EPLIN-ß is a novel substrate of ornithine decarboxylase antizyme 1 and mediates cellular migration.

Polyamines promote cellular proliferation. Their levels are controlled by ornithine decarboxylase antizyme 1 (Az1, encoded by OAZ1), through the proteasome-mediated, ubiquitin-independent degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Az1-mediated degradation of other substrates such as cyclin D1 (CCND1), DNp73 (TP73) or Mps1 regulates cell growth and centrosome amplification, and the currently known six Az1 substrates are all linked with tumorigenesis. To understand whether Az1-mediated protein degradation might play a role in regulating other cellular processes associated with tumorigenesis, we employed quantitative proteomics to identify novel Az1 substrates. Here, we describe the identification of LIM domain and actin-binding protein 1 (LIMA1), also known as epithelial protein lost in neoplasm (EPLIN), as a new Az1 target. Interestingly, between the two EPLIN isoforms (α and ß), only EPLIN-ß is a substrate of Az1. The interaction between EPLIN-ß and Az1 appears to be indirect, and EPLIN-ß is degraded by Az1 in a ubiquitination-independent manner. Az1 absence leads to elevated EPLIN-ß levels, causing enhanced cellular migration. Consistently, higher LIMA1 levels correlate with poorer overall survival of colorectal cancer patients. Overall, this study identifies EPLIN-ß as a novel Az1 substrate regulating cellular migration.

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.

Inhibitory Effect of Ursolic Acid on the Migration and Invasion of Doxorubicin-Resistant Breast Cancer.

The cause of death in most breast cancer patients is disease metastasis and the occurrence of multidrug resistance (MDR). Ornithine decarboxylase (ODC), which is involved into multiple pathways, is closely related to carcinogenesis and development. Ursolic acid (UA), a natural triterpenoid compound, has been shown to reverse the MDR characteristics of tumor cells. However, the effect of UA on the invasion and metastasis of tumor cells with MDR is not known. Therefore, we investigated the effects of UA on invasion and metastasis, ODC-related polyamine metabolism, and MAPK-Erk-VEGF/MMP-9 signaling pathways in a doxorubicin-resistant breast cancer cell (MCF-7/ADR) model. The obtained results showed that UA significantly inhibited the adhesion and migration of MCF-7/ADR cells, and had higher affinities with key active cavity residues of ODC compared to the known inhibitor di-fluoro-methyl-ornithine (DFMO). UA could downregulate ODC, phosphorylated Erk (P-Erk), VEGF, and matrix metalloproteinase-9 (MMP-9) activity. Meanwhile, UA significantly reduced the content of metabolites of the polyamine metabolism. Furthermore, UA increased the intracellular accumulation of Dox in MCF-7/ADR cells. Taken together, UA can inhibit against tumor progression during the treatment of breast cancer with Dox, and possibly modulate the Erk-VEGF/MMP-9 signaling pathways and polyamine metabolism by targeting ODC to exert these effects.

Discovery of ancestral L-ornithine and L-lysine decarboxylases reveals parallel, pseudoconvergent evolution of polyamine biosynthesis.

Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5'-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable.

Oesophageal squamous cell carcinoma-associated IL-33 rewires macrophage polarization towards M2 via activating ornithine decarboxylase.

