Inhibitors of microsomal oxidations in insect homogenates.
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ABSTRACT:
SUBMITTER: Chakroborty J
PROVIDER: S-EPMC1270271 | biostudies-other | 1967 Feb
REPOSITORIES: biostudies-other
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Project description:Enzymes are complex biological catalysts and are critical to life. Most oxidations of chemicals are catalyzed by cytochrome P450 (P450, CYP) enzymes, which generally utilize mixed-function oxidase stoichiometry, utilizing pyridine nucleotides as electron donors: NAD(P)H + O2 + R → NAD(P)+ + RO + H2O (where R is a carbon substrate and RO is an oxidized product). The catalysis of oxidations is largely understood in the context of the heme iron-oxygen complex generally referred to as Compound I, formally FeO3+, whose basis was in peroxidase chemistry. Many X-ray crystal structures of P450s are now available (≥ 822 structures from ≥146 different P450s) and have helped in understanding catalytic specificity. In addition to hydroxylations, P450s catalyze more complex oxidations, including C-C bond formation and cleavage. Enzymes derived from P450s by directed evolution can even catalyze more unusual reactions, e.g. cyclopropanation. Current P450 questions under investigation include the potential role of the intermediate Compound 0 (formally FeIII-O2 -) in catalysis of some reactions, the roles of high- and low-spin forms of Compound I, the mechanism of desaturation, the roles of open and closed structures of P450s in catalysis, the extent of processivity in multi-step oxidations, and the role of the accessory protein cytochrome b 5. More global questions include exactly how structure drives function, prediction of catalysis, and roles of multiple protein conformations.
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Project description:A critical review on the approaches to assess the infectivity of the Hepatitis E virus (HEV) in food recommended that a cell culture-based method should be developed. Due to the observations that viral loads in food may be low, it is important to maximise the potential for detection of HEV in a food source in order to fully assess infectivity. To do so, would require minimal processing of any target material. In order to proceed with the development of an infectivity culture method that is simple, robust and reproducible, there are a number of points to address; one being to assess if food homogenates are cytotoxic to HEV susceptible target cells. Food matrices previously shown to have detectable HEV nucleic acid were selected for analysis and assessed for their effect on the percentage survival of three cell lines commonly used for infectivity assays. Target cells used were A549, PLC/PRF/5 and HepG2 cells. The results showed that, as expected, various food homogenates have differing effects on cells in vitro. In this study, the most robust cell line over a time period was the A549 cell line in comparison to HepG2, with PLC/PRF/5 cells being the most sensitive. Overall, this data would suggest that FH can be left in contact with A549 cells for a period of up to 72 h to maximise the potential for testing infection. Using food homogenates directly would negate any concerns over losing virus as a result of any additional processing steps.
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Project description:Microsomal prostaglandin E(2) synthase-1 (mPGES-1) catalyzes prostaglandin E(2) formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC(50) values between 0.4 and 7.9 ?M, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC(50) values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis.
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Project description:1. A potent inhibitor of microsomal mixed-function oxidation has been isolated from the gut contents of larvae of the southern armyworm (Prodenia eridania). 2. Progressive inhibition is associated with a soluble proteinase with a molecular weight of approx. 26000. 3. Although the proteolytic activity of the inhibitor can be reduced by phenylmethanesulphonyl fluoride and other reagents, these materials are only partially effective in protecting the microsomal enzymes. 4. Inhibitory activity is decreased in the presence of bovine serum albumin.
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Project description:Glutathione S-transferase is present in rat liver microsomal fraction, but its activity is low relative to the transferase activity present in the soluble fraction of the hepatocyte. We have found, however, that the activity of microsomal glutathione S-transferase is increased 5-fold after treatment with small unilamellar vesicles made from phosphatidylcholine. The increase in activity is due to the removal of an inhibitor of the enzyme from the microsomal membrane. The inhibitor is present in the organic layer of a washed Folch extract of the microsomal fraction. When this fraction of the microsomal extract is reconstituted in the form of small unilamellar vesicles, it inhibits microsomal glutathione S-transferase that had been activated by prior treatment with small unilamellar vesicles of pure phosphatidylcholine, but does not affect the activity of unactivated microsomal glutathione S-transferase. The inhibitor did not seem to be formed during the isolation of the microsomal fraction, and hence may be a physiological regulator of microsomal glutathione S-transferase. In this regard, both free fatty acid (palmitate) and lysophosphatidylcholine were shown to inhibit the enzyme reversibly. The results indicate that the activity of microsomal glutathione S-transferase is far greater than appreciated until now, and that this form of the enzyme may be an important factor in the hepatic metabolism of toxic electrophiles.
| S-EPMC1153823 | biostudies-other
Project description:Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn- and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.
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Project description:Background and purposeMicrosomal PGE synthase-1 (mPGES-1), the inducible synthase that catalyses the terminal step in PGE2 biosynthesis, is of high interest as therapeutic target to treat inflammation. Inhibition of mPGES-1 is suggested to be safer than traditional NSAIDs, and recent data demonstrate anti-constrictive effects on vascular tone, indicating new therapeutic opportunities. However, there is a lack of potent mPGES-1 inhibitors lacking interspecies differences for conducting in vivo studies in relevant preclinical disease models.Experimental approachPotency was determined based on the reduction of PGE2 formation in recombinant enzyme assays, cellular assay, human whole blood assay, and air pouch mouse model. Anti-inflammatory properties were assessed by acute paw swelling in a paw oedema rat model. Effect on vascular tone was determined with human ex vivo wire myography.Key resultsWe report five new mPGES-1 inhibitors (named 934, 117, 118, 322, and 323) that selectively inhibit recombinant human and rat mPGES-1 with IC50 values of 10-29 and 67-250 nM respectively. The compounds inhibited PGE2 production in a cellular assay (IC50 values 0.15-0.82 μM) and in a human whole blood assay (IC50 values 3.3-8.7 μM). Moreover, the compounds blocked PGE2 formation in an air pouch mouse model and reduced acute paw swelling in a paw oedema rat model. Human ex vivo wire myography analysis showed reduced adrenergic vasoconstriction after incubation with the compounds.Conclusion and implicationsThese mPGES-1 inhibitors can be used as refined tools in further investigations of the role of mPGES-1 in inflammation and microvascular disease.
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Project description: Not available
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