A QSAR study that compares the ability of bisdioxopiperazine analogs of the doxorubicin cardioprotective agent dexrazoxane (ICRF-187) to protect myocytes with DNA topoisomerase II inhibition.

The cardiotoxicity caused by doxorubicin and extravasation injury caused by anthracyclines is reduced by the clinically approved bisdioxopiperazine drug dexrazoxane. Dexrazoxane is a rings-closed analog of EDTA and is hydrolyzed in vivo to a form that strongly binds iron. Its protective effects were originally thought to be due to the ability of its metabolite to remove iron from the iron-doxorubicin complex, thereby preventing oxygen radical damage to cellular components. More recently it has been suggested that dexrazoxane may exert its protective effects by inhibiting topoisomerase IIß in the heart and inducing a reduction in its protein levels through induction of proteasomal degradation. The ability of dexrazoxane, other bisdioxopiperazines, and mitindomide to protect against doxorubicin-induced damage was determined in primary neonatal rat myocytes. This QSAR study showed that the protection that a series of bisdioxopiperazine analogs of dexrazoxane and the bisimide mitindomide offered against doxorubicin-induced myocyte damage was highly correlated with the ability of these compounds to catalytically inhibit the decatenation activity of topoisomerase II. The structural features of the dexrazoxane analogs that contribute to the binding and inhibition of topoisomerase II have been identified. These results suggest that the inhibition of topoisomerase II in myocytes by dexrazoxane is central to its role in its activity as an anthracycline cardioprotective agent. Additionally, sequence identity analysis of the amino acids surrounding the dexrazoxane binding site showed extremely high identity, not only between both invertebrate topoisomerase II isoforms, but also with yeast topoisomerase II as well.

Mechanisms of the Cardiac Myocyte-Damaging Effects of Dasatinib.

The anticancer drug dasatinib (Sprycel) is a BCR-ABL1-targeted tyrosine kinase inhibitor used in treating chronic myelogenous leukemia that has been shown in clinical trials to display cardiovascular toxicities. While dasatinib potently inhibits BCR-ABL1, it is not a highly selective kinase inhibitor and may have off-target effects. A neonatal rat cardiac myocyte model was used to investigate potential mechanisms by which dasatinib damaged myocytes. The anthracycline cardioprotective drug dexrazoxane was shown to be ineffective in preventing dasatinib-induced myocyte damage. Dasatinib treatment increased doxorubicin accumulation in myocytes and doxorubicin-induced myocyte damage, likely through its ability to bind to one or more ABC-type efflux transporters. Dasatinib induced myocyte damage either after a brief treatment that mimicked the clinical situation, or more potently after continuous treatment. Dasatinib slightly induced apoptosis in myocytes as evidenced by increases in caspase-3/7 activity. Dasatinib treatment reduced pERK levels in myocytes most likely through inhibition of RAF, which dasatinib strongly inhibits. Thus, inhibition of the RAF/MEK/ERK pro-survival pathway in the heart may be, in part, a mechanism by which dasatinib induces cardiovascular toxicity.

The Role of Topoisomerase IIß in the Mechanisms of Action of the Doxorubicin Cardioprotective Agent Dexrazoxane.

Dexrazoxane is clinically used to reduce doxorubicin cardiotoxicity and anthracycline-induced extravasation injury. Dexrazoxane is a strong catalytic inhibitor of topoisomerase II. It can also undergo metabolism to form an iron-binding analog of EDTA. Dexrazoxane was originally thought to act by reducing iron-dependent doxorubicin-based oxidative stress. However, a competing hypothesis posits that dexrazoxane may be protective through its ability to inhibit and reduce topoisomerase IIß protein levels in the heart. A primary neonatal rat myocyte model was used to study the mechanism by which dexrazoxane protects against doxorubicin-induced myocyte damage. This study characterized the kinetics of the rapid and nearly complete dexrazoxane-induced loss of topoisomerase IIß protein from neonatal rat cardiac myocytes. Immunofluorescent staining of attached myocytes for topoisomerase IIß revealed that most of the topoisomerase IIß was localized to the nucleus, although it was also present in the cytoplasm. Dexrazoxane treatment resulted in an almost complete reduction of topoisomerase IIß in the nucleus and a lesser reduction in the cytoplasm. The recovery of topoisomerase IIß levels after a pulse topoisomerase IIß inhibitory concentration of dexrazoxane occurred slowly, with partial recovery only occurring after 24 h. The ability of dexrazoxane to reduce doxorubicin-induced damage to myocytes was greatest when topoisomerase IIß levels were at their lowest.