BACKGROUND: The tumour microenvironment primarily constitutes macrophages in the form of an immunosuppressive M2 phenotype, which promotes tumour growth. Thus, the development of methodologies to rewire M2-like tumour-associated macrophages (TAMs) into the M1 phenotype, which inhibits tumour growth, might be a critical advancement in cancer immunotherapy research. METHODS: The expressions of IL-33 and indicators related to macrophage polarization in oesophageal squamous cell carcinoma (ESCC) tissues and peripheral blood mononuclear cell (PBMC)-derived macrophages were determined. Inhibition of ornithine decarboxylase (ODC) with small interfering RNA was used to analyse the phenotype of macrophage polarization and polyamine secretory signals. CCK-8, wound-healing and Transwell assays were used to detect the proliferation and migration of ECA109 cells in vitro. The tumour xenograft assay in nude mice was used to examine the role of IL-33 in ESCC development in vivo. RESULTS: This study showed the substantially elevated IL-33 expression in ESCC tissues compared with the normal tissues. Additionally, enhanced infiltration of M2-like macrophages into the ESCC tumour tissue was also observed. We observed a strong correlation between the IL-33 levels and the infiltration of M2-like macrophages in ESCC tumours locally. Mechanistically, IL-33 induces M2-like macrophage polarization by activating ODC, a key enzyme that catalyses the synthesis of polyamines. Inhibition of ODC suppressed M2-like macrophage polarization. Finally, in vivo, we confirmed that IL-33 promotes tumour progression. CONCLUSIONS: This study revealed an oncogenic role of IL-33 by actively inducing M2-like macrophage differentiation; thus, contributing to the formation of an immunosuppressive ESCC tumour microenvironment. Thus, IL-33 could act as a novel target for cancer immunotherapies.

The rational discovery of multipurpose inhibitors of the ornithine decarboxylase.

Metabolic reprograming is a hallmark of cancer, and the polyamine metabolic network is dysregulated in many cancers. Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis in the polyamine metabolic network. In many cancer cells, ODC is over-expressed, so this enzyme has been an attracting anti-cancer drug target. In the catalysis axis (pathway), ODC converts ornithine to putrescine. Meanwhile, ODC's activity is regulated by protein-protein interactions (PPIs), including the ODC-OAZ1-AZIN1 PPI axis and its monomer-dimer equilibrium. Previous studies showed that when ODC's activity is inhibited, the PPIs might counteract the inhibition efficiency. Therefore, we proposed that multipurpose inhibitors that can simultaneously inhibit ODC's activity and perturb the PPIs would be very valuable as drug candidates and molecular tools. To discover multipurpose ODC inhibitors, we established a computational pipeline by combining positive screening and negative screening. We used this pipeline for the forward screening of multipurpose ligands that might inhibit ODC's activity, block ODC-OAZ1 interaction and enhance ODC non-functional dimerization. With a combination of different experimental assays, we identified three multipurpose ODC inhibitors. At last, we showed that one of these inhibitors is a promising drug candidate. This work demonstrated that our computational pipeline is useful for discovering multipurpose ODC inhibitors, and multipurpose inhibitors would be very valuable. Similar with ODC, there are a lot of proteins in human proteome that act as both enzymes and PPI components. Therefore, this work is not only presenting new molecular tools for polyamine study, but also providing potential insights and protocols for discovering multipurpose inhibitors to target more important protein targets.

Allicin, a Potent New Ornithine Decarboxylase Inhibitor in Neuroblastoma Cells.

The natural product allicin is a reactive sulfur species (RSS) from garlic (Allium sativum L.). Neuroblastoma (NB) is an early childhood cancer arising from the developing peripheral nervous system. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are oncometabolites that contribute to cell proliferation in NB and other c-MYC/MYCN-driven cancers. Both c-MYC and MYCN directly transactivate the E-box gene ODC1, a validated anticancer drug target. We identified allicin as a potent ODC inhibitor in a specific radioactive in vitro assay using purified human ODC. Allicin was ∼23 000-fold more potent (IC50 = 11 nM) than DFMO (IC50 = 252 µM), under identical in vitro assay conditions. ODC is a homodimer with 12 cysteines per monomer, and allicin reversibly S-thioallylates cysteines. In actively proliferating human NB cells allicin inhibited ODC enzyme activity, reduced cellular polyamine levels, inhibited cell proliferation (IC50 9-19 µM), and induced apoptosis. The natural product allicin is a new ODC inhibitor and could be developed for use in conjunction with other anticancer treatments, the latter perhaps at a lower than usual dosage, to achieve drug synergism with good prognosis and reduced adverse effects.

