1. Introduction

2. Thiosemicarbazones as Inhibitors of Topoisomerases

2.1. TSC Ligand or Bis(TSC) Ligand Inhibition of Topoisomerases

TSCs have been studied for decades for antiviral, antifungal and antiproliferation activity. In recent years, Triapine (3-AP, 3-aminopyridine-2carboxaldehyde thiosemicarbazone), a member of TSC family, has been studied in clinical trial phase I and II [14,15,16,17]. The major molecular target of Triapine was identified as ribonucleotide reductase [17,18]. Triapine did show some inhibition of Top2A but only in the presence of Cu(II) [19]. Previous work on Triapine did not show any inhibition of relaxation or poisoning of plasmid DNA cleavage with Top2α. [20]

In a series of TSC compounds, Top2 was discovered as one of the molecular targets for TSC24 [21]. The pattern of TSC antiproliferative effects against the human cancer cell line (NCI-60) is similar to those found with recognized anticancer treatments [21]. These investigations revealed TSC24’s profile is comparable to known Top2 agents. TSC24 was further investigated and found that it hindered DNA relaxing and decatenation by inhibiting the Top2α ATPase domain [21].

Another TSC ligand that has been well studied was Dp44mT. In relaxation and cleavage experiments, Dp44mT was shown to preferentially target Top2α, with minimal effect on Top2β and little inhibition of Top1 [22]. But this result is controversial as an additional study found that the ligand Dp44mT did not inhibit Top2α or increase cellular cleavage complexes [20]. A more recent study on the ligand Dp44mT also found little effect of the compound alone, but they show inhibition when combined with Cu(II) [19]. It is unclear whether it was the presence of Cu(II) alone or if the Cu(II) formed a complex with the ligand leading to the effect in this case. As is discussed below, it is possible that the ligand forms alone show little activity, while metal-chelated forms have varying levels of activity depending on the metal ion.

Figure 1. Structures of selected TSC compounds.

Figure 1. Structures of selected TSC compounds.

Computational docking and surface plasmon resonance studies support the ability of TSC24 to bind near the ATP binding pocket, but it is unclear if this is generalizable to other TSCs and whether this has been biochemically validated. Both TSC24 and Dp44mT appear to act as catalytic inhibitors, and there is an increase in DNA cleavage seen with Dp44mT [21,22]. TSC24 does not appear to increase strand breaks in cells, nor block the effects of VP16 [21].

A series of thiosemicarbazones and 4-thiazolindinones have been synthesized and some were identified with activity against Top2. The thiosemicarbazone (E)-2-(1H-indol-3ylmethylene)-N-(naphthalen-1-yl) hydrazinecarbothioamide (compound 2b) was shown to disrupt cell growth of HT29 cells and appear to weakly inhibit plasmid DNA relaxation by Top2α [23]. Compound 3a [(Z)-2-(Acridin-9-ylmethylene)-N-phenyl-hydrazinecarbothioamide)] and (compound 3h) [(Z)-2-(Acridin-9-ylmethylene)-N-(naphtalen-1-yl) hydrazine-carbothioamide] also had limited ability to inhibit relaxation [24]. Detailed studies on the structure-activity relationship of TSC against Top2α showed that most of TSC ligands inhibit Top2α very weakly or not at all [25,26,27,28].

Despite the fact that TSC ligands were initially found to target Top2, the majority of the ligands produced had little or no effect on Top1 [29] or Top2 [26,27,28,30,31]. Metal-TSCs, on the other hand, showed profound inhibition on Top, as discussed below.

Table 1. TSC ligand inhibition on Tops.

Table 1. TSC ligand inhibition on Tops.

