ABSTRACT
Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of ß-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial ß-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 µM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.
Subject(s)
Bacteria/drug effects , Colon/microbiology , Diarrhea/prevention & control , Enzyme Inhibitors/pharmacology , Gastrointestinal Microbiome/drug effects , Glucuronidase/antagonists & inhibitors , Imino Pyranoses/pharmacology , Irinotecan , Uronic Acids/pharmacology , Animals , Bacteria/enzymology , Cell Line , Diarrhea/chemically induced , Diarrhea/microbiology , Disease Models, Animal , Female , Glucuronidase/metabolism , Humans , Mice, Inbred BALB CABSTRACT
Selective inhibitors of gut bacterial ß-glucuronidases (GUSs) are of particular interest in the prevention of xenobiotic-induced toxicities. This study reports the first structure-activity relationships on potency and selectivity of several iminocyclitols (2-7) for the GUSs. Complex structures of Ruminococcus gnavus GUS with 2-7 explained how charge, conformation, and substituent of iminocyclitols affect their potency and selectivity. N1 of uronic isofagomine (2) made strong electrostatic interactions with two catalytic glutamates of GUSs, resulting in the most potent inhibition (Ki ≥ 11 nM). C6-propyl analogue of 2 (6) displayed 700-fold selectivity for opportunistic bacterial GUSs (Ki = 74 nM for E. coli GUS and 51.8 µM for RgGUS). In comparison with 2, there was 200-fold enhancement in the selectivity, which was attributed to differential interactions between the propyl group and loop 5 residues of the GUSs. The results provide useful insights to develop potent and selective inhibitors for undesired GUSs.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Cyclitols/chemistry , Gastrointestinal Microbiome/drug effects , Glucuronidase/antagonists & inhibitors , Piperidines/chemistry , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Cattle , Clostridiales/enzymology , Clostridium perfringens/enzymology , Crystallography, X-Ray , Cyclitols/chemical synthesis , Cyclitols/metabolism , Enzyme Assays , Escherichia coli/enzymology , Glucuronidase/chemistry , Glucuronidase/metabolism , Molecular Conformation , Piperidines/chemical synthesis , Piperidines/metabolism , Protein Binding , Structure-Activity RelationshipABSTRACT
Crosslinking of B-cell receptor (BCR) sets off an apoptosis programme, but the underlying pathways remain obscure. Here we decipher the molecular mechanisms bridging B-cell activation and apoptosis mediated by post-translational modification (PTM). We find that O-GlcNAcase inhibition enhances B-cell activation and apoptosis induced by BCR crosslinking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identifies 313 O-GlcNAcylation-dependent phosphosites on 224 phosphoproteins. Among these phosphoproteins, temporal regulation of the O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggers apoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1 is a prerequisite for the recruitment of its kinase, PKC-ß1, to induce S243 phosphorylation, leading to ERK activation and downregulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Calcium-Binding Proteins/metabolism , Acetylglucosamine/metabolism , Animals , Apoptosis/drug effects , Apoptosis/immunology , B-Lymphocytes/cytology , Calcium-Binding Proteins/chemistry , Enzyme Inhibitors/pharmacology , Glycosylation , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Microfilament Proteins , Phosphorylation , Protein Kinase C beta/metabolism , Pyrans/pharmacology , Signal Transduction , Thiazoles/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , beta-N-Acetylhexosaminidases/metabolismABSTRACT
Don't interrupt! Protein serotonylation has been implicated in living cells, yet its role remains poorly defined because of the lack of characterization tools. We synthesized a serotonin derivative to enable selective tagging of serotonylation and to investigate its effect on Ras; the latter displayed undisrupted interaction with Raf-1 at the Ras binding domain.
Subject(s)
Protein Processing, Post-Translational , Serotonin/metabolism , Transglutaminases/metabolism , ras Proteins/metabolism , Cell Line, Tumor , Enzyme Activation , Humans , Serotonin/analogs & derivatives , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , ras Proteins/chemistryABSTRACT
Upon treatment with phenyl dichlorophosphate (PhOP=OCl(2)) in acetonitrile at ambient temperature, a variety of ketoximes underwent a Beckmann rearrangement in an effective manner to afford the corresponding amides in moderate to high yields.
Subject(s)
Organophosphorus Compounds/chemistry , Oximes/chemistry , Acetonitriles/chemistry , Acetophenones/chemistry , Amides/chemical synthesis , Models, Chemical , Solvents/chemistry , TemperatureABSTRACT
Cellular redox conditions affect Gsp amidase activity in Escherichia coli. Guided by the structure and catalytic mechanism of the amidase, we designed and synthesized an acyloxymethyl ketone-based activity probe containing a biotin handle. This probe was used to monitor Gsp amidase activity in E. coli lysates that had been subjected to oxidative or methylglyoxal-induced stress.
Subject(s)
Amidohydrolases/metabolism , Enzyme Assays/methods , Escherichia coli/enzymology , Glutathione/analogs & derivatives , Ketones/metabolism , Spermidine/analogs & derivatives , Binding Sites , Glutathione/metabolism , Ketones/chemistry , Models, Molecular , Oxidation-Reduction , Spermidine/metabolismABSTRACT
Bicyclic isoxazolines and isoxazoles are obtained in good yields by proceeding through a convenient one-pot, two-step procedure utilizing 2,4,6-trichloro-1,3,5-triazine (TCT) as a dehydrating agent.