Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effects of thiosemicarbazones (TSC) and metal–TSC complexes have been demonstrated to target several biological processes, including DNA metabolism. Human topoisomerases were discovered among the molecular targets for TSCs, and metal-chelated TSCs specifically displayed significant inhibition of topoisomerase II. The processes by which metal–TSCs or TSCs inhibit topoisomerases are still being studied. In this brief review, we summarize the TSCs and metal–TSCs that inhibit various types of human topoisomerases, and we note some of the key unanswered questions regarding this interesting class of diverse compounds.

Topoisomerase IIα is a nuclear enzyme needed for dealing with topological entanglements in the DNA arising from replication and transcription. The N-terminal region and core of the protein are utilized in the catalytic cycle of the enzyme, which generates a transient double-stranded break in one segment of DNA and passes another segment through the break. The C-terminal domain is a large, intrinsically disordered region that appears to be involved in regulating the function of the enzyme both in terms of substrate selection and the level of activity of the enzyme. In a previous study, we explored eleven targeted mutations to the C-terminal domain. This present study explores six of these mutants to determine whether there are any defects in closure of the N-terminal clamp and whether an experimental compound known as a Cu(II)-thiosemicarbazone affects DNA cleavage with the mutants. Based upon our results, the mutants are able to close the N-terminal clamp, but some of the mutants that displayed the least clamp closing activity also had the lowest catalytic activity. Further, Cu-APY-ETSC did impact the ability of the enzymes to cleave DNA to similar levels as seen with the WT enzyme. These results lay the groundwork for additional analyses of the C-terminal domain and indicate the C-terminal domain regions tested did not influence the action of Cu-APY-ETSC except at the level of coordination between the two active sites.

Thiosemicarbazones (TSCs) has been shown as noncompetitive inhibitors of human Topoisomerase IIα (TopoIIα). Metal‐TSC complexes demonstrated higher activities than their ligands. Metal‐TSC was shown to bind on TopoIIα near the ATP binding pocket. Thus in the absence of ATP, metal‐TSC complexes can increase TopoIIα‐induced cleavage complexes, unlike other inhibitors of TopoIIα. The metal ions of TSC play a predominant role in the activity of TSCs. Cu‐TSC complexes are the most active ones compared to their Pd and Pt counterpart. In this study, we study the activity of metal‐TSC effects on TopoIIα in the presence and absence of ATP molecule and try to understand the mechanism how metal‐TSC interact with TopoIIα.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Through experimentation, it has been discovered that thiosemicarbazone ligands and their metal complexes can be used for medicinal uses by inhibiting human topoisomerase IIα, as an alternate treatment for chemotherapy. In this research, acetyl-pyrazine methyl thiosemicarbazone [APZ-MTCS] and acetyl-pyrazine ethyl thiosemicarbazone [APZ-ETSC] ligands were synthesized and purified. After the synthesis and purification of [APZ-MTCS] and [APZ-ETSC] ligands, the 1H NMR and 13C NMR spectra were obtained using a new 500 MHz NMR spectrometer. Structural information was obtained by running 2D HSQC (heteronuclear single quantum coherence) 1H-13C NMR and HSQC 1H-15NNMR experiments, which provide evidence of our predicted structures. After the analysis was complete, we reacted the ligands with Pd(II) and two synthesis routes of Pt(II), using dichlorobis (benzonitrile) platinum(II) and potassium tetrachloroplatinate(II), to form their metal complexes. Similar NMR experiments were performed on the Pd(II) and Pt(II) complexes. A topoisomerase assay ran with an inhibition concentration between 2-6micro molar on the metal complexes. This poster will present the synthesis of [APZ-MTSC] and [APZ-ETSC] and the reactions with their metal complexes, the NMR characterization, and discussion of the topoisomerase assay results.

A new series of Isatin Thiosemicarbazone (I-TSC) ligands were synthesized. The ligands were synthesized with the following substituent groups: methyl thiosemicarbazone, ethyl thiosemicarbazone, tert-butyl thiosemicarbazone, phenyl thiosemicarbazone, and benzyl thiosemicarbazone. These ligands were then characterized by NMR spectroscopy in order to verify their structures. The following test were ran on the compounds: 1H, 13C, gradient selected COSY, 1H-13C multiplicity, and 1H-13C HMBC. The compounds were then reacted with CuCl to form metal complex [Cu(I-TSC)Cl]. An inhibition assay study on these Isatin ligands and copper compounds for evidence of inhibition of Topoisomerase II α will be presented.

