Examination of the Effect of Copper (II) α‐(N)‐Heterocyclic Thiosemicarbazone Complexes on DNA Topoisomerase IIβ

Abstract

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.