Inhibitor Of Ornithine Decarboxylase Research Articles

Eflornithine (Iwilfin) for high-risk neuroblastoma.

Eflornithine (Iwilfin – US WorldMeds), an oral ornithine decarboxylase inhibitor, has been approved by the FDA to reduce the risk of relapse in children and with high-risk neuroblastoma who had at least a partial response to prior multiagent, multimodality therapy, including anti-GD2 immunotherapy.

Integrative metabolomics-genomics analysis identifies key networks in a stem cell-based model of schizophrenia.

Schizophrenia (SCZ) is a neuropsychiatric disorder, caused by a combination of genetic and environmental factors. The etiology behind the disorder remains elusive although it is hypothesized to be associated with the aberrant response to neurotransmitters, such as dopamine and glutamate. Therefore, investigating the link between dysregulated metabolites and distorted neurodevelopment holds promise to offer valuable insights into the underlying mechanism of this complex disorder. In this study, we aimed to explore a presumed correlation between the transcriptome and the metabolome in a SCZ model based on patient-derived induced pluripotent stem cells (iPSCs). For this, iPSCs were differentiated towards cortical neurons and samples were collected longitudinally at various developmental stages, reflecting neuroepithelial-like cells, radial glia, young and mature neurons. The samples were analyzed by both RNA-sequencing and targeted metabolomics and the two modalities were used to construct integrative networks in silico. This multi-omics analysis revealed significant perturbations in the polyamine and gamma-aminobutyric acid (GABA) biosynthetic pathways during rosette maturation in SCZ lines. We particularly observed the downregulation of the glutamate decarboxylaseencoding genes GAD1and GAD2, as well astheir protein product GAD65/67 and their biochemical product GABA in SCZ samples. Inhibition of ornithine decarboxylase resulted in further decrease of GABA levels suggesting a compensatory activation of the ornithine/putrescine pathway as an alternative route for GABA production. These findings indicate an imbalance of cortical excitatory/inhibitory dynamics occurring during early neurodevelopmental stages in SCZ. Our study supports the hypothesis of disruption of inhibitory circuits to be causative for SCZ and establishes a novel in silico approach that enables for integrative correlation of metabolic and transcriptomic data of psychiatric disease models.

Abstract 5122: Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma

Abstract Neuroblastoma (NB) is an often-lethal childhood solid tumor. Anti-GD2 immunotherapy improves survival but not all responses are durable. Transforming growth factor-beta (TGFβ) has been implicated in solid tumor immunoresistance. High-risk NBs harbor activated MYC or MYCN to coordinately expand intratumoral polyamines (PA), oncometabolites with immunomodulatory roles, to support tumor progression. Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the rate-limiting enzyme in PA synthesis, and has shown activity in preclinical NB models, including TH-MYCN+/+ transgenic mice, and is in clinical trials in children. We sought to augment anti-GD2 therapy using DFMO and the small molecule TGFβ-receptor inhibitor, galunisertib. We randomized TH-MYCN+/+ mice in two experiments (10 therapy arms; n=6-10 mice/arm) to receive combinations of DFMO, galunisertib, and anti-GD2 antibody m14G2a and to assess impact on survival. We measured 770 genes using nCounter Immuno-Oncology 360 (Nanostring), 36 cytokines using xMap bead-arrays (Luminex), and tumor-infiltrating leukocytes using flow cytometry in harvested tumors. Cell line TGFβ signaling was evaluated in tissue culture. In vitro, galunisertib consistently suppressed the upregulated TGFβ-Smad2/3 signaling cascade in MYCN-amplified neuroblastoma cell lines. In vivo, DFMO alone extends mouse survival and increases intra-tumoral neutrophils and natural killer (NK) cells. These NK cells increased surface expression of activation receptors like NKG2D (p<0.05), while tumor cells increased surface expression of NK-ligands. Tumor cells also expressed increased latency-associated peptide (LAP; p<0.05), a TGFβ precursor, suggesting NK cells were poised to kill but restrained by TGFβ. Cytotoxicity assays showed reversal of this inhibition with TGFβR1 inhibitor galunisertib in vitro. In a proof-of-concept trial, DFMO and galunisertib (DFMO/gal) demonstrated a clinical anti-tumor synergistic effect. DFMO/gal treated tumors had upregulated NK cell activity genes and downregulated TGFβ signaling genes. Cytokine analysis confirmed DFMO induced upregulation of TGFβ1 and TGFβ2 (p<0.01) that was modulated by galunisertib. IL6, GCSF, and GMCSF were markedly downregulated in tumors treated with both DFMO and galunisertib (p<0.05). An expanded trial showed DFMO/gal added to m14G2a showed no significant survival benefit over the DFMO and m14G2a combination. Ultimately, DFMO creates a pro-inflammatory cellular and cytokine milieu in TH-MYCN+/+ NBs. DFMO and TGFβR1 inhibition via galunisertib activate NK cells further and decrease the anti-inflammatory cytokine IL6. Ongoing studies are further characterizing the gene expression and cytokine levels of the NB TME under these treatment conditions. Citation Format: Om H. Gandhi, Christina S. Turn, Kangning Liu, Annette T. Vu, Hamid Bassiri, Michael D. Hogarty. Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5122.

