Characterization of chemical inhibitors of brefeldin A-activated mono- ADP-ribosylation

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 μg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the V(max) of the reaction, whereas it does not influence the K(m) of the enzyme for NAD⁺ (154 ± 13 μM). 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.