Name	Inhibition of Top	Reference
TSC24	Inhibit Top2α DNA relaxation at 25 µM Inhibit Top2 at decatenation at 100 µM	[21]
Dp44mT	Inhibit human Top2α in relaxation assay with 5’ labeled 161-bp fragment from pBluescript SK phagemid DNA Do not inhibit human Top2β or human TopoI	[22]
Dp44mT	Do not inhibit Top2α in decatenation Do not increase cleavage complex by human Top2α	[20]
Triapine
Compound 2b	Inhibit human Top2α weakly at 100 µM	[23]
Compound 3a	Inhibit human Top2α-mediated DNA relaxation at 100 µM	[24]
Compound 3h
Triapine	Do not inhibit Top2α-mediated DNA relaxation at 50 µM	[19]
Dp44mT
Compound 24	Do not inhibit isolated Top2 from L1210	[12]
Compound 36
NQTS	Do not inhibit Top2α	[30]
HFp4mT, HFp4pyrrT	Inhibit Top2α at 100 µM	[31]
HFp4eT, HFp4ipT, HFp4alT, HAp4mT, HAp4-eT, HFp4bzT, HFpT, HAp4alT, and HApz4mT.	Do not inhibit Top2α at 100 µM	[31]
BZP-TSC ligands series	Inhibits Top2α slightly at 50 µM.	[14]
ATZ ligand series	Do not inhibit Top2α at 10µM, do not increase cleavage complex	[27]
BZP ligands series
APY	Do not inhibit Top2α at 100 µM	[26]
APY	Do not inhibit Top2β at 200 µM	[28]
APZ	Inhibit Top2 α at 100µM	[26]
BZP	Inhibit Top2β at 200µM weakly	[28]
HPyCT4BrPh	Do not inhibit human Top1B at 50 µM	[29]

2.2. Metal-TSC or Metal-Bis(TSC) Inhibition of Topoisomerases

TSCs can chelate with various metal ions. Metal-TSC complexes have been produced from copper (Cu), nickel (Ni), palladium (Pd), ruthenium (Ru), tin (Sn), gallium (Ga), gold (Au), and cobalt (Co) [25,30,32,33,34,35,36]. Metal-TSCs inhibit Top more strongly than their TSC ligand [19,25,26,27,28,31]. As will be discussed more below, metal-TSCs appear to be active against both Type I and Type II Top.

2.2.1. Inhibition of Type I Top

dHuman Top1B belongs to the Type I Top family. It relaxes the DNA supercoil during DNA replication, recombination and transcription by cutting one strand of DNA [1,2]. Top1B inhibitors are a class of compounds that target the enzyme and preventing from relaxing DNA and leading to the accumulation of DNA damages. These inhibitors have potential as antitumor agents since tumor cells are under fast proliferation and DNA replication, making them more susceptible to DNA damage. Several classes of Top1B inhibitors have been developed, including camptothecin analogs and indolocarbazoles [1,2,37]. There are a couple of metal-TSC compounds that have been studied against Top1B activity.

Table 2. Metal-TSC inhibition of TopI.

Table 2. Metal-TSC inhibition of TopI.

Name	Inhibition of TopI	Reference
Cu(PyCT4BrPh)Cl	Inhibit TopI The inhibition is severe when pre-incubation of the compound to TopI Inhibited the cleavage step and partially inhibited religation.	[29]
Pd-pyrene-TSC	Inhibited human Top1B at 12.5 µM	[38]
Ga(III)-TSC complex (C4)	Inhibited TopI	[36]
Au(III)(TSC)Cl (complex 1)	Inhibited human Top1B activity starting at 1.5 μM Pre-incubation of Top1B with Complex 1 increased the inhibition	[25]
Ni-bis(TSC)	No inhibition of E. coli TopI	[39]
Nine copper complexes	Inhibited TopI	[40]
Cobalt (III)-TSC (Complex 4)	Inhibited TopI-induced DNA relaxation	[35]

Cu(PyCT4BrPh)Cl [Cu(3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-one-thiosemi-carbazone)Cl] was studied against human Top1B [29]. It inhibited Topo1B by partially blocking ligation of the cleaved DNA [29]. The complex also reduced enzyme-DNA binding according to an EMSA assay [29]. But additional studies would be needed to clarify the exact mechanism.