Preventing Cancer by Inhibiting Ornithine Decarboxylase: A Comparative Perspective on Synthetic vs. Natural Drugs.

This perspective delves into the investigation of synthetic and naturally occurring inhibitors, their patterns of inhibition, and the effectiveness of newly utilized natural compounds as inhibitors targeting the Ornithine decarboxylase enzyme. This enzyme is known to target the MYC oncogene, thereby establishing a connection between polyamine metabolism and oncogenesis in both normal and cancerous cells. ODC activation and heightened polyamine activity are associated with tumor development in numerous cancers and fluctuations in ODC protein levels exert a profound influence on cellular activity for inhibition or suppressing tumor cells. This perspective outlines efforts to develop novel drugs, evaluate natural compounds, and identify promising inhibitors to address gaps in cancer prevention, highlighting the potential of newly designed synthetic moieties and natural flavonoids as alternatives. It also discusses natural compounds with potential as enhanced inhibitors.

Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase.

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.

Inhibition of polyamine biosynthesis preserves β cell function in type 1 diabetes

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing β cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with β cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during β cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve β cell function in T1D through islet cell-autonomous effects.

THU277 Deletion Or inhibition Of Ornithine Decarboxylase Protects Islet β-cell Health And Is Associated With Preserved Residual C-peptide In Persons With Recent Onset Type 1 Diabetes

