DL-a-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase.
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ABSTRACT:
SUBMITTER: Kallio A
PROVIDER: S-EPMC1158419 | biostudies-other | 1982 Jun
REPOSITORIES: biostudies-other
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Project description:Human African trypanosomiasis, caused by the eukaryotic parasite Trypanosoma brucei, is a serious health problem in much of central Africa. The only validated molecular target for treatment of human African trypanosomiasis is ornithine decarboxylase (ODC), which catalyzes the first step in polyamine metabolism. Here, we describe the use of an enzymatic high throughput screen of 316,114 unique molecules to identify potent and selective inhibitors of ODC. This screen identified four novel families of ODC inhibitors, including the first inhibitors selective for the parasitic enzyme. These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme. Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites.
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Project description:Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.
| S-EPMC4066113 | biostudies-literature
Project description:BackgroundHuman ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention.MethodsWe examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies.ResultsBaicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC50 values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor ?-DL-difluoromethylornithine (IC50, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells.ConclusionsTherefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.
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Project description:Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials.
| S-EPMC4775427 | biostudies-literature
Project description:The ornithine decarboxylase antizyme gene of Escherichia coli was identified by immunological screening of an E. coli genomic library. A 6.4-kilobase fragment containing the antizyme gene was subcloned and sequenced. The open reading frame encoding the antizyme was identified on the basis of its ability to direct the synthesis of immunoreactive antizyme. Antizyme shares significant homology with bacterial transcriptional activators of the two-component regulatory system family; these systems consist of a "sensor" kinase and a transcriptional regulator. The open reading frame next to antizyme is homologous to sensor kinases. Antizyme overproduction inhibits the activities of both ornithine and arginine decarboxylases without affecting their protein levels. Extracts from E. coli bearing an antizyme gene-containing plasmid exhibit increased antizyme activity. These data strongly suggest that (i) the cloned gene encodes the ornithine decarboxylase antizyme and (ii) antizyme is a bifunctional protein serving as both an inhibitor of polyamine biosynthesis as well as a transcriptional regulator of an as yet unknown set of genes.
| S-EPMC47089 | biostudies-other
Project description:Polyamines are small cationic molecules that in adult brain are connected to neuronal signaling by regulating inward-rectifier K(+)-channels and different glutamate receptors. Antizyme inhibitors (AZINs) regulate the cellular uptake of polyamines and activate ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis. Elevated levels of ODC activity and polyamines are detected in various brain disorders including stroke and Alzheimer's disease (AD). We originally reported a novel brain- and testis-specific AZIN, called AZIN2, the distribution of which we have now studied in normal and diseased human brain by in situ hybridization and immunohistochemistry. We found the highest accumulation of AZIN2 in a pearl-on-the-string-like distribution along the axons in both the white and gray matter. AZIN2 was also detected in a vesicle-like distribution in the somas of selected cortical pyramidal neurons. Double-immunofluorescence staining revealed co-localization of AZIN2 and N-methyl D-aspartate-type glutamate receptors (NMDARs) in pyramidal neurons of the cortex. Moreover, we found accumulation of AZIN2 in brains affected by AD, but not by other neurodegenerative disorders (CADASIL or Lewy body disease). ODC activity is mostly linked to cell proliferation, whereas its regulation by AZIN2 in post-mitotically differentiated neurons of the brain apparently serves different purposes. The subcellular distribution of AZIN2 suggests a role in vesicular trafficking.
| S-EPMC8094758 | biostudies-literature
Project description:A macromolecular factor that inhibits the activity of the antizyme to ornithine decarboxylase (ODC) was found in rat liver extracts. The factor, 'antizyme inhibitor', was heat-labile, non diffusable and of similar molecular size to ODC. The antizyme inhibitor re-activated ODC that had been inactivated by antizyme, apparently by replacing ODC in a complex with antizyme. Therefore the antizyme inhibitor can be used to assay the amount of inactive ODC-antizyme complex formed in vitro. When assayed by this method, the complex was shown to be eluted before ODC from a Sephadex G-100 column. Significant increase in ODC activity was observed when the antizyme inhibitor was added to crude liver extracts from rats that had been injected with 1,3-diaminopropane to cause decay of ODC activity, suggesting the presence of inactive ODC-antizyme complex in the extracts.