In another study, pyrene TSCs were complexed with Pd (Complex 1) and examined for inhibition of human Top1B [38]. Pd-pyrene-TSC complexes inhibited relaxation of supercoiled plasmid by human Top1B at 12.5 µM [38]. Additionally, the Pd pyrene-TSC complex displayed the ability to inhibit ligation of cleaved DNA with Top1B, similar to Cu(PyCT4BrPh)Cl [38].

One group reported the use of a Ga(III)-TSC complex, [N,N-diethyl-2-[1-(2-pyridinyl) ethylidene]hydrazinecarbothioamide-N,N,S-gallium(III)]bis(chloride), referred to as C4 in the study [36]. Based upon their results, human Top1B cleavage activity was inhibited by the Ga(III)-TSC complex while the ligand alone did not show significant inhibition [36].

The Au(III) TSC complex [(3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-enone thiosemicarbazonato)chlorogold(III)] chloride, [Au(PyCT4BrPh)Cl]Cl, was studied with human Top1B and found to inhibit relaxation at 1.5 μM [25]. In contrast, HAuCl₄·3H₂O did not inhibit until 200 µM. Pre-incubation of Top1B with this compound increased the inhibition, which suggests Gold(III)-TSC binds and inhibits the activity of Topo1B [25].

In another study, Ni chelated with testosterone TSC to form a distorted square planar with ligand as a bidentate NS donor---Ni-bisTSC [39]. Ni-bis(TSC) did not inhibit E. coli TopI, but it showed DNA binding affinity similar to ethidium bromide [39].

In summary, the research on metal-TSCs inhibiting Top1B is limited. Some compounds displayed catalytic inhibition, such as [Au(PyCT4BrPh)Cl]Cl, others are interfacial poisons by inhibiting ligation, including Cu(PyCT4BrPh)Cl, Pd-Pyrene-TSC, and Ga(III)-TSC.

2.2.2. Inhibition of Type II Top

Type II Top is the primary target for studies of TSCs antitumor activity. Multiple metal-TSC complexes showed higher inhibition compared with their ligand counterpart. Cu-TSCs are the most studied and have demonstrated the highest inhibition of Top2.

• Ni chelated with bis(TSC)

Ni-TSCs were discovered to block a variety of metabolic pathways, including purine synthesis, DNA polymerase, PRPP-amino transferase, IMP dehydrogenase, dihydrofolate reductase, TMP-kinase, and thymidylate synthetase activities, against the L1210 cell line in 1997 [32]. Despite the fact that Ni-TSCs demonstrated several cellular pathways for inhibition, research suggests that Ni-TSCs do not efficiently inhibit Top enzymes [13,32]. Ni(II) coordinates with two TSC ligands, Ni-bis(TSC), which lack the essential square planar structure for Topo inhibition [26,32].

There are some controversies on the inhibition of Ni-TSCs against Top2. Ni-NQTS showed inhibition of Top2α-mediated DNA relaxation assay using a TopoGEN kit [30]. However, the data are inconsistent with other reported results. For example, a yeast screen did not show Ni-bisTSC interferes with Top [13]. Our unpublished results showed that when bisTSC chelate with metal ions, the metal-bisTSC compounds do not inhibit Top2α (Beckett and Jiang, unpublished). Ni-NQTS were also tested in DNA cleavage assays with Top2α [30]. The results seem to show that Ni-NQTS does not stabilize double-stranded DNA cleavage, but there was a low amount of nicking observed, though it was not quantified [30]. In another study, several Ni-TSC complexes were examined alongside Cu analogs discussed below [41]. Interestingly, Ni(L1)(HL1)Cl, Ni(HL2)₂Cl₂, Ni(L3)₂, Ni(L4)₂, and Ni(L5)₂Cl₂ did not appreciably inhibit Top2α from TopoGEN [41]. Although Ni-TSCs performed profound inhibition against cell proliferation, Top2 may not be the target (or the primary target) for Ni-TSCs [8,9,10].

2.