Abstract Disclosure: B. Hammoud: None. L.A. DiMeglio: None. A. Kulkarni: None. A. Hull: None. S. Woerner: None. S. Cabrera: None. L.D. Mastrandrea: None. F. Ouyang: None. S.N. Perkins: None. C. Evans-Molina: None. T.L. Mastracci: None. S.A. Tersey: None. S. Sarkar: None. E. Nakayasu: None. B. Webb-Robertson: None. E. Gerner: None. R.G. Mirmira: None. E.K. Sims: None. Inhibition of polyamine biosynthesis using α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, reduces β cell stress and type 1 diabetes (T1D) incidence in preclinical animal models. However, the cellular and molecular mechanisms by which polyamines promote β-cell stress and the tolerability and effectiveness of their depletion by DFMO in humans with T1D remain unknown. We hypothesized that β-cell protection from stress occurs via cell-autonomous effects of polyamine depletion. To test this hypothesis, we generated β-cell specific knockout of ornithine decarboxylase in mice, performed comprehensive proteomics and transcriptomics on human islets treated with DFMO under inflammatory conditions in vitro, and performed a clinical study of DFMO tolerability, safety, and efficacy in persons with new-onset T1D. β-cell-specific deletion of the gene encoding ornithine decarboxylase in mice demonstrated protection of these animals against toxin (streptozotocin)-induced β-cell loss and hyperglycemia, implicating a cell-autonomous role for ornithine decarboxylase in β-cell stress. RNA sequencing and proteomics analyses of human islets exposed to a cytokine mix (1000 U/mL IFNγ + 50 U/mL IL-1β) +/- 5 mM DFMO identified 130 differentially regulated transcripts and 701 differentially regulated proteins. Pathway analysis implicated endosomal transport, vesicle organization and regulation of exocytosis, mRNA processing, and antigen cross presentation as mechanisms underlying DFMO effects on the islet response. To test DFMO in humans, we performed a 3-site randomized, placebo-controlled, double masked dose-ranging study of 3 months daily oral DFMO in persons with recent-onset T1D (<100 days). Dosing at 125, 250, 500, 750 or 1000 mg/m2/day was tested in 41 persons (12-34 y/o, 59% male, mean HbA1c 7.3%), with 6-10 participants/dosing group. Only mild and moderate adverse effects (AEs) were noted. Possible drug-related AEs included mild-moderate gastrointestinal symptoms, headache, upper respiratory infections, a pump site infection, and anemia. Urinary levels of the ODC product putrescine were decreased (p=0.05) in the 1000 mg/m2 dosing group. Compared to placebo, individuals receiving 750 and 1000 mg/m2 doses had significantly higher mixed-meal C-peptide area under the curve values at the 6-month post-randomization visit (p<0.05 vs. placebo). In conclusion, β-cell ODC inhibition protects β-cell health and function in the context of the proinflammatory T1D milieu. A 3-month course of oral DFMO was well tolerated with a favorable AE profile in persons with recent-onset T1D. Higher doses were associated with greater β-function compared to placebo. DFMO may have utility as a β-cell preservation therapy in recent-onset T1D. Presentation: Thursday, June 15, 2023

The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: implications for retinal ischemic injury

The enzyme arginase 1 (A1) hydrolyzes the amino acid arginine to form L-ornithine and urea. Ornithine is further converted to polyamines by the ornithine decarboxylase (ODC) enzyme. We previously reported that deletion of myeloid A1 in mice exacerbates retinal damage after ischemia/reperfusion (IR) injury. Furthermore, treatment with A1 protects against retinal IR injury in wild-type mice. PEG-A1 also mitigates the exaggerated inflammatory response of A1 knockout (KO) macrophages in vitro. Here, we sought to identify the anti-inflammatory pathway that confers macrophage A1-mediated protection against retinal IR injury. Acute elevation of intraocular pressure was used to induce retinal IR injury in mice. A multiplex cytokine assay revealed a marked increase in the inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the retina at day 5 after IR injury. In vitro, blocking the A1/ODC pathway augmented IL-1β and TNF-α production in stimulated macrophages. Furthermore, A1 treatment attenuated the stimulated macrophage metabolic switch to a pro-inflammatory glycolytic phenotype, whereas A1 deletion had the opposite effect. Screening for histone deacetylases (HDACs) which play a role in macrophage inflammatory response showed that A1 deletion or ODC inhibition increased the expression of HDAC3. We further showed the involvement of HDAC3 in the upregulation of TNF-α but not IL-1β in stimulated macrophages deficient in the A1/ODC pathway. Investigating HDAC3 KO macrophages showed a reduced inflammatory response and a less glycolytic phenotype upon stimulation. In vivo, HDAC3 co-localized with microglia/macrophages at day 2 after IR in WT retinas and was further increased in A1-deficient retinas. Collectively, our data provide initial evidence that A1 exerts its anti-inflammatory effect in macrophages via ODC-mediated suppression of HDAC3 and IL-1β. Collectively we propose that interventions that augment the A1/ODC pathway and inhibit HDAC3 may confer therapeutic benefits for the treatment of retinal ischemic diseases.

A first-in-human pharmacodynamic evaluation of dual polyamine depletion for patients with high-grade gliomas.