Evaluation of presumptive identification of Enterobacterales using CHROMagar Orientation medium and rapid biochemical tests.

BACKGROUND: The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is gradually spreading among large-scale laboratories; however, this method is impractical for small-scale laboratories. In laboratories without access to these rapid identification methods, problems related to them remain unsolved. In this study, we aimed to develop a rapid and inexpensive method to presumptively identify Enterobacterales using CHROMagar Orientation medium. METHODS: The algorithm for presumptive identification of Enterobacteriaceae using CHROMagar Orientation medium was based on our previous studies. Modified property tests for indole, lysine decarboxylase, ornithine decarboxylase, and hydrogen sulfide were performed to evaluate the differentiation of the bacterial species. RESULTS: Using the type strains and clinical isolates, it was possible to conduct the property tests at a low cost, within 4 hours. The spot indole test was performed without any nonspecific reactions for the bacteria forming colored colonies. The presumptive identification of bacteria was thereby possible within 24 hours after specimen submission. CONCLUSION: All these results suggest that the rapid presumptive identification of Enterobacterales is possible with this new identification method using CHROMagar Orientation medium. This is therefore a prompt and economical method that can be used in routine laboratory work.

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis.

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence1-3. To survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryogenic electron microscopy structure of an Escherichia coli ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor 1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

Off-pathway 3D-structure provides protection against spontaneous Asn/Asp isomerization: shielding proteins Achilles heel.

Spontaneous deamidation prompted backbone isomerization of Asn/Asp residues resulting in - most cases - the insertion of an extra methylene group into the backbone poses a threat to the structural integrity of proteins. Here we present a systematical analysis of how temperature, pH, presence of charged residues, but most importantly backbone conformation and dynamics affect isomerization rates as determined by nuclear magnetic resonance in the case of designed peptide-models. We demonstrate that restricted mobility (such as being part of a secondary structural element) may safeguard against isomerization, but this protective factor is most effective in the case of off-pathway folds which can slow the reaction by several magnitudes compared to their on-pathway counterparts. We show that the geometric descriptors of the initial nucleophilic attack of the isomerization can be used to classify local conformation and contribute to the design of stable protein drugs, antibodies or the assessment of the severity of mutations.At any ­Asn/AspGly­ sites in proteins a spontaneous backbone isomerization occurs within days under physiological conditions leading to various forms of proteopathy. This unwanted transformation especially harmful to long-lived proteins (e.g. hemoglobin and crystallins), can be slowed down, though never stopped, by a rigid three-dimensional protein fold, if it can delay in the conformational maze, on-pathway intermediates from occurring.

Engineering Tropane Alkaloid Production Based on Metabolic Characterization of Ornithine Decarboxylase in Atropa belladonna.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and ß-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A. belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A. belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A. belladonna.

Unforeseen Possibilities To Investigate the Regulation of Polyamine Metabolism Revealed by Novel C-Methylated Spermine Derivatives.

The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me2Spm), 3,10-dimethylspermine (3,10-Me2Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me2Spm was a poor substrate of spermine oxidase and spermidine/spermine-N1-acetyltransferase (SSAT) when compared with 2,11-Me2Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me2Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.

Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors.

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4-7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10-5 mM-1·s-1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Crystal Structure of d-Ornithine/d-Lysine Decarboxylase, a Stereoinverting Decarboxylase: Implications for Substrate Specificity and Stereospecificity of Fold III Decarboxylases.