Cu chelated TSCs

In cell toxicity studies, copper (Cu²⁺) chelated TSCs are one of the most active groups of metal-TSCs [8,10]. When Copper chelates with TSC, it forms a square planar structure with Cu in the middle, which seems to be the crucial structure element for Top2 inhibition [26,27,28], Cu(TSC)s demonstrated greater inhibition compared with their ligands. For example, Cu(TSC)Cl (Compound 1 and 2) inhibited Top2 while the corresponding TSC ligands (Compound 24 and 36) did not [12]. In general, Cu(TSC) complexes act on Top2 as catalytic inhibitors through inhibiting the ATPase function and inhibiting relaxation.

Another study showed that Cu(TSC)Cl complexes (Compounds 1-3) reduced DNA cleavage observed in the presence of etoposide, and these compounds alone did not show any stabilization of cleavage complexes [42]. Cu-NQTS inhibited Top2α-mediated DNA relaxation assays [30].

Cu(Fp4alT)Cl completely inhibits Top2α without promoting the formation of linear DNA products [31]. Similar results were observed with the other Cu(TSC)Cl complexes in the study [31]. Thus, Cu(Fp4alT)Cl and its family of Cu(TSC)Cl complexes are catalytic inhibitors of Top2α rather than poisons of the enzyme [31]. Cu(L1)Cl, Cu(L2)Cl, Cu(L3)Cl, Cu(L4)Cl, and Cu(L5)Cl₂ all showed inhibition of Top2α from TopoGEN [41]. Cu(TSC) cation (Complex 1) increase DNA cleavage complex and inhibited DNA relaxation [43].

In another study, the complexes [Cu(S,R)-L] and [Cu(R,S)-L] showed inhibition of Top2α relaxation at 300 µM [44]. However, the concentration of inhibition is similar to ligand TSC and much higher (10-100+-fold) than other Cu(TSC)Cl [44].

Our collaboration works on a series of Cu(TSC)Cl complexes that demonstrated their inhibition of both human Top2α and Top2β [26,27,28]. The structure-activity relationship of metal-TSC showed that Cu(II) played a predominant role in inhibition of Top2 [26,27,28]. The mechanism of Cu(TSC)Cl inhibition on Top2 is complicated. Cu(TSC)Cl inhibited ATP hydrolysis and plasmid DNA relaxation by Top2α and Top2β, which is consistent with these compounds acting as catalytic inhibitors. However, unlike other catalytic inhibitors, Cu(TSC)Cl complexes stabilize the DNA cleavage complexes and increase levels of DNA cleavage, which is the characteristic of interfacial poisons [26,27,28]. In addition, the complexes we tested lead to higher levels of double-stranded breaks implying an increase in coordination between the two active sites [26]. The increase in DNA cleavage was not seen in a mutant lacking the ATPase domain [26]. Further, incubation of Cu(TSC)Cl complexes with Top2α or Top2β prior to DNA leads to a progressive inactivation of the enzyme [26,28]. Consistent with this data is the observation that the Cu(TSC)Cl stabilize a closed N-terminal region (ATPase domain) of Top2α or Top2β [28]. The significance of this particular aspect is that the ATPase domains of each half of the homodimer close around DNA in the presence of ATP. Our results demonstrate that the Cu(TSC)Cl complexes that were studied were able to induce closure of this N-terminal gate in a way that stabilized the gate similar to what is seen with a non-hydrolyzable ATP analog (AMP-PNP) [28].

Although Top2α has been widely used as the molecular target to study Cu(TSC)Cl inhibition, our research found that Cu(TSC)Cl complexes inhibited ATPase and relaxation activity of both Top2α and Top2β [28]. Taken together, the data support the idea that these Cu(TSC)Cl complexes act on or near the ATPase domain, which is highly similar between both isoforms. Using N-terminally and C-terminally truncated versions of Top2α or Top2β both resulted in a lack of increased DNA cleavage [26,28]. Interestingly, some Cu(TSC)Cl showed inhibition both of Top1 and Top2, as will be discussed below [40].

Other metal-TSC also showed inhibition of Top2. Pd-NQTS inhibited Top2α-mediated DNA relaxation assay [30]. When chelated with the same ligand, Pd(TSC)Cl seemed to be less active compared with its Copper counterpart [27]. Ru(TSC)Cl {[(η-6-p-cymene)Ru(EtATSC)Cl]+ cation}, with a big substrate ring structure inhibited human Top2α in relaxation assay [33]. Ruthenium complex of TSC has been tested in a Top2α-mediated DNA relaxation assay and found to inhibit relaxation [33]. Sn(II) chelated TSC complex (C5) inhibited Top2 at 20 µM [34].