TPS2082 Background: No drug has improved survival for patients with high-grade gliomas (HGGs) since temozolomide in 2005. Relatively little is known about the live response of in situ gliomas to therapeutic intervention. Phase 0 studies that leverage access to the live human glioma in situ, including via microdialysis to sample the live glioma microenvironment, provide an attractive and feasible solution to this problem. We performed intraoperative microdialysis to evaluate the extracellular metabolome of divergent regions in 15 HGG resections. Our data revealed a conserved metabolic signature of HGGs and a novel glioma-associated metabolite, guanidinoacetate (GAA). Pathways enriched in this tumor signature included methionine and polyamine metabolism. Although GAA is typically converted to creatine, we hypothesize that the high flux of ornithine through ornithine decarboxylase (ODC) results in excess GAA accumulation. As such, in a Phase 0 trial, we will evaluate in situ glioma responses to polyamine depletion via ODC inhibition (Difluoromethylornithine, DFMO), with or without blockade of polyamine uptake (AMXT 1501), to identify candidate extracellular biomarkers of target engagement and cytotoxicity. Methods: Patients will be recruited who are undergoing a clinically indicated subtotal resection for a known or suspected high-grade glioma (NCT05717153). At the end of the surgery, two microdialysis catheters will be implanted for post-operative microdialysis, one in residual tumor and one in brain adjacent to tumor for an internal control. Beginning on post-operative day (POD) 1, patients will be randomized to one of the following three groups, including administration of vehicle, DFMO (500 mg, PO, BID) and AMXT 1501 (600 mg, PO, BID): 1) DFMO + AMXT 1501 (POD1-5); 2) Vehicle (POD1–POD2), followed by DFMO + AMXT 1501 (POD3-POD5); 3) DFMO alone (POD1–POD2), followed by DFMO + AMXT 1501 (POD3- POD5). Throughout this time, extracellular metabolite levels of polyamines, guanidinoacetate, and glutamate will be quantified from microdialysate collected every two hours from intraoperatively implanted 100 kDA microdialysis catheters. The primary outcome of this study is to determine how polyamine depletion impacts extracellular intratumoral guanidinoacetate abundance. The secondary outcomes of this study are to (1) determine the impact of polyamine depletion on polyamine abundance and the global extracellular metabolome within live human gliomas, in situ, and (2) assess the feasibility of longitudinal microdialysis for pharmacodynamic analysis within live human gliomas in situ in the post-operative setting. Clinical trial information: NCT05717153 .

Glioma Metabolic Feedback In Situ: A First-In-Human Pharmacodynamic Trial of Difluoromethylornithine + AMXT-1501 Through High-Molecular Weight Microdialysis.

No new drug has improved survival for glioblastoma since temozolomide in 2005, due in part to the relative inaccessibility of each patient's individualized tumor biology and its response to therapy. We have identified a conserved extracellular metabolic signature of enhancing high-grade gliomas enriched for guanidinoacetate (GAA). GAA is coproduced with ornithine, the precursor to protumorigenic polyamines through ornithine decarboxylase (ODC). AMXT-1501 is a polyamine transporter inhibitor that can overcome tumoral resistance to the ODC inhibitor, difluoromethylornithine (DFMO). We will use DFMO with or without AMXT-1501 to identify candidate pharmacodynamic biomarkers of polyamine depletion in patients with high-grade gliomas in situ . We aim to determine (1) how blocking polyamine production affects intratumoral extracellular guanidinoacetate abundance and (2) the impact of polyamine depletion on the global extracellular metabolome within live human gliomas in situ. DFMO, with or without AMXT-1501, will be administered postoperatively in 15 patients after clinically indicated subtotal resection for high-grade glioma. High-molecular weight microdialysis catheters implanted into residual tumor and adjacent brain will be used for postoperative monitoring of extracellular GAA and polyamines throughout therapeutic intervention from postoperative day (POD) 1 to POD5. Catheters will be removed on POD5 before discharge. We anticipate that GAA will be elevated in tumor relative to adjacent brain although it will decrease within 24 hours of ODC inhibition with DFMO. If AMXT-1501 effectively increases the cytotoxic impact of ODC inhibition, we expect an increase in biomarkers of cytotoxicity including glutamate with DFMO + AMXT-1501 treatment when compared with DFMO alone. Limited mechanistic feedback from individual patients' gliomas hampers clinical translation of novel therapies. This pilot Phase 0 study will provide in situ feedback during DFMO + AMXT-1501 treatment to determine how high-grade gliomas respond to polyamine depletion.