| S-EPMC1158403 | biostudies-other
Project description:The synthesis of a new inhibitor, N-phosphonacetyl-L-isoasparagine (PALI), of Escherichia coli aspartate transcarbamoylase (ATCase) is reported, as well as structural studies of the enzyme.PALI complex. PALI was synthesized in 7 steps from beta-benzyl L-aspartate. The KD of PALI was 2 microM. Kinetics and small-angle X-ray scattering experiments showed that PALI can induce the cooperative transition of ATCase from the T to the R state. The X-ray structure of the enzyme.PALI complex showed 22 hydrogen-bonding interactions between the enzyme and PALI. The kinetic characterization and crystal structure of the ATCase.PALI complex also provides detailed information regarding the importance of the alpha-carboxylate for the binding of the substrate aspartate.
| S-EPMC2538380 | biostudies-literature
Project description:Ornithine decarboxylase (ODC) catalyzes ornithine decarboxylation to yield putrescine, a key precursor of polyamines, and tropane alkaloids (TAs). Here, to investigate in depth the role of ODC in polyamine/TA biosynthesis and to provide a candidate gene for engineering polyamine/TA production, the ODC gene (HnODC) was characterized from Hyoscyamus niger, a TA-producing plant. Our phylogenetic analysis revealed that HnODC was clustered with ODC enzymes of plants. Experimental work showed HnODC highly expressed in H. niger roots and induced by methyl jasmonate (MeJA). In the MeJA treatment, the production of both putrescine and N-methylputrescine were markedly promoted in roots, while contents of putrescine, spermidine, and spermine were all significantly increased in leaves. By contrast, MeJA did not significantly change the production of either hyoscyamine or scopolamine in H. niger plants. Building on these results, the 50-kDa His-tagged HnODC proteins were purified for enzymatic assays. When ornithine was fed to HnODC, the putrescine product was detected by HPLC, indicating HnODC catalyzed ornithine to form putrescine. Finally, we also investigated the enzymatic kinetics of HnODC. Its K m, V max, and K cat values for ornithine were respectively 2.62 ± 0.11 mM, 1.87 ± 0.023 nmol min-1 ?g-1 and 1.57 ± 0.015 s-1, at pH 8.0 and at 30°C. The HnODC enzyme displays a much higher catalytic efficiency than most reported plant ODCs, suggesting it may be an ideal candidate gene for engineering polyamine/TA biosynthesis.
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Project description:Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues. This work focuses on Escherichia coli ornithine transcarbamoylase (OTC), which plays a central role in amino acid metabolism. OTC has been reported to undergo an induced-fit conformational change upon binding its first substrate, carbamoyl phosphate (CP), and several residues important for activity have been identified. Using computational methods based on the computed chemical properties from theoretical titration curves, sequence-based scores derived from evolutionary history, and protein surface topology, residues important for catalytic activity were predicted. The roles of these residues in OTC activity were tested by constructing mutations at predicted positions, followed by steady-state kinetics assays and substrate binding studies with the variants. First-layer mutations R57A and D231A, second-layer mutation H272L, and third-layer mutation E299Q, result in 57- to 450-fold reductions in kcat/KM with respect to CP and 44- to 580-fold reductions with respect to ornithine. Second-layer mutations D140N and Y160S also reduce activity with respect to ornithine. Most variants had decreased stability relative to wild-type OTC, with variants H272L, H272N, and E299Q having the greatest decreases. Variants H272L, E299Q, and R57A also show compromised CP binding. In addition to direct effects on catalytic activity, effects on overall protein stability and substrate binding were observed that reveal the intricacies of how these residues contribute to catalysis.
| S-EPMC7004355 | biostudies-literature