Topoisomerase IIα (TopoIIα) is an essential enzyme for cell proliferation. Cancer cells are rapidly dividing cells. Thus, TopoIIα is a primary target for many anticancer drugs. Thiosemicarbazones (TSCs) have been known to possess anticancer activity by inhibiting topoisomerase‐II. Recent studies of our lab found the structure‐activity relationship of TSCs and their inhibition on TopoIIα. Our results suggest that the metal ion of TSC metal complexes and the terminal nitrogen side chain play important roles in increasing TopoIIα‐mediated cleavage complexes. The direct interaction between TSCs and TopoIIα remains unknown. It is hypothesized by our lab that the metal ions and the terminal nitrogen side chains interact with TopoIIα directly. This hypothesis is being tested by studying the interaction of 2‐acetylthiazole (ATZ) TSC, 2‐acetyl‐4‐methylthiazole (AMT) TSC, and benzoylpyridine (BZP) TSC metal complexes with TopoIIα by saturation transfer difference nuclear magnetic resonance (STD‐NMR) and diffusion ordered spectroscopy nuclear magnetic resonance (DOSY‐NMR). Recent STD‐NMR experiments suggest that the aliphatic region of 2‐acetylthiazole (ATZ) TSC and benzoylpyridine (BZP) TSC interact with TopoIIα.Support or Funding InformationThis research is supported by the Faculty Research Grant (XJ) and the NSF MRI project (1531870).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Our previous research has shown that alpha-(N)-heterocyclic thiosemicarbazone (TSC) metal complexes inhibit human topoisomerase II alpha (TopoII alpha), while the ligands without metals do not. To find out the structural elements of TSC that are important for inhibiting TopoII alpha, we have synthesized two series of alpha-(N)-heterocyclic TSCs with various substrate ring segments, side chain substitutions, and metal ions, and we have examined their activities in TopoII alpha-mediated plasmid DNA relaxation and cleavage assays. Our goal is to explore the structure-activity relationship of alpha-(N)-heterocyclic TSCs and their effect on TopoII alpha. Our data suggest that, similar to Cu(II)-TSCs, Pd(II)-TSC complexes inhibit plasmid DNA relaxation mediated by TopoII alpha. In TopoII alpha-mediated plasmid DNA cleavage assays, the Cu(II)-TSC complexes induce higher levels of DNA cleavage than their Pd(II) counterparts. The Cu(II)-TSC complexes with methyl, ethyl, and tert-butyl substitutions are slightly more effective than those with benzyl and phenyl groups. The alpha-(N)-heterocyclic ring substrates of the TSCs, including benzoylpyridine, acetylpyridine, and acetylthiazole, do not exhibit a significant difference in TopoII alpha-mediated DNA cleavage. Our data suggest that the metal ion of TSC complexes plays a predominant role in inhibition of TopoII alpha, the side chain substitution of the terminal nitrogen plays a secondary role, while the substrate ring segment has the least effect. Our molecular modeling data support the biochemical data, which together provide a mechanism by which Cu(II)-TSC complexes stabilize TopoII alpha-mediated cleavage complexes.

Two new thiosemicarbazone ligands, 2-propionylthiazole ethylthiosemicarbazone (PTZ-ETSC), and 2-propionylthiazole tert-butylthiosemicarbazone (PTZ-tBTSC), along with their two copper(II) complexes, [Cu(PTZ-ETSC)Cl] and [Cu(PTZ-tBTSC)Cl], are reported here for the first time. Once characterized by NMR and MS, these mono-anionic tridentate ligands were reacted with Cu2+ to form the square planar metal complexes [Cu(PTZ-ETSC)Cl] and [Cu(PTZ-tBTSC)Cl]. The x-ray crystal structure of the [Cu(PTZ-tBTSC)Cl] complex shows that the complex adopts a square planar arrangement around the copper(II) ion, but forms a sulfur-bridged dimer in the solid state. Both of the copper complexes displayed strong inhibition of human topoisomerase IIα at activities between 2-4 μM for [Cu(PTZ-ETSC)Cl], and between 8-10 μM for the [Cu(PTZ-tBTSC)Cl] complex. The EC50 values for the MDA-MB-231 breast cancer cell line were 82.6 μM for (PTZ-ETSC), 17.9 μM for [Cu(PTZ- ETSC)Cl], 97.8 μM for (PTZ-tBTSC), and 1.41 μM for [Cu(PTZ-tBTSC)Cl]. The EC50 values for the MCF7 breast cancer cell lines were 9.36 μM for (PTZ-ETSC), 0.13 μM for [Cu(PTZ-ETSC)Cl], 0.333 μM for (PTZ-tBTSC), and 0.093 μM for [Cu(PTZ-tBTSC)Cl].