Drug repositioning to discover novel ornithine decarboxylase inhibitors against visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by Leishmania donovani (Ld), and most cases occur in Brazil, East Africa, and India. The treatment for VL is limited and has many adverse effects. The development of safer and more efficacious drugs is urgently needed. Drug repurposing is one of the best processes to repurpose existing drugs. Ornithine decarboxylase (ODC) is an important target against L. donovani in the polyamine biosynthesis pathway. In this study, we have modeled the 3D structure of ODC and performed high-throughput virtual screening of 8630 ZINC database ligands against Leishmania donovani ornithine decarboxylase (Ld ODC), selecting 45 ligands based on their high binding score. It is further validated through molecular docking simulation and the selection of the top two lead molecules (ceftaroline fosamil and rimegepant) for Molecular Dynamics (MD) simulation, Density functional theory (DFT), and molecular mechanics generalized born surface area (MMGBSA) analysis. The results showed that the binding affinities of ceftaroline fosamil, and rimegepant are, respectively, -10.719 and 10.159 kcal/mol. The docking complexes of the two lead compounds, ceftaroline fosamil, and rimegepant, with the target ODC, were found stable during molecular dynamics simulations. Furthermore, the analysis of MMGBSA revealed that these compounds had a high binding free energy. The DFT analysis showed that the top lead molecules were more reactive than the standard drug (pentamidine). In-silico findings demonstrated that ceftaroline fosamil, and rimegepant might be recognized as potent antagonists against ODC for the treatment of VL.

Abstract 6725: Targeting urea cycle dysfunction to prevent and treat osteosarcoma

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents and young adults. Intensive multimodal treatment cures almost 75% of patients who present with localized disease; however, only 25% of patients who present with metastases become long term survivors, and those who suffer a metastatic relapse are almost never cured. Polyamine metabolism and signaling play important roles in multiple cancers but have not previously been studied in osteosarcoma. D, L-alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the initial, rate-limiting enzyme within the polyamine biosynthetic pathway, and has been studied in a number of different cancers as either a therapeutic or a chemopreventive agent. We investigated the role of polyamines in OS proliferation and metastasis and whether blocking the polyamine synthetic pathway with DFMO had therapeutic potential in the treatment of OS. Methods: We evaluated proliferation, apoptosis, clonogenic growth and the ability to grow under nonadherent conditions in vitro using established OS lines. To investigate the effect of DFMO on tumor growth in vivo, we implanted fragments of patient derived xenograft into the tibias of NOD/SCID/IL-2Rγ null mice. Upon confirmation of tumor growth, mice were randomized to either receive drinking water or drinking water supplemented with 2% DFMO. A third cohort of mice received water supplemented with 2% DFMO only after hindlimb amputation. Results: DFMO has a profound effect on the proliferation of OS cell lines in vitro. Utilizing caspase assays, we determined that DFMO does not induce apoptosis, but is cytostatic and inhibits proliferation. We found that DFMO prevents the clonogenic growth of 3 different OS cell lines in soft agar and prevents spheroid formation under non-adherent conditions. Interestingly, once established, spheroidss were not disrupted by the addition of DFMO. In vivo, administration of 2% DFMO after hindlimb amputation decreased local recurrence and limited metastasis (p=0.02). Conclusion: DFMO inhibits proliferation and clonogenic growth in vitro, and decreases distant metastasis in vivo. Since pediatric dosing and safety data are already established, our findings are readily translatable to clinical trials. Citation Format: Rachel Offenbacher, Paul Ciero, David Loeb. Targeting urea cycle dysfunction to prevent and treat osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6725.

