhan et al, Cancer Cell 2009, 16:309-323;Ray et al, Leukemia 2015, 29:1441-1444). A direct interruption of pDC-MM and pDC-T cells interactions with novel agents will enhance cytotoxicity and anti-tumor immunity. In this context, our DNA microarray analysis identified upregulation of tryptophan catabolic kynurenine (Kyn) pathway during pDC-MM interactions. To date, the role of Kyn pathway in MM is unclear, but two rate-limiting enzymes of Kyn pathway, kynurenine 3-monooxygenase (KMO) and indoleamine 2,3-dioxygenase 1 (IDO1), have been implicated in various cancers. Overexpression of these enzymes causes hyper-accumulation of terminal metabolites, thereby triggering immune suppression via disruption of bidirectional signaling axes between antigen-presenting cells and T cells. In our study, we found that KMO and IDO1 are increased in both pDCs and MM cells during pDC-MM interactions. To assess the functional significance of these findings, we used our pDC/MM or pDC/T cells co-culture models, and show that targeting Kyn pathway generates MM-specific CTL activity. Moreover, the combination of KMO/IDO1 inhibitors with anti-PD-L1 Ab enhances anti-tumor immunity and cytotoxicity in MM.MethodsMicroArray: MM cells were cocultured with pDCs for 72h; separated using CD138 Microbeads; poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared ( >1.5-fold change was considered significant, CI > 95%). pDC-induced overexpression of target proteins: MM cells were cocultured with pDCs for 24h; analyzed by flow to detect KMO/IDO1 expression. CTL or NK activity assays: Minimally cytotoxic concentrations of inhibitors were used to assess immune functions. MM-BM CD8+ T or NK- cells were cultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of KMO (Ro 61-8048: 100 nM) or IDO1 (INCB 024360: 0.1 µM) inhibitors (Selleck Chemicals) for 5 days; drug washed, pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T/NK:MM), followed by quantification of viable MM cells by FACS. Degranulation assay was quantified using cell surface CD107a. Osteoclast and osteoblast formations were assessed from TRAP and Alizarin Red staining, respectively.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of KMO in MM cells (2.153 fold vs MM alone; n=3; CI > 95%). 2) Both pDCs and MM cells expressed KMO and IDO1; pDC-MM coculture further increased KMO expression in MM cells (2-3-fold vs MM; p = 0.006); KMO+ MM cell populations also increased after coculture (~1.2 fold; p <0.05). Importantly, IDO1 expression in MM cells is also enhanced after co-culture; validating the importance of Kyn pathway in the context of pDC-MM interactions (MFI: 1.5-2 fold; p=0.006); IDO1+ MM cell populations increased by ~1.5 fold after coculture (p <0.05). 3) KMOi directly activates pDCs and upregulates functional markers on pDCs (CD80: 1.19 fold; CD83: 1.4 fold; p <0.05). 4) KMOi activates patient BM-CD8+T cells in autologous pDCs/T cell coculture; triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0 fold vs control; p = 0.009). 5) In agreement with above findings, KMOi also increased surface CD107a expression on NK and CD8+T cells (CD56+NK: 1.41 fold; p = 0.004; CD8+T: 1.6 fold; p= 0.001). 6) Importantly, the combination of KMOi or IDO1i and anti-PD-L1 Ab (5 µg/ml) triggers a more robust MM-specific CD8+ CTL activity than single agent (%MM viability: KMOi: 72%; KMOi + anti-PD-L1 Ab: 57%; IDOi: 82%; IDOi + anti-PD-L1 Ab: 70%; n=7; p = 0.01). Finally, 7) Both KMOi and IDO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.Conclusions pDC-MM interactions upregulate immunosuppressive enzymes in Kyn pathway; and importantly, blockade of Kyn pathway enzymes induces MM-specific CD8+ CTL activity. Our preclinical data therefore provides provides the basis for novel immune-based therapeutic approaches targeting kynurenine (Kyn) pathway enzymes KMO and IDO1 to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Equity Ownership; Oncopep: Equity Ownership; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Takeda Millennium: Consultancy.

Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds

Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z′ value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

Diclofenac Identified as a Kynurenine 3-Monooxygenase Binder and Inhibitor by Molecular Similarity Techniques.

In this study, we apply a battery of molecular similarity techniques to known inhibitors of kynurenine 3-monooxygenase (KMO), querying each against a repository of approved, experimental, nutraceutical, and illicit drugs. Four compounds are assayed against KMO. Subsequently, diclofenac (also known by the trade names Voltaren, Voltarol, Aclonac, and Cataflam) has been confirmed as a human KMO protein binder and inhibitor in cell lysate with low micromolar KD and IC50, respectively, and low millimolar cellular IC50. Hit to drug hopping, as exemplified here for one of the most successful anti-inflammatory medicines ever invented, holds great promise for expansion into new disease areas and highlights the not-yet-fully-exploited potential of drug repurposing.

Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase

Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens(pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It actsas a competitive inhibitor in scKMO and as anon-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.