The new ligand, 2-acetylpyrazine-tertbutylthiosemicarbazone (APZ-tBTSC), and its Cu(II), Pd(II) and Pt(II) complexes have been synthesized.This ligand coordinates to the metal ions in a tridentate monoanionic fashion forming monometallic complexes with the formula [M(APZ-tBTSC)Cl].The ligand and the three metal complexes [Cu(APZ-tBTSC)Cl], [Pd(APZ-tBTSC)Cl], and [Pt(APZ-tBTSC)Cl] were tested for anti-proliferative biological behavior with a panel of seven microbes, and the copper and palladium complexes were found to be highly active against Gram positive bacteria.The 4 compounds were also tested in human topoisomerase IIα DNA relaxation assays and all three metal complexes had topoisomerase inhibition at a concentration between 4 -6 micro-molar.The 4 compounds were also tested for activity with the HEK293T cell line and also the breast cell cancer line, MDA-MB-231.The most effective compound for activity against the HEK293T cell line was the [Cu(APZ-tBTSC)Cl] complex, and the MDA-MB-231 breast cancer cell line was the [Pt(APZ-tBTSC)Cl] complex.

Thiosemicarbazone ligands and their metal complexes have been seen to inhibit Topoisomerase II-α, a popular target of chemotherapy, as has been described in the literature. The new thiosemicarbazone ligands, pyruvic aldehyde-1-oxime [x]-thiosemicarbazone (PAO-xTSC’s) and their metal complexes with Cu(II) have been synthesized. The ligands were characterized and analyzed by 1H Nuclear Magnetic Resonance spectrometry (NMR), 13C NMR, 1H13C HSQC, and 1H15N HSQC. The compounds were then tested in a Topoisomerase II-α relaxation assay, and this poster will present our findings.

We have previously showed that Cu(II) acetylpyrazine methylthiosemicarbazone chloride ([Cu(APZ‐MTSC)Cl]) inhibits the ATP hydrolysis of human topoisomerase (Topo II) as catalytic inhibitor and share some common features as covalent poisons such as increasing TopoII‐mediate DNA cleavage which can be abolished by the presence of DTT. To further test if acteylpyrazine thiosemicarbazone metal complexes all have the similar characteristics, we synthesized a series of acetylpyrazine thiosemicarbazone metal (copper and palladium) complexes with different side chain substitutions including methyl, ethyl, turt‐butyl, benzyl, phenyl and dimethyl functional groups on the terminal nitrogen. We examined their effect on TopoII‐mediated DNA relaxation and DNA cleavage assays. Our data suggest that all metal acetylpyrazine thiosemicarbazones display similar characteristics as [Cu(APZ‐MTSC)Cl], but metal ions play a very important role in inhibiting TopoII. Support or Funding Information This project is funded by Faculty Development Research Grant from Tennessee Board of Regents and Faculty Research Grant from Tennessee Technological University.

Topoisomerase IIα is known to play a vital role in cell proliferation of all cells, especially cancer cells. It has been a popular target for the production of new drugs to combat cancer. This study looks at the inhibition of Topoisomerase IIα with a variety of thiosemicarbazone, such as ATZ ligands with different metals complexes. We studied three groups of ATZ compounds including ATZ‐ligand without metal, ATZ‐Copper, and ATZ‐Palladium complexes. In each of those groups, six different modifications on the side chain are examined. The compounds were tested using cleavage assays for stimulation of the relative DNA cleavage by Topoisomerase, which is a very important indicator for the potentiality to be an anticancer drug. Compounds that showed promising stimulation were selected and tested further in a dose dependent cleavage assay. Our results show that ATZ‐Copper complexes exhibited greater stimulation on Topoisomerase II. And Cu(ATZ‐ETSC)Cl is twice as effective as Etoposide‐ a leading anticancer drug in targeting Topoisomearse II. Support or Funding Information This project is funded by URECA! Undergraduate research program at Tennessee Tech University

The focus of this research is on the study of a series of copper (II) benzoylpyridine thiosemicarbazone complexes. Of the six benzoylpyridine thiosemicarbazone ligands used in this study, two are reported for the first time; 2-benzoylpyridine tert-butyl thiosemicarbazone (BZP-tBTSC), and 2-benzoylpyridine benzyl thiosemicarbazone (BZP-BzTSC). Once characterized by NMR, melting point, and MS, these mono-anionic tridentate ligands were then reacted with Cu2+ to form the new square planar metal complexes [Cu(BZP-tBTSC)Cl] and [Cu(BZP-BzTSC)Cl]. All of the copper complexes display marked inhibition of human topoisomerase IIα. The [Cu(BZP-tBTSC)Cl] complex shows marked activity against human breast cancer cell lines.