Combined inhibition of polyamine metabolism and eIF5A hypusination suppresses colorectal cancer growth through a converging effect on MYC translation

A key mechanism driving colorectal cancer (CRC) development is the upregulation of MYC and its targets, including ornithine decarboxylase (ODC), a master regulator of polyamine metabolism. Elevated polyamines promote tumorigenesis in part by activating DHPS-mediated hypusination of the translation factor eIF5A, thereby inducing MYC biosynthesis. Thus, MYC, ODC and eIF5A orchestrate a positive feedback loop that represents an attractive therapeutic target for CRC therapy.Here we show that combined inhibition of ODC and eIF5A induces a synergistic antitumor response in CRC cells, leading to MYC suppression. We found that genes of the polyamine biosynthesis and hypusination pathways are significantly upregulated in colorectal cancer patients and that inhibition of ODC or DHPS alone limits CRC cell proliferation through a cytostatic mechanism, while combined ODC and DHPS/eIF5A blockade induces a synergistic inhibition, accompanied to apoptotic cell death in vitro and in mouse models of CRC and FAP. Mechanistically, we found that this dual treatment causes complete inhibition of MYC biosynthesis in a bimodal fashion, by preventing translational elongation and initiation.Together, these data illustrate a novel strategy for CRC treatment, based on the combined suppression of ODC and eIF5A, which holds promise for the treatment of CRC.

Inhibition of chikungunya virus replication by N-ω-Chloroacetyl-L-Ornithine in C6/36, Vero cells and human fibroblast BJ.

Polyamines are involved in several cellular processes and inhibiting their synthesis affects chikungunya virus (CHIKV) replication and translation, and, therefore, reduces the quantity of infectious viral particles produced. In this study, we evaluated the inhibition of CHIKV replication by N-ω-chloroacetyl-L-ornithine (NCAO), a competitive inhibitor of ornithine decarboxylase, an enzyme which is key in the biosynthesis of polyamines (PAs). The cytotoxicity of NCAO was evaluated by MTT in cell culture. The inhibitory effect of CHIKV replication by NCAO was evaluated in Vero and C6/36 cells. The intracellular polyamines were quantified by HPLC in CHIKV-infected cells. We evaluated the yield of CHIKV in titres via the addition of PAs in Vero, C6/36 cells and human fibroblast BJ treated with NCAO. We found that NCAO inhibits the replication of CHIKV in Vero and C6/36 cells in a dose-dependent manner, causing a decrease in the PFU/mL of at least 4 logarithms (p < 0.01) in both cell lines. Viral yields were restored by the addition of exogenous polyamines, mainly putrescine. The HPLC analyses showed that NCAO decreases the content of intracellular PAs, even though it is predominantly spermidines and spermines which are present in infected cells. Inhibition of CHIKV replication was observed in human fibroblast BJ treated with 100μM NCAO 24h before and 48h after the infection at a MOI 1. NCAO inhibits CHIKV replication by depleting the intracellular polyamines in Vero, C6/36 cells and human fibroblast BJ, suggesting that this compound is a possible antiviral agent for CHIKV.

The Polyamine Putrescine Is a Positive Regulator of Group 3 Innate Lymphocyte Activation.

Group 3 innate lymphocytes (ILC3s) rapidly respond to invading pathogens or inflammatory signals, which requires shifting cellular metabolic demands. Metabolic adaptations regulating ILC3 function are not completely understood. Polyamines are polycationic metabolites that have diverse roles in cellular functions and in immunity regulate immune cell biology, including Th17 cells. Whether polyamines play a role in ILC3 activation is unknown. In this article, we report that the polyamine synthesis pathway is important for ILC3 activation. IL-23-activated mouse ILC3s upregulate ornithine decarboxylase, the enzyme catalyzing the rate-limiting step of the conversion of ornithine to putrescine in polyamine synthesis, with a subsequent increase in putrescine levels. Inhibition of ornithine decarboxylase via a specific inhibitor, α-difluoromethylornithine, reduced levels of IL-22 produced by steady-state or IL-23-activated ILC3s in a putrescine-dependent manner. Thus, the polyamine putrescine is a positive regulator of ILC3 activation. Our results suggest that polyamines represent a potential target for therapeutic modulation of ILC3 activation during infection or inflammatory disorders.

