scholarly journals A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor

2017 ◽  
Vol 22 (7) ◽  
pp. 906-914 ◽  
Author(s):  
Mohamed Nilam ◽  
Philip Gribbon ◽  
Jeanette Reinshagen ◽  
Kathrin Cordts ◽  
Edzard Schwedhelm ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
High Throughput Screening ◽  
Enzymatic Reaction ◽  
Epigallocatechin Gallate ◽  
Cancer Chemoprevention ◽  
Decarboxylase Activity ◽  
Label Free ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
Formed Product

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M−1) than to the substrate L-ornithine (340 M−1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s−1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z′ factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

scholarly journals Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth

1994 ◽  
Vol 304 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Y Murakami ◽  
S Matsufuji ◽  
Y Miyazaki ◽  
S Hayashi
Keyword(s):  
Cell Growth ◽  
Ornithine Decarboxylase ◽  
Polyamine Metabolism ◽  
Decarboxylase Activity ◽  
Inhibitory Protein ◽  
Polyamine Biosynthesis ◽  
Rapid Degradation ◽  
Physiological Importance ◽  
Key Enzyme

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. It is a short-lived protein and negatively regulated by its products, polyamines. Its degradation is accelerated by the binding of antizyme, an ODC-inhibitory protein induced by polyamines. To evaluate the physiological importance of antizyme we examined the effect of forced expression of antizyme on cellular ODC and polyamine levels and cell growth. Antizyme almost completely abolished the induction of ODC by growth stimuli. This may have been caused by antizyme-induced rapid degradation of newly synthesized ODC, since the half-life of ODC complexes with antizyme was less than 5 min. Forced expression of antizyme caused reductions of cellular putrescine and spermidine levels, and inhibited cell growth, which was partially restored by the addition of putrescine. These observations suggested a critically important role of antizyme in polyamine metabolism.

scholarly journals The ornithine decarboxylase gene of Caenorhabditis elegans: cloning, mapping and mutagenesis.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 517-525 ◽  
Author(s):  
M Macrae ◽  
R H Plasterk ◽  
P Coffino
Keyword(s):  
Enzymatic Activity ◽  
Ornithine Decarboxylase ◽  
In Vitro Translation ◽  
In Vitro Assays ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis ◽  
Eukaryotic Species ◽  
Key Enzyme ◽  
Stage 1

Abstract The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 422 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5' RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved stage 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory.

scholarly journals Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity

1977 ◽  
Vol 166 (1) ◽  
pp. 81-88 ◽  
Author(s):  
A E Pegg
Keyword(s):  
Ornithine Decarboxylase ◽  
Pyridoxal Phosphate ◽  
Vitamin B ◽  
Decarboxylase Activity ◽  
Deficient Diet ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Ornithine Decarboxylase Activity

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1′-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.

scholarly journals Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction

2018 ◽  
Vol 23 (6) ◽  
pp. 561-573 ◽  
Author(s):  
Martin Winter ◽  
Tom Bretschneider ◽  
Carola Kleiner ◽  
Robert Ries ◽  
Jörg P. Hehn ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput Screening ◽  
Tyrosine Phosphatase ◽  
Enzymatic Reaction ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Ionization Time ◽  
Maldi Tof ◽  
Ptp1b Inhibitors ◽  
Promising Perspective

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

scholarly journals The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 740-745 ◽  
Author(s):  
E Niskanen ◽  
A Kallio ◽  
PP McCann ◽  
DG Baker
Keyword(s):  
Ornithine Decarboxylase ◽  
Colony Formation ◽  
Calf Serum ◽  
Decarboxylase Activity ◽  
Irreversible Inhibitor ◽  
Host Animal ◽  
Polyamine Biosynthesis ◽  
Diffusion Chambers

Abstract Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2–4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

scholarly journals Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine

2003 ◽  
Vol 375 (2) ◽  
pp. 465-470 ◽  
Author(s):  
Ning QU ◽  
Natalia A. IGNATENKO ◽  
Phillip YAMAUCHI ◽  
David E. STRINGER ◽  
Corey LEVENSON ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Enzyme Inhibitor ◽  
Human Colon ◽  
Common Mechanism ◽  
Decarboxylase Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Polyamine Biosynthesis ◽  
Irreversible Reaction ◽  
Kd Values

Racemic difluoromethylornithine (d/l-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. d/l-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either l- or d-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (KD) values for the formation of enzyme–inhibitor complexes were 28.3±3.4, 1.3±0.3 and 2.2±0.4 μM respectively for d-, l- and d/l-DFMO. The differences in these KD values were statistically significant (P<0.05). The inhibitor inactivation constants (Kinact) for the irreversible step were 0.25±0.03, 0.15±0.03 and 0.15±0.03 min−1 respectively for d-, l- and d/l-DFMO. These latter values were not statistically significantly different (P>0.1). d-DFMO was a more potent inhibitor (IC50~7.5 μM) when compared with d-ornithine (IC50~1.5 mM) of ODC-catalysed l-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either l- or d-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme–inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for l-DFMO when compared with d-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the l- and d-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the α-substituent of the inhibitor. The d-enantiomer may have advantages, such as decreased normal tissue toxicity, over l- or d/l-DFMO in some clinical applications.

