Human topoisomerase IIα (TOP2A) is a vital nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. TOP2A is a homodimer with a symmetrical, multidomain structure. While the N-terminal and core regions of the protein are well-studied, the C-terminal domain is poorly understood but is involved in enzyme regulation and is predicted to be intrinsically disordered. In addition, it appears to be a major region of post-translational modification and includes several Ser and Thr residues, many of which have not been studied for biochemical effects. Therefore, we generated a series of human TOP2A mutants where we changed specific Ser and Thr residues in the C-terminal domain to Ala, Gly, or Ile residues. We designed, purified, and examined 11 mutant TOP2A enzymes. The amino acid changes were made between positions 1272 and 1525 with 1−7 residues changed per mutant. Several mutants displayed increased levels of DNA cleavage without displaying any change in plasmid DNA relaxation or DNA binding. For example, mutations in the regions 1272−1279, 1324−1343, 1351−1365, and 1374− 1377 produced 2−3 times more DNA cleavage in the presence of etoposide than wild-type TOP2A. Further, several mutants displayed changes in relaxation and/or decatenation activity. Together, these results support previous findings that the C-terminal domain of TOP2A influences catalytic activity and interacts with the substrate DNA. Furthermore, we hypothesize that it may be possible to regulate the enzyme by targeting positions in the C-terminal domain. Because the C-terminal domain differs between the two human TOP2 isoforms, this strategy may provide a means for selectively targeting TOP2A for therapeutic inhibition. Additional studies are warranted to explore these results in more detail.

During replication, transcription, and cell division, knots and tangles can form in DNA. To relieve this stress, type II topoisomerases are employed, which use a transient enzyme‐linked double strand break to remove the knots and tangles formed within the DNA. Furthermore, because the cell has an immediate need for topoisomerase II activity in order to properly perform cellular functions, topoisomerase II has become a target of interest for anticancer therapy. Over the past several years, a class of compounds termed α‐(N)‐heterocyclic thiosemicarbazones have been identified as a possible anticancer therapy option due to their ability to impact topoisomerase II activity. Moreover, literature evidence suggests that copper complexes [Cu(II)] of α‐(N)‐heterocyclic thiosemicarbazones act as catalytic inhibitors of topoisomerase II similar to the topoisomerase II inhibitor Dexrazoxane. Previously, our lab demonstrated activity against human topoisomerase IIα, but there is a second isoform in humans, topoisomerase IIβ, which has been largely unexplored with thiosemicarbazones. Therefore, we set out to determine the mechanism of two Cu(II) complexes of α‐(N)‐heterocyclic thiosemicarbazones as inhibitors of topoisomerase IIβ. The Cu(II) complexes, copper(II) acetylpyridine‐ethylthiosemicarbazone [Cu(APY‐ETSC)Cl] and copper(II)benzoylpyridine‐ethylthiosemicarbazone [Cu(BZP‐ETSC)Cl] were examined for their ability to alter the catalytic activity of topoisomerase IIβ. Both Cu(II) complexes were effective at inhibiting DNA relaxation at around 10–25 μM. Additionally, both Cu(II) complexes increased double‐stranded DNA cleavage peaking around 25–50 μM. When narrowing the focus to just Cu(APY‐ETSC)Cl and Cu(BZP‐ETSC)Cl, it was determined that both compounds interfere with ATP hydrolysis of topoisomerase IIβ, which is required for strand passage. Additional experiments determined that ATP cannot outcompete Cu(APY‐ETSC)Cl or Cu(BZP‐ETSC)Cl for binding to topoisomerase IIβ, which suggests that these compounds bind outside the ATP binding pocket but allosterically impact ATP hydrolysis. Lastly, we examined whether the Cu(II) complexes could stabilize the N‐terminal ATPase domain in a closed conformation similar to ATP. Both compounds stabilize the N‐terminal closed conformation of the ATPase domain, suggesting a mechanism for topoisomerase II inhibition by these compounds. Taken together, these results provide evidence that Cu(II) complexes of α‐(N)‐heterocyclic thiosemicarbazones catalytically inhibit topoisomerase IIβ similar to topoisomerase IIα. Additionally, the mechanism involves binding to the ATPase domain outside of the ATP pocket and inducing a closed N‐terminal gate of topoisomerase II. These results also provide a possible explanation for the increase in topoisomerase II‐mediated DNA cleavage observed in the presence of Cu(II) thiosemicarbazone complexes. Support or Funding Information JMK and JED were supported by LUCOPHS. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associ...

