The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison.

2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) 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 DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (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 since both occur even under conditions of depleted ubiquitin. Like etoposide, since 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 (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.

A comprehensive structural analysis of the ATPase domain of human DNA topoisomerase II beta bound to AMPPNP, ADP, and the bisdioxopiperazine, ICRF193.

Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.