A newly discovered Fold III pyridoxal 5'-phosphate (PLP)-dependent decarboxylase, d-ornithine/lysine decarboxylase (DOKDC), catalyzes decarboxylation of d-lysine and d-ornithine with inversion of stereochemistry. The X-ray crystal structure of DOKDC has been determined to 1.72 Å. DOKDC has a low level of sequence identity (<30%) with meso-diaminopimelate decarboxylase (DAPDC) and l-lysine/ornithine decarboxylase (LODC), but its three-dimensional structure is very similar. The distal binding site of DAPDC contains a conserved arginine that forms an ion pair with the l-carboxylate end of DAP. In both LODC and DOKDC, this distal site is modified by replacement of the arginine with aspartate, changing the substrate specificity. l-Ornithine decarboxylase (ODC) and LODC have a conserved phenylalanine on the re-face of the PLP complex that has been found to play a key role in the decarboxylation mechanism. We have found that both DAPDC and DOKDC have tyrosine instead of phenylalanine at this position, which precludes the binding of l-amino acids. Because the PLP-binding lysine in ODC, LODC, DAPDC, and DOKDC is located on the re-face of the PLP, we propose that this is the acid group responsible for protonation of the product, thus resulting in the observed retention of configuration for decarboxylation of l-amino acids and inversion for decarboxylation of d-amino acids. The reactions of DAPDC and DOKDC are likely accelerated by positive electrostatics on the re-face by the lysine ε-ammonium ion and on the si-face by closure of the lid over the active site, resulting in desolvation and destabilization of the d-amino acid carboxylate.

Virtual screening of natural inhibitors targeting ornithine decarboxylase with pharmacophore scaffolding of DFMO and validation by molecular dynamics simulation studies.

Ornithine decarboxylase (ODC) is an enzyme that initiates polyamine synthesis in human. Polyamines play key roles in cell-cell adhesion, cell motility and cell cycle regulation. Higher synthesis of polyamines also occurs in rapidly proliferating cancer cells are mediated by ODC. As per earlier studies, di-flouro-methyl-orninthine (DFMO) is a proven efficient inhibitor ODC targeting the catalytic activity, however, its usage is limited due to side effects. Targeting ODC is considered as a potential therapeutic modality in the treatment of cancer. In this study, it is attempted to use DFMO scaffold to build a ligand-based pharmocophore query using MOE to screen similar active compounds from Universal Natural Products Database with better ADMET properties. The identified compounds were virtually screened against the active cavity of ODC using Glide. Further, potential natural hits targeting ODC were shortlisted based on Molecular Mechanics/Generalized-Born/Surface Area (MM-GBSA) score. Finally, molecular dynamics simulations were performed for the natural molecule hit and DFMO in complex with ODC using Desmond. Among the hits shortlisted, 2-amino-5, 9, 13, 17-tetramethyloctadeca-8, 16-diene-1, 3, 14-triol (UNPD208110) was found to be highly potential, as it showed a higher binding affinity in terms of interactions with key active cavity residues, and also showed better ADMET property, HUMO-LUMO gap energy and more stable complex formation with ODC compared to DFMO. Hence, the proposed molecule (UNPD208110) shall be favourably considered as a potential natural inhibitor targeting ODC-mediated disease conditions.

The antizyme family for regulating polyamines.

The polyamines spermidine, spermine, and their precursor putrescine are organic polycations involved in various cellular processes and are absolutely essential for cellular proliferation. Because of their crucial function in the cell, their intracellular concentration must be maintained at optimal levels. To a large extent, this regulation is achieved through the activity of an autoregulatory loop that involves two proteins, antizyme (Az) and antizyme inhibitor (AzI), that regulate the first enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), and polyamine uptake activity in response to intracellular polyamine levels. In this Minireview, I will discuss what has been learned about the mechanism of Az expression and its physical interaction with both ODC and AzI in the regulation of polyamines.

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2'-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.

Inhibition of ornithine decarboxylase 1 facilitates pegylated arginase treatment in lung adenocarcinoma xenograft models.