Cannabidiol and related cannabinoids are under exploration for the treatment of a number of disease states. The cannabinoid-quinone HU-331 has been studied as a potential anticancer therapeutic. Previous studies provide evidence that HU-331 displays anticancer activity without some of the known adverse events associated with traditional anticancer agents. In this brief review, we will explore the literature related to the activity of HU-331 in purified systems, cancer cell lines, and animal models. For example, HU-331 displays inhibitory activity against human topoisomerase IIα, a known anticancer drug target. Further, in multiple cell model systems, the IC50 value for HU-331 was less than 10 μM. In addition, mouse model systems demonstrate the ability of HU-331 to shrink tumors without causing cardiotoxicity. In addition, we will briefly review the activity of some key analogs and derivatives of HU-331 for various disease states. Taken together, the published studies support further exploration of HU-331 for the treatment of cancer and possibly other disease states.

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During replication, transcription, and cell division, knots and tangles can form in DNA. To relieve this stress, type II topoisomerases are employed, which use a transient enzyme‐linked double strand break to remove the knots and tangles formed within the DNA. Furthermore, because the cell has an immediate need for topoisomerase II activity in order to properly perform cellular functions, topoisomerase II has become a target of interest for anticancer therapy. Over the past several years, a class of compounds termed α‐(N)‐heterocyclic thiosemicarbazones have been identified as a possible anticancer therapy option due to their ability to impact topoisomerase II activity. Moreover, literature evidence suggests that copper complexes [Cu(II)] of α‐(N)‐heterocyclic thiosemicarbazones act as catalytic inhibitors of topoisomerase II similar to the topoisomerase II inhibitor Dexrazoxane. Previously, our lab demonstrated activity against human topoisomerase IIα, but there is a second isoform in humans, topoisomerase IIβ, which has been largely unexplored with thiosemicarbazones. Therefore, we set out to determine the mechanism of two Cu(II) complexes of α‐(N)‐heterocyclic thiosemicarbazones as inhibitors of topoisomerase IIβ. The Cu(II) complexes, copper(II) acetylpyridine‐ethylthiosemicarbazone [Cu(APY‐ETSC)Cl] and copper(II)benzoylpyridine‐ethylthiosemicarbazone [Cu(BZP‐ETSC)Cl] were examined for their ability to alter the catalytic activity of topoisomerase IIβ. Both Cu(II) complexes were effective at inhibiting DNA relaxation at around 10–25 μM. Additionally, both Cu(II) complexes increased double‐stranded DNA cleavage peaking around 25–50 μM. When narrowing the focus to just Cu(APY‐ETSC)Cl and Cu(BZP‐ETSC)Cl, it was determined that both compounds interfere with ATP hydrolysis of topoisomerase IIβ, which is required for strand passage. Additional experiments determined that ATP cannot outcompete Cu(APY‐ETSC)Cl or Cu(BZP‐ETSC)Cl for binding to topoisomerase IIβ, which suggests that these compounds bind outside the ATP binding pocket but allosterically impact ATP hydrolysis. Lastly, we examined whether the Cu(II) complexes could stabilize the N‐terminal ATPase domain in a closed conformation similar to ATP. Both compounds stabilize the N‐terminal closed conformation of the ATPase domain, suggesting a mechanism for topoisomerase II inhibition by these compounds. Taken together, these results provide evidence that Cu(II) complexes of α‐(N)‐heterocyclic thiosemicarbazones catalytically inhibit topoisomerase IIβ similar to topoisomerase IIα. Additionally, the mechanism involves binding to the ATPase domain outside of the ATP pocket and inducing a closed N‐terminal gate of topoisomerase II. These results also provide a possible explanation for the increase in topoisomerase II‐mediated DNA cleavage observed in the presence of Cu(II) thiosemicarbazone complexes.Support or Funding InformationJMK and JED were supported by LUCOPHS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.