Thiacetazone (TAZ) and ethionamide (ETA) are, respectively, thiourea and thioamide-containing second line antitubercular prodrugs for which there is an extensive clinical history of cross-resistance in Mycobacterium tuberculosis. EtaA, a recently identified flavin-containing monooxygenase (FMO), is responsible for the oxidative activation of ETA in M. tuberculosis. We report here that EtaA also oxidizes TAZ and identify a sulfinic acid and a carbodiimide as the isolable metabolites. Both of these metabolites are derived from an initial sulfenic acid intermediate. Oxidation of TAZ by EtaA at basic pH favors formation of the carbodiimide, whereas neutral or acidic conditions favor formation of the sulfinic acid. The same metabolites are formed from TAZ by human FMO1 and FMO3. The sulfenic acid and carbodiimide metabolites, but not the sulfinic acid product, readily react with glutathione, the first to regenerate the parent drug and the second to give a glutathione adduct. These reactions that may contribute to the antitubercular activity and/or toxicity of TAZ.
Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1 Cb1 , the first intron from the constant region of the mouse T-cell receptor-b (TCR-b) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1 Cb1 and its pre-mRNA had half-lives of 6.0 6 1.4 min and 3.7 6 1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1 Cb1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1 Cb1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1 Cb1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1 Cb1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1 Cb1 also appeared to be selectively exported to the cytoplasm, whereas TCR-b pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.
Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions both with G-protein coupled seven transmembrane receptors and with the nuclear hormone receptor PPARgamma. Synthesis and biological activities of fluorinated analogues of LPA are still relatively unknown. In an effort to identify receptor-selective LPA analogues and to document in detail the structure-activity relationships of fluorinated LPA isosteres, we describe a series of monofluorinated LPA analogues in which either the sn-1 or the sn-2 hydroxy group was replaced by fluorine, or the bridging oxygen in the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) moiety. The sn-1 or sn-2 monofluorinated LPA analogues were enantiospecifically prepared from chiral protected glycerol synthons, and the alpha-monofluoromethylene-substituted LPA analogues were prepared from a racemic epoxide with use of a hydrolytic kinetic resolution. The sn-2 and sn-1 fluoro LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. The alpha-monofluoromethylene LPA analogues were unique new nonhydrolyzable ligands with surprising enantiospecific and receptor-specific biological readouts, with one compound showing a 1000-fold higher activity than native LPA for one receptor subtype.
The metabolically stabilized LPA analogue, 1-oleoyl-2-O-methyl-rac-glycerophosphothioate (OMPT), is a potent agonist for the LPA(3) G-protein-coupled receptor. A new enantiospecific synthesis of both (2R)-OMPT and (2S)-OMPT is described. Calcium release assays in both LPA(3)-transfected insect Sf9 and rat hepatoma Rh7777 cells showed that (2S)-OMPT was 5- to 20-fold more active than (2R)-OMPT. Similar results were found for calcium release, MAPK and Akt activation, and IL-6 release in human OVCAR3 ovarian cancer cells.
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