PT  - JOURNAL ARTICLE
AU  - Ian G. Cowell
AU  - Elise M Ling
AU  - Rebecca L Swan
AU  - Matilda LW Brooks
AU  - Caroline A Austin
TI  - The deubiquitinating enzyme inhibitor PR-619 is a potent DNA topoisomerase II poison
AID  - 10.1124/mol.119.117390
DP  - 2019 Jan 01
TA  - Molecular Pharmacology
PG  - mol.119.117390
4099  - http://molpharm.aspetjournals.org/content/early/2019/09/12/mol.119.117390.short
4100  - http://molpharm.aspetjournals.org/content/early/2019/09/12/mol.119.117390.full
AB  - PR-619 (2,6-diaminopyridine-3,5-bis(thiocyanate)) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent TOP2 poison, inducing both TOP2A and TOP2B covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination, as both occur even under conditions of depleted ubiquitin. Since like etoposide, PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20μM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20μM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.SIGNIFICANCE STATEMENT PR-619 is a broad acting deubiquitinating enzyme (DUB) inhibitor and is a useful tool in studies on protein ubiquitination. We report that PR-619 is surprisingly also a novel type of TOP2 poison, that uniquely directs TOP2 activity to the nucleolar compartment and robustly induces TOP2-DNA covalent complexes independently of its DUB inhibitor activity.