Growth hormone inhibition of lipogenesis in sheep adipose tissue: requirement for gene transcription and polyamines

1994 ◽  
Vol 142 (2) ◽  
pp. 235-243 ◽  
Author(s):  
C A Borland ◽  
M C Barber ◽  
M T Travers ◽  
R G Vernon
Keyword(s):  
Growth Hormone ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
Ornithine Decarboxylase ◽  
Gene Transcription ◽  
Actinomycin D ◽  
Total Activity ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis

Abstract The chronic inhibitory effect of growth hormone (GH) on lipogenesis in sheep adipose tissue explants was investigated in an in vitro tissue culture system. In the absence of other hormones, GH caused a decrease in the rate of lipogenesis after 6 h of culture. In contrast, when lipogenesis was stimulated by the presence of insulin plus dexamethasone, GH again decreased lipogenesis but after a lag of at least 12 h. Actinomycin D, an inhibitor of gene transcription, prevented the effect of GH on lipogenesis in both the absence and presence of insulin plus dexamethasone. Actinomycin D added to tissue previously incubated for 6 h in the presence of GH alone prevented further decline in lipogenesis over the next 5 h, suggesting that transcription of a short-lived mediator protein is required for the GH effect to occur. An increase in ornithine decarboxylase activity was detected in explants exposed to GH, reaching a peak after 12 h incubation; this was prevented by actinomycin D. Methylglyoxal bis-(guanylhydrazone), an inhibitor of polyamine biosynthesis, partially alleviated the effect of GH on lipogenesis; this was reversed by addition of spermidine. However, spermidine did not reverse the effects of actinomycin D, implicating a short-lived protein in addition to ornithine decarboxylase in the action of GH. In the absence of other hormones GH had no effect on either the expressed (initial) or total activity of acetyl-CoA carboxylase, but GH prevented the increase in both expressed and total activities of the enzyme induced by insulin plus dexamethasone. Varying lipolysis and fatty acid accumulation in adipose tissue by addition of adenosine deaminase plus indomethacin or bovine serum albumin to the culture medium had no effect on lipogenesis and these agents partly alleviated GH inhibition of lipogenesis. No effect of GH was found on the amount of glycerol released by cultured tissue. GH also had no effect on fatty acid esterification. Thus the chronic inhibitory effects of GH on lipogenesis involve a protein with a very short half-life. The effect also requires polyamines but does not appear to involve changes in fatty acid concentrations in the cell. In addition GH appears to inhibit lipogenesis and to antagonise insulin-stimulation of lipogenesis by different mechanisms. Journal of Endocrinology (1994) 142, 235–243

Inhibition of phorbol ester--induced human epidermal ornithine decarboxylase activity by oral compounds: a possible role in human chemoprevention studies.

1985 ◽  
Vol 3 (6) ◽  
pp. 751-757 ◽  
Author(s):  
C L Loprinzi ◽  
A K Verma ◽  
R K Boutwell ◽  
P P Carbone
Keyword(s):  
Ornithine Decarboxylase ◽  
Human Cancer ◽  
Mouse Skin ◽  
Cancer Chemoprevention ◽  
Tumor Formation ◽  
Tumor Promoter ◽  
Decarboxylase Activity ◽  
Activity Levels ◽  
Animal Data ◽  
Human Volunteers

Extensive animal data have suggested that, in some systems, the induction of ornithine decarboxylase (ODC) is an essential, although not sufficient, aspect of tumor promotion and that compounds that inhibit ODC can inhibit tumor formation. Using fasting human volunteers, we report that human epidermal and dermal ODC are consistently induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in a manner similar to that seen in mouse skin. There is a marked intersubject variation in TPA-induced epidermal ODC activity levels. Orally administered compounds significantly inhibited TPA-caused human epidermal ODC induction. These data may be useful in the further development of drugs, doses, and dose schedules for use in human cancer chemoprevention studies.

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