Evaluation of Nitrobenzyl Derivatives of Camptothecin as Anti-Cancer Agents and Potential Hypoxia Targeting Prodrugs.

As part of our initial efforts into developing a tumor-targeting therapy, C-10 substituted derivatives of a camptothecin analog (SN-38) have been synthesized (2-, 3- and 4-nitrobenzyl) for use as potential hypoxia-activated prodrugs and evaluated for their cytotoxicity, topoisomerase I inhibition and electrochemical (reductive) properties. All three derivatives were found to possess reduced toxicity towards human leukemia K562 cells compared to SN-38, validating a condition for prodrug action. Using an MTS assay, IC50's were found to be 3.0, 25.9, 12.2 and 58.0 nM for SN-38, 2-nitro-, 3-nitro- and 4-nitrobenzyl-C10-substituted-SN-38, respectively, representing an 8-, 4- and 19-fold decrease in cytotoxicity. Using a topoisomerase I assay, one of the analogs (4-nitrobenzyl) was shown to inhibit the ability of this enzyme to relax supercoiled pBR322 DNA, at a similar concentration to the clinically-approved active metabolite SN-38. Cyclic voltammetry detailed the reductive nature of the analogs, and was used to infer the potential of these compounds to serve as hypoxia-targeting prodrugs. The electrochemical results also validated the quasi-reversible nature of the first reduction step, and served as a proof-of-principle that hypoxia-targeting prodrugs of SN-38 can participate in a redox-futile cycle, the proposed mechanism of activation and targeting. Chemical reduction of the 4-nitrobenzyl analog led to the formation/release of SN-38 and validated the prodrug ability of the C-10 substituted derivative.

Myocyte-Damaging Effects and Binding Kinetics of Boronic Acid and Epoxyketone Proteasomal-Targeted Drugs.

The proteasome inhibitors bortezomib, carfilzomib, and ixazomib, which are used in the treatment of multiple myeloma have greatly improved response rates. Several other proteasome inhibitors, including delanzomib and oprozomib, are in clinical trials. Carfilzomib and oprozomib are epoxyketones that form an irreversible bond with the 20S proteasome, whereas bortezomib, ixazomib, and delanzomib are boronic acids that form slowly reversible adducts. Several of the proteasome inhibitors have been shown to exhibit specific cardiac toxicities. A primary neonatal rat myocyte model was used to study the relative myocyte-damaging effects of five proteasome inhibitors with a view to identifying potential class differences and the effect of inhibitor binding kinetics. Bortezomib was shown to induce the most myocyte damage followed by delanzomib, ixazomib, oprozomib, and carfilzomib. The sensitivity of myocytes to proteasome inhibitors, which contain high levels of chymotrypsin-like proteasomal activity, may be due to inhibition of proteasomal-dependent ongoing sarcomeric protein turnover. All inhibitors inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the low nanomolar concentration range and exhibited time-dependent inhibition kinetics characteristic of slow-binding inhibitors. Progress curve analysis of the inhibitor concentration dependence of the slow-binding kinetics was used to measure second-order "on" rate constants for binding. The second-order rate constants varied by 90-fold, with ixazomib reacting the fastest, and oprozomib the slowest. As a group, the boronic acid drugs were more damaging to myocytes than the epoxyketone drugs. Overall, inhibitor-induced myocyte damage was positively, but not significantly, correlated with their second-order rate constants.

Progress curve analysis of the kinetics of slow-binding anticancer drug inhibitors of the 20S proteasome.

Bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib are anticancer drugs that target the proteasomal system. Carfilzomib and oprozomib are epoxyketones that form an irreversible covalent bond with the 20S proteasome, whereas bortezomib, ixazomib, and delanzomib are boronic acids that form slowly reversible adducts. The binding kinetics of some of these drugs have either not been well characterized, or have been studied under a variety of different conditions. Utilizing a fluorogenic substrate the kinetics of the slow-binding inhibition of the chymotrypsin-like proteasomal activity of human 20S proteasome was determined under a standard set of conditions in order to compare the kinetic and equilibrium properties of these drugs. Progress curve analysis was used to obtain second order "on" and first-order "off" rate constants, and equilibrium- and kinetically-determined inhibitor dissociation constants. Oprozomib inhibited the 20S proteasome with a second-order binding "on" rate constant that was 60-fold slower than for ixazomib, the fastest binding drug. Delanzomib dissociated from its complex with the 20S proteasome with a half-time that was more than 20-fold slower than for ixazomib, the fastest dissociating drug. The differences in the binding and the dissociation of these drugs may, in part, explain some of their pharmacological and toxicological properties.

Disulfiram is a slow-binding partial noncompetitive inhibitor of 20S proteasome activity.

The alcohol abuse drug disulfiram has also been shown to exhibit potent cell growth inhibitory and anticancer activity. While a number of cellular and animal studies have suggested that disulfiram exhibits its anticancer activity through interaction with the proteasome, direct evidence for inhibition of proteasome activity is lacking. In this study we show that disulfiram potently inhibits the chymotrypsin-like activity of purified human 20S proteasome at low micromolar pharmacological concentrations. The enzyme progress curves displayed characteristics of a slow-binding reaction, similar to that observed for the FDA-approved proteasomal-targeted anticancer drugs bortezomib and carfilzomib. The apparent second order rate constant for reaction with 20s proteasome that was derived from an analysis of the progress curves was about 250-fold smaller than for bortezomib and carfilzomib. The concentration dependence of the enzyme kinetics was consistent with partial noncompetitive inhibition, whereby the putative disulfiram-proteasome adduct retains, partial but decreased enzyme activity. Disulfiram, which is known to have a high affinity for protein thiols, likely reacted with a non-critical cysteine residue, and not at the proteasome substrate binding site.

Molecular Mechanisms of the Cardiotoxicity of the Proteasomal-Targeted Drugs Bortezomib and Carfilzomib.

Bortezomib and carfilzomib are anticancer drugs that target the proteasome. However, these agents have been shown to exhibit some specific cardiac toxicities by as yet unknown mechanisms. Bortezomib and carfilzomib are also being used clinically in combination with doxorubicin, which is also cardiotoxic. A primary neonatal rat myocyte model was used to study these cardiotoxic mechanisms. Exposure to submicromolar concentrations of bortezomib and carfilzomib resulted in significant myocyte damage and induced apoptosis. Both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the low nanomolar concentration range and exhibited time-dependent inhibition kinetics. The high sensitivity of myocytes, which were determined to contain high specific levels of chymotrypsin-like proteasomal activity, to the damaging effects of bortezomib and carfilzomib was likely due to the inhibition of proteasomal-dependent ongoing sarcomeric protein turnover. A brief preexposure of myocytes to non-toxic nanomolar concentrations of bortezomib or carfilzomib greatly increased doxorubicin-mediated damage, which suggests that the combination of doxorubicin with either bortezomib or carfilzomib may produce more than additive cardiotoxicity. The doxorubicin cardioprotective agent dexrazoxane partially protected myocytes from doxorubicin plus bortezomib or carfilzomib treatment, in spite of the fact that bortezomib and carfilzomib inhibited the dexrazoxane-induced decreases in topoisomerase IIß protein levels in myocytes. These latter results suggest that the doxorubicin cardioprotective effects of dexrazoxane and the doxorubicin-mediated cardiotoxicity were not exclusively due to targeting of topoisomerase IIß.

The Myocyte-Damaging Effects of the BCR-ABL1-Targeted Tyrosine Kinase Inhibitors Increase with Potency and Decrease with Specificity.