Table 3. Metal-TSC inhibition of Top2.

Table 3. Metal-TSC inhibition of Top2.

Name	Inhibition of Top2	Reference
Nine compounds and their copper complexes	Inhibited human Top2α	[40]
Cobalt (III) chelated with TSC ligands. Complex 4	Inhibited human Topo2α-induced DNA relaxation	[35]
Ni-bis(TSC) complex 1	Did not inhibit isolated Top2 from L1210 cells at 100 µM	[32]
Cu-TSCs (Compound 1 and 2)	Inhibited isolated Top2 from L1210 cells	[12]
Copper TSC	Inhibited Top2 of L1210 cells with IC50 value of 6.25-12.2 µM. antagonize the DNA break affect by etoposide.	[42]
Ni-NQTS	Inhibited DNA relaxation	[30]
Cu-NQTS	Inhibited DNA relaxation (TOPOGEN kit)	[30]
Pd-NQTS
Cu(Fp4alT)Cl and its family of Cu(TSC)Cl	Inhibited relaxation by Top2 at 10 µM Completely inhibits Top2α without promoting the formation of linear DNA products	[31]
Ni-bis(TSC)	No effect in stabilizing DNA breaks	[45]
Cu(TSC)	Stabilizes DNA breaks	[45]
Ni(L1)(H1)Cl, Ni(HL2)2Cl2, Ni(L3)2,Ni(L4)2 Ni(L5)2Cl2	No inhibition of Top2 (TopoGEN)	[41]
Cu(L1)Cl, Cu(L2)Cl, Cu(L3)Cl, Cu(L4)Cl, Cu(L5)Cl2	Inhibited Top2 (TopoGEN)	[41]
complex 1 (CuTSC cation)	inhibited DNA relaxation and increase DNA cleavage	[43]
Sn(II)-TSC—(C5)	Inhibited Topo2α at 20 µM.	[34]
Cu(S,R)L and Cu(R,S)L	Inhibited Top2α relaxation at 300 µM	[44]
Cu(APY)Cl]	Inhibited Top2α from 0.5 µM, Increased DNA cleavage Inhibited Top2α ATP hydrolysis No inhibition of ligation by Top2α Pre-incubate compounds with Top2α inactivate the enzyme	[26]
Cu(APZ)Cl]
[Cu(APY)Cl]	Inhibited Top2β at 5 µM, Increased DNA cleavage by Top2β, Inhibited Top2β ATP hydrolysis, Inhibited ligation by Top2β Pre-incubate compounds with Top2β inactivate the enzyme Stabilized closure of N-terminal Top2α and Top2β clamp	[28]
[Cu(BZP)Cl]
Cu(BZP)Cl series	Inhibited Top2α relaxation and increased DNA cleavage	[27]
Cu(ATZ)Cl series
Pd(BZP)Cl series	Inhibited Top2α relaxation and increased DNA cleavage	[27]

2.2.3. Inhibition of Type I and Type II Top

A few studies have compared the inhibition on Top1B and Top2α. Nine compounds and their copper complex were investigated against human Top1B and Top2α from TopoGEN [40]. Relaxation assays were quantified to generate an IC₅₀. The Cu-TSC complexes were at least 10-fold more effective than the ligand alone [40]. They displayed greater inhibition of Top2α than Top1B [40]. Interestingly, the larger side chain substitutions generally displayed better inhibition of Top2α [40].

Complex 4 of Co(III)-TSC complexes inhibited Top1B-induced and Top2α-induced DNA relaxation, but the ligand nor its precursor were not able to inhibit either enzyme [35]. Complex 4 did not cause significant increase in DNA complexes with Top1B or Top2α, which suggests that Complex 4 is a catalytic inhibitor not a poison [35].

3. Discussion

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