Nutraceutical Potential of Seven “Quelites” Harvested in the Northern Highlands of Puebla-México

The northern highlands of Puebla harbor several plant foods consumed since pre-Columbian times. Most of the native vegetables from this geographical region are still uncharacterized at the nutraceutical level. This investigation was focused on the elucidation of chemical and biochemical properties of the edible organs from Rhamnus pompana, Solanum nigrescens, Sechium edule, Yucca aloifolia, Piper auritum, Amaranthus hybridus and Rumex obtusifolius, which are used as traditional foods in this region. The content of basic nutrients (including vitamins of the B complex), nutraceuticals (carotenoids, flavonoids, and fatty acids), phenolics (including antioxidant capacity), and inhibitory properties of these plants on specific enzymes linked to carbohydrate and lipid metabolism were determined. In addition, extracts of these plants were evaluated against pathogenic microorganisms of the gastrointestinal tract. Our results demonstrated statistically significant divergences (p &lt; 0.05) in the content protein (12.5 g/100 g), fiber (9.8 g/100 g), fat (81.2 mg/100 g), beta-carotene (2.48 mg/100 g), beta-cryptoxanthin (1.24 mg/100 g), thiamine (159.6 µg/100 g), riboflavin (106.4 µg/100 g), and vitamin B6 between Y. aloifolia and other plants studied. All plants showed low amounts of reducing sugars (&lt;150 mg/100 g) and sodium (&lt;70 mg/100 g), but the berries of R. pompana exhibited substantial differences (p &lt; 0.05) in the levels of vitamin C (14.6 mg/100 mg). The edible leaves of R. obtusifolius were particularly rich in quercetin (4.38 mg/100 g), kaempferol (2.38 mg/100 g) and lutein (2.52 mg/100 g), whereas the aerial parts of S. edule contained high concentrations of folate (50.6 µg/100 g). All plants showed variable phenolic content and antioxidant capacity; however, Y. aloifolia had the highest values (23 GAE/mg/g and 440 TEAC/µM/g). The ethanolic extracts from Y. aloifolia efficiently inhibited pancreatic lipase (IC50, 43.76 µg/mL) and alpha-glucosidase (IC50, 60.04 µg/mL), whereas those from S. edule inhibited alpha-glucosidase (IC50, 47.4 µg/mL) and HMG-CoA reductase (IC50, 33.6 µg/mL). Only the ethanolic extracts of R. pompana (IC50, 35.36 µg/mL) and Y. aloifolia (IC50, 53.28 µg/mL) inhibited ornithine decarboxylase. All plant extracts exerted moderated antimicrobial activity in at least one species associated with the gastrointestinal tract.

Antizyme Inhibitor 2-Deficient Mice Exhibit Altered Brain Polyamine Levels and Reduced Locomotor Activity.

Alterations in the neural polyamine system are known to be associated with different brain pathological conditions. In addition, the regulation of enzymes involved in polyamine metabolism such as ornithine decarboxylase (ODC), antizymes (AZs), and antizyme inhibitors (AZINs) is critical during brain development. However, while most studies focus on ODC and AZs, less is known about AZIN expression and function in the brain. Thus, our aim was to analyze the expression pattern of AZIN2 during postnatal development, its brain distribution, and its possible implication in phenotypical alterations. The expression pattern of Azin2 and other genes related to polyamine metabolism was analyzed by RT-qPCR. β-D-galactosidase staining was used to determine the anatomical distribution of AZIN2 in a Azin2 knockout model containing the βGeo marker. Brain polyamine content was determined by HPLC. The Rota-Rod and Pole functional tests were used to evaluate motor skills in Azin2-lacking mice. Our results showed that expression of genes codifying for AZs and AZINs showed a similar increasing pattern over time that coincided with a decrease in ODC activity and putrescine levels. The analysis of AZIN2 distribution demonstrated that it is strongly expressed in the cerebellum and distributed along the neuron body and dendrites. The ablation of Azin2 showed a decrease in putrescine levels and is related to reduced motor skills. Our study revealed that AZIN2 expression in the brain is particularly limited to the cerebellum. In addition, the ablation of Azin2 leads to a reduction in putrescine that relates to alterations in motor function, suggesting the role of AZIN2 in the functioning of dopaminergic neurons.