Five clinically approved BCR-ABL1-targeted tyrosine kinase inhibitors (bosutinib, dasatinib, imatinib, nilotinib, and ponatinib) used for treating chronic myelogenous leukemia have been studied in a neonatal rat myocyte model for their relative ability to induce myocyte damage. This was done in order to determine if kinase inhibitor-induced myocyte damage was a consequence of inhibiting ABL1 (on-target effects), or due to a lack of kinase selectivity (off-target effects) since previous studies have come up with conflicting conclusions about whether imatinib-induced cardiotoxicity results directly from inhibition of ABL1. The most specific and least potent inhibitors, imatinib and nilotinib, induced the least myocyte damage, while the least specific and most potent inhibitors, ponatinib and dasatinib, induced the most damage. Inhibitor-induced myocyte damage also correlated with clinically observed cardiovascular toxicity. Growth inhibition of the erythroleukemic K562 human cell line with a constitutively active BCR-ABL1 kinase was negatively correlated with inhibitor-induced myocyte damage, which suggests that inhibition of ABL1 causes myocyte damage. Myocyte damage was also negatively correlated with inhibitor dissociation binding constants and with inhibition of enzymatic ABL1 kinase activity. Myocyte damage was also positively correlated with two measures of inhibitor selectivity, which suggests that a lack of inhibitor selectivity is responsible for myocyte damage. In conclusion, myocyte damage, and thus the cardiovascular toxicity of the BCR-ABL1-targeted tyrosine kinase inhibitors, is due to direct inhibition of ABL1 and/or their lack of inhibitor selectivity.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the ß isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIß.

Structure-based design, synthesis and biological testing of piperazine-linked bis-epipodophyllotoxin etoposide analogs.

Drugs that target DNA topoisomerase II, such as the epipodophyllotoxin etoposide, are a clinically important class of anticancer agents. A recently published X-ray structure of a ternary complex of etoposide, cleaved DNA and topoisomerase IIß showed that the two intercalated etoposide molecules in the complex were separated by four DNA base pairs. Thus, using a structure-based design approach, a series of bis-epipodophyllotoxin etoposide analogs with piperazine-containing linkers was designed to simultaneously bind to these two sites. It was hypothesized that two-site binding would produce a more stable cleavage complex, and a more potent anticancer drug. The most potent bis-epipodophyllotoxin, which was 10-fold more growth inhibitory toward human erythroleukemic K562 cells than etoposide, contained a linker with eight methylene groups. All of the mono- and bis-epipodophyllotoxins, in a variety of assays, showed strong evidence that they targeted topoisomerase II. COMPARE analysis of NCI 60-cell GI50 endpoint data was also consistent with these compounds targeting topoisomerase II.

Cellular mechanisms of the cytotoxicity of the anticancer drug elesclomol and its complex with Cu(II).

The potent anticancer drug elesclomol, which forms an extremely strong complex with copper, is currently undergoing clinical trials. However, its mechanism of action is not well understood. Treatment of human erythroleukemic K562 cells with either elesclomol or Cu(II)-elesclomol caused an immediate halt in cell growth which was followed by a loss of cell viability after several hours. Treatment of K562 cells also resulted in induction of apoptosis as measured by annexin V binding. Elesclomol or Cu(II)-elesclomol treatment caused a G1 cell cycle block in synchronized Chinese hamster ovary cells. Elesclomol and Cu(II)-elesclomol induced DNA double strand breaks in K562 cells, suggesting that they may also have exerted their cytotoxicity by damaging DNA. Cu(II)-elesclomol also weakly inhibited DNA topoisomerase I (5.99.1.2) but was not active against DNA topoisomerase IIα (5.99.1.3). Elesclomol or Cu(II)-elesclomol treatment had little effect on the mitochondrial membrane potential of viable K562 cells. NCI COMPARE analysis showed that Cu(II)-elesclomol exerted its cytotoxicity by mechanisms similar to other cytotoxic copper chelating compounds. Experiments with cross-resistant cell lines overexpressing several ATP-binding cassette (ABC) type efflux transporters showed that neither elesclomol nor Cu(II)-elesclomol were cross-resistant to cells overexpressing either ABCB1 (Pgp) or ABCG2 (BCRP), but that cells overexpressing ABCC1 (MRP1) were slightly cross-resistant. In conclusion, these results showed that elesclomol caused a rapid halt in cell growth, induced apoptosis, and may also have inhibited cell growth, in part, through its ability to damage DNA.

Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA.

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI(50) in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.

The cytotoxicity of the anticancer drug elesclomol is due to oxidative stress indirectly mediated through its complex with Cu(II).