Arginine depletion has shown anticancer effects among arginine auxotrophic cancers. An anti­proliferative effect of pegylated arginase (BCT­100) has been shown in acute myeloid leukaemia, hepatocellular carcinoma and mesothelioma. The aim of the present study was to evaluate the effect of BCT­100 in lung adenocarcinoma. A panel of lung adenocarcinoma cell lines and xenograft models were used to investigate the effect of BCT­100. Protein expression, arginine level, putrescine level, spermidine level and apoptosis were analyzed by western blotting, ELISA, high performance liquid chromatography, dot blot and TUNEL assay, respectively. BCT­100 converts arginine to ornithine. BCT­100 reduced in vitro cell viability across different lung adenocarcinoma cell lines and suppressed tumour growth in an HCC4006 xenograft, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 xenografts. Upon BCT­100 treatment, ornithine decarboxylase 1 (ODC1) was induced in two solid tumour xenografts (H1650 and H1975). It was postulated that the accumulated ornithine could be channeled via ODC1 to produce polyamines that promoted tumour growth. The action of an ODC1 inhibitor (α­difluoromethylornithine, DFMO) was studied in the restoration of the anticancer effects of BCT­100 in lung adenocarcinoma. In both H1650 and H1975 xenografts, a combination of DFMO and BCT­100 significantly suppressed tumour growth, resulting in doubled median survival compared with the control. Putrescine was decreased in almost all treatment arms in the H1650, H1975 and HCC4006 xenografts. Nonetheless spermidine was reduced only following DFMO/BCT­100 treatment in the H1650 and H1975 xenografts. Apoptosis was enhanced in the combined treatment arm in both H1650 and H1975 xenografts. In the HCC4006 xenograft, addition of DFMO did not alter the tumour suppressive effect of BCT­100. In conclusion, inhibition of ODC1 by DFMO was crucial in facilitating BCT­100 treatment in lung adenocarcinoma that was partially mediated by depleting arginine and polyamines with consequent apoptosis.

Molecular Modeling and Virtual Screening Approach to Discover Potential Antileishmanial Inhibitors Against Ornithine Decarboxylase.

BACKGROUND: Visceral leishmaniasis (VL) is a tropical neglected disease, which encounters poorest of poor people living in Asia, Africa and Latin America; causing the mortality of more than 30,000 people worldwide. The armamentarium for the treatment of VL cases is limited and continuously facing decreasing of efficacy for existing drugs. Ornithine decarboxylase (ODC) is one of the interesting drug targets in Leishmania donovani, due to its association with redox metabolism. OBJECTIVE: To search an antileishmanial compound showing the inhibitory effect against ornithine decarboxylase of Leishmania donovani Method: In this study, we have modelled the three dimensional structure of ODC using Phyre2 (Protein Homology/analog Y Recognition Engine V 2.0), followed by validation using VADAR (Volume, Area, Dihedral Angle Reporter), RAMPAGE, ERRAT, Verify3D and ProSA (Protein Structure Analysis). In order to develop potential antileishmanial, we conducted a high throughput virtual screening of ZINC database ligands comprising of 135,966 compounds. Furthermore, QikProp, ADMET predictor and MM-GBSA was performed for ADME (Absorption, Distribution, Metabolism and Elimination), toxicity and binding energy prediction for top ligands, respectively. Finally, molecular dynamics simulation was performed to get potential antileishmanial compounds. RESULT: Screening of zinc database compounds using high throughput virtual screening has given twelve compounds with good inhibition activity against ornithine decarboxylase. Furthermore, the molecular dynamics simulation work reveals that ZINC67909154 could be a potent inhibitor and this compound can be used to combat VL disease Conclusion: This study concludes that ZINC67909154 has the great potential to inhibit L. donovani ODC and would add to the drug discovery process against visceral leishmaniasis.

Endoplasmic Reticulum Stress is Involved in DFMO Attenuating Isoproterenol-Induced Cardiac Hypertrophy in Rats.

BACKGROUND/AIMS: Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear. METHODS: Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis. RESULTS: DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine. CONCLUSION: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.