title: Characterization of chemical inhibitors of brefeldin A-activated mono-ADP-ribosylation.
abstract: Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 +/- 3.1 microg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the Vmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+ (154 +/- 13 microM). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.
authors.affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy. weigert@cmns.mnegri.it
authors.forename: R
authors.initials: R
authors.lastname: Weigert
authors.forename: A
authors.initials: A
authors.lastname: Colanzi
authors.forename: A
authors.initials: A
authors.lastname: Mironov
authors.forename: R
authors.initials: R
authors.lastname: Buccione
authors.forename: C
authors.initials: C
authors.lastname: Cericola
authors.forename: M G
authors.initials: MG
authors.lastname: Sciulli
authors.forename: G
authors.initials: G
authors.lastname: Santini
authors.forename: S
authors.initials: S
authors.lastname: Flati
authors.forename: A
authors.initials: A
authors.lastname: Fusella
authors.forename: J G
authors.initials: JG
authors.lastname: Donaldson
authors.forename: M
authors.initials: M
authors.lastname: Di Girolamo
authors.forename: D
authors.initials: D
authors.lastname: Corda
authors.forename: M A
authors.initials: MA
authors.lastname: De Matteis
authors.forename: A
authors.initials: A
authors.lastname: Luini
mesh_terms: D000244:Adenosine Diphosphate; D000818:Animals; D020126:Brefeldin A; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D008297:Male; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012266:Ribose; D013329:Structure-Activity Relationship; D014018:Tissue Distribution
chemical_list: D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D000244:Adenosine Diphosphate; D012266:Ribose; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019204:GTP-Binding Proteins