Elesclomol is an anticancer drug that is currently undergoing clinical trials. Elesclomol forms a strong 1:1 complex with Cu(II) and may exert its anticancer activity through the induction of oxidative stress and/or its ability to transport copper into the cell. A UV-vis spectrophotometric titration showed that Cu(I) also formed a 1:1 complex with elesclomol. Ascorbic acid, but not glutathione or NADH, potently reduced the Cu(II)-elesclomol complex to produce hydrogen peroxide. Even though hydrogen peroxide mediated reoxidation of the copper(I) produced by ascorbic acid reduction has the potential to lead to hydroxyl radical formation, electron paramagnetic resonance spin trapping experiments, either with or without added hydrogen peroxide, showed that the ascorbic acid-reduced Cu(II)-elesclomol complex could not directly generate damaging hydroxyl radicals. Both Cu(II)-elesclomol and elesclomol potently oxidized dichlorofluorescin in K562 cells. The highly specific copper chelators tetrathiomolybdate and triethylenetetramine were found to greatly reduce the cytotoxicity of both elesclomol and Cu(II)-elesclomol complex towards erythroleukemic K562 cells, consistent with a role for copper in the cytotoxicity of elesclomol. The superoxide dismutating activity of Cu(II)-elesclomol was much lower than that of Cu(II). Depletion of glutathione levels in K562 cells by treatment with buthionine sulfoximine sensitized cells to both elesclomol and Cu(II)-elesclomol. In conclusion, these results showed that elesclomol indirectly inhibited cancer cell growth through Cu(II)-mediated oxidative stress.

Molecular mechanisms of the biological activity of the anticancer drug elesclomol and its complexes with Cu(II), Ni(II) and Pt(II).

The bis(thiohydrazide) amide elesclomol has extremely potent antiproliferative activity and is currently in clinical trials as an anticancer agent. Elesclomol strongly binds copper and may be exerting its cell growth inhibitory effects by generating copper-mediated oxidative stress. Nickel(II) and platinum(II) complexes of elesclomol were synthesized and characterized in order to investigate if these biologically redox inactive metal complexes could also inhibit cell growth. The nickel(II)-elesclomol and platinum(II) elesclomol complexes were 34- and 1040-fold less potent than the copper(II)-elesclomol complex towards human leukemia K562 cells. These results support the conclusion that a redox active metal is required for elesclomol to exert its cell growth inhibitory activity. Copper(II)-elesclomol was also shown to efficiently oxidize ascorbic acid at physiological ascorbic acid concentrations. Reoxidation of the copper(I) thus produced would lead to production of damaging reactive oxygen species. An X-ray crystallographic structure determination of copper(II)-elesclomol showed that it formed a 1:1 neutral complex with a distorted square planar structure. The kinetics and equilibria of the competition reaction of the strong copper(II) chelator TRIEN with copper(II)-elesclomol were studied spectrophotometrically under physiological conditions. These results showed elesclomol bound copper(II) with a conditional stability constant 24-fold larger than TRIEN. A log stability constant of 24.2 was thus indirectly determined for the copper(II)-elesclomol complex.

The dual-targeted HER1/HER2 tyrosine kinase inhibitor lapatinib strongly potentiates the cardiac myocyte-damaging effects of doxorubicin.

The anticancer drug lapatinib (Tykerb) is a dual tyrosine kinase inhibitor targeting the HER2 (ERBB2) and EGFR (ERBB1, HER1) pathways that have been shown in clinical trials to display some cardiotoxicity. Because trastuzumab also targets HER2 receptors, the lapatinib/doxorubicin combination provides a good model to probe the mechanism of the increased cardiotoxicity caused by the concurrent use of trastuzumab and doxorubicin. Using a neonatal rat cardiac myocyte model, we have investigated the ability of lapatinib alone and in combination with doxorubicin to damage myocytes. Lapatinib treatment alone only slightly induced myocyte damage. However, doxorubicin-induced myocyte damage was greatly potentiated by the addition of nanomolar lapatinib concentrations. Lapatinib alone treatment decreased phosphorylated ERK (MAPK), which may have, in part, contributed to the increased myocyte damage. As measured by flow cytometry, lapatinib-treated myocytes displayed an increased accumulation of doxorubicin. As lapatinib is a strong inhibitor of several ATP-dependent ABC-type efflux transporters, this likely occurred because lapatinib blocked doxorubicin efflux, thereby increasing intracellular doxorubicin concentrations and, thus, increasing myocyte damage. These results suggest that the clinical use of concurrent doxorubicin and lapatinib should be approached with care due to the possibility of lapatinib increasing doxorubicin cardiotoxicity.