Topoisomerase II (TopoII) regulates DNA topology during DNA metabolism and is active in replicating cells. Thus, it is the target of several FDA approved anticancer drugs such as Etoposide. Current topoisomerase II inhibiting drugs do not specifically target the alpha form of the enzyme, affecting normal cells and causing serious health problems. There has been evidence that α‐(N)‐heterocyclic thiosemicarbazone (TSC) compounds have demonstrated antiproliferation effects on tumor lines. Our experiment investigates the inhibition of specific BZP thiosemicarbazone compounds on TopoIIα and stimulation of the cleavage complex of this enzyme. Metal‐free as well as Cu(II) and Pd(II) complexes of BZP derivatives of TSCs were examined against the full TopoII relaxation and its ability to create double strand breaks. The Cu(II) complexes showed the highest inhibitory activity against the enzyme and highest stimulation of the cleavage complexes in the absence of ATP. Among the Cu(II) complexes of BZP derivatives, Cu(BZP‐tBTSC)Cl exhibited a significantly high increase in the cleavage complex of TopoIIα compared with Etoposide. In addition, DTT abolished the effect of BZP TSCs on TopoIIα The mechanism of inhibition is not clear, but seems to be different from that of Etoposide.Support or Funding InformationResearch supported by Tennessee Technological University through Undergraduate Research and Creative Activity (URECA) grants.

Type II DNA topoisomerases resolve topological knots and tangles in DNA that result from routine cellular processes and are effective targets for anticancer therapeutics. To this end, thiosemicarbazones have been identified as having the ability to kill cancer cells from several cell lines. Literature evidence suggests that at least some thiosemicarbazones have an impact on topoisomerase II activity. However, the mechanism is not as clearly defined. Therefore, we set out to analyze the activity of four α-(N)-heterocyclic thiosemicarbazone compounds against topoisomerase IIα. The ligands, acetylpyridine-ethylthiosemicarbazone (APY-ETSC) and acetylpyrazine-methylthiosemicarbazone (APZ-MTSC), and their copper(II) [Cu(II)] complexes [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] were examined for the ability to impact the catalytic cycle of human topoisomerase IIα. Both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] were more effective at inhibiting DNA relaxation compared with the ligands alone. Further, both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] increased double-stranded DNA cleavage levels without inhibiting topoisomerase IIα-mediated DNA ligation. The Cu(II) complexes inactivate enzyme activity over time suggesting a critical interaction with the enzyme. Additionally, we found that the Cu(II)-thiosemicarbazone complexes do not significantly impact DNA cleavage by the catalytic core of the enzyme. This evidence is supported by the fact that both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl], and to a lesser extent the ligands, inhibit topoisomerase IIα-mediated ATP hydrolysis. Based upon kinetic analysis, the Cu(II) complexes appear to be noncompetitive inhibitors of the ATPase domain of topoisomerase IIα. Taken together, our results provide evidence that Cu(II) complexes of α-(N)-heterocyclic thiosemicarbazones catalytically inhibit the enzyme through the ATPase domain but also promote double-stranded DNA cleavage by the enzyme.

DNA is the blueprint which contains the information necessary for a cell to maintain normal structure and function. This essential function of DNA means the cell must necessarily preserve the integrity of its blueprint. Harmful mutations must be minimalized where they cannot be completely eliminated. One such method for maintaining the integrity of DNA is through the function of human polymerase η, a replication enzyme which gains its utility in its ability to correct thymine dimerization mutations to DNA as a result of exposure to ultraviolet radiation. Mutations in human polymerase η lead to a xeroderma pigmentosum variant (XPV) which is limited in its ability to repair damage done to DNA. This research will include introducing the A264P mutation to the catalytic core of the enzyme through site‐directed mutagenesis, transformation of the resultant DNA into E. coli cells, eventually to overexpress and purify the protein, and run translesion synthesis assays in order to determine how restricted the function of the A264P mutant is when compared to the wild‐type protein.