MicroRNA-378a-3p prevents initiation and growth of colorectal cancer by fine tuning polyamine synthesis

BackgroundInhibitors of ornithine decarboxylase (ODC) are effective at preventing colorectal cancer (CRC). However, their high toxicity limits their clinical application. This study was aimed to explore the potential of microRNAs (miRNAs) as an inhibitor of ODC.MethodsmiRNA array was used to identify dysregulated miRNAs in CRC tumors of mice and patients. Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) were used to induce CRC in mice. miRNA function in carcinogenesis was determined by soft-agar colony formation, flow cytometry, and wound healing of CRC cells. Mini-circle was used to deliver miRNA into colons.ResultsMiRNA profiling identified miR-378a-3p (miR-378a) as the most reduced miRNA in CRC tumors of patients and mice treated with AOM/DSS. Pathway array analysis revealed that miR-378a impaired c-MYC and ODC1 pathways. Further studies identified FOXQ1 (forkhead box Q1) and ODC1 as two direct targets of miR-378a. FOXQ1 activated transcription of c-MYC, a transcription activator of ODC1. In addition to directly targeting ODC1, miR-378a also inhibited expression of ODC1 via the FOXQ1-cMYC axis, thereby inhibiting polyamine synthesis in human CRC cells. Phenotypically, by reducing polyamine synthesis, miR-378a induced apoptosis and inhibited proliferation and migration of CRC cells, while disrupting the association of miR-378a with FOXQ1 and ODC1 offset the effects of miR-378a, suggesting that FOXQ1 and ODC1 were required for miR-378a to inhibit CRC cell growth. MiR-378a treatment robustly prevented growth of HCC by inhibiting polyamine synthesis in AOM/DSS mice.ConclusionMiR-378a prevents CRC by inhibiting polyamine synthesis, suggesting its use as a novel ODC inhibitor against CRC.

Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function.

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1-FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Analytical assays to evaluate enzymatic activity and screening of inhibitors for ornithine decarboxylase

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to produce putrescine, the first step in the metabolism of polyamines (putrescine, spermidine, and spermine), which are essential growth factors in eukaryotic cells. ODC is active as a homodimer and depends on pyridoxal 5′-phosphate (PLP) as a cofactor. An increase in the concentration of polyamines has been associated with carcinogenesis. Therefore, there is much interest in identifying inhibitors of this pathway as potential chemotherapeutic and chemopreventive agents. The best-known inhibitor of mammalian ODC is α-difluoromethylornithine (DFMO), a highly selective compound that alkylates Cys-360 (a residue of the ODC active site). Although DFMO was initially developed for the treatment of cancer, the World Health Organization recommends its use in combination with nifurtimox for the treatment of human African trypanosomiasis. Considering the importance of ODC as a promising target for the treatment of various types of cancer and other infectious diseases, choosing the right method for screening potential inhibitors can help to accelerate the discovery of new drugs. Several methods for the determination of ODC activity are found in the literature. Among these, we can mention analysis with radioactive markers, colorimetric assays using auxiliary enzymes to detect CO2 or H2O2 release, chromatographic separations with putrescine derivatization, mass spectrometry, and spectroscopic techniques. In this review, the main analysis methods used will be described, highlighting their advantages and disadvantages, as well as identifying the most promising methods for high-throughput screening.