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II.

Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.

The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα.

The thiosemicarbazones Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and triapine have potent antiproliferative activity and have been evaluated as anticancer agents. While these compounds strongly bind iron and copper, their mechanism(s) of action are incompletely understood. A recent report (Rao et al., Cancer Research 69:948-57, 2009) suggested that Dp44mT may, in part, exert its cytotoxicity through poisoning of DNA topoisomerase IIα. In the present report, a variety of assays were used to determine whether Dp44mT and triapine target topoisomerase IIα. Neither of these compounds inhibited topoisomerase IIα decatenation or induced cleavage of pBR322 DNA in the presence of enzyme. In cells, Dp44mT did not stabilize topoisomerase IIα covalent binding to DNA using an immunoblot band depletion assay, an ICE (immunodetection of complexes of enzyme-to-DNA) assay, and a protein-DNA covalent complex forming assay. Dp44mT did not display cross resistance to etoposide resistant K562 cells containing reduced topoisomerase IIα levels. Synchronized Dp44mT-treated CHO cells did not display a G2/M cell cycle block expected of a topoisomerase II inhibitor. A COMPARE analysis of Dp44mT using the NCI 60-cell line data indicated that inhibition of cell growth was poorly correlated with DNA topoisomerase IIα mRNA levels. In summary, we found no support for the conclusion that Dp44mT inhibits cell growth through the targeting of topoisomerase IIα. Since clinical trials of triapine are underway, it will be important to better understand the intracellular targeting and mechanisms of action of the thiosemicarbazones to support forward development of these agents and newer analogs.

Chemical reactivity and microbicidal action of bethoxazin.

Bethoxazin is a new broad spectrum industrial microbicide with applications in material and coating preservation. However, little is known of its reactivity profile and mechanism of action. In this study, we examined the reactivity of bethoxazin toward biologically important nucleophilic groups using UV-vis spectroscopy and LC-MS/MS techniques and found the molecule to be highly electrophilic. Bethoxazin reacted with molecules containing free sulfhydryl groups such as GSH and human serum albumin to form covalent adducts that were detectable by MS, but did not react with amino, carboxylic, phenolic, amino oxo, alcoholic, and phosphate functional groups. Bethoxazin potently inhibited the catalytic activity of yeast DNA topoisomerase II and the growth of yeast BY4742 cells at low micromolar concentrations. However, the reduced form of bethoxazin and GSH-treated bethoxazin were both inactive in these assays. The experimentally determined relative reactivity of bethoxazin and its reduced form analog correlated with their biological activities as well as their quantum-mechanically calculated electrophilicity properties. Taken together, the results suggest that bethoxazin may exert its microbicidal action by reacting with sensitive endogenous sulfhydryl biomolecules of microbial cells. Consistent with this view, the inhibitory activity of bethoxazin on topoisomerase II may be due to its ability to react with critical free cysteine sulfhydryl groups on the enzyme. Our studies have provided for the first time a better understanding of the reactivity of bethoxazin, as well as some insights into the mechanism by which the compound exerts its microbicidal action.

Design, synthesis, and biological evaluation of a novel series of bisintercalating DNA-binding piperazine-linked bisanthrapyrazole compounds as anticancer agents.

A series of bisintercalating DNA binding bisanthrapyrazole compounds containing piperazine linkers were designed by molecular modeling and docking techniques. Because the anthrapyrazoles are not quinones they are unable to be reductively activated like doxorubicin and other anthracyclines and thus they should not be cardiotoxic. The concentration dependent increase in DNA melting temperature was used to determine the strength of DNA binding and the bisintercalation potential of the compounds. Compounds with more than a three-carbon linker that could span four DNA base pairs achieved bisintercalation. All of the bisanthrapyrazoles inhibited human erythroleukemic K562 cell growth in the low to submicromolar concentration range. They also strongly inhibited the decatenation activity of topoisomerase IIα and the relaxation activity of topoisomerase I. However, as measured by their ability to induce double strand breaks in plasmid DNA, the bisanthrapyrazole compounds did not act as topoisomerase IIα poisons. In conclusion, a novel group of bisanthrapyrazole compounds were designed, synthesized, and biologically evaluated as potential anticancer agents.