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.

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.

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.

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.

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.

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.

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.

The cardiotoxicity and myocyte damage caused by small molecule anticancer tyrosine kinase inhibitors is correlated with lack of target specificity.

The use of the new anticancer tyrosine kinase inhibitors (TKI) has revolutionized the treatment of certain cancers. However, the use of some of these results in cardiotoxicity. Large-scale profiling data recently made available for the binding of 7 of the 9 FDA-approved tyrosine kinase inhibitors to a panel of 317 kinases has allowed us to correlate kinase inhibitor binding selectivity scores with TKI-induced damage to neonatal rat cardiac myocytes. The tyrosine kinase selectivity scores, but not the serine-threonine kinase scores, were highly correlated with the myocyte damaging effects of the TKIs. Additionally, we showed that damage to myocytes gave a good rank order correlation with clinical cardiotoxicity. Finally, strength of TKI binding to colony-stimulating factor 1 receptor (CSF1R) was highly correlated with myocyte damage, thus possibly implicating this kinase in contributing to TKI-induced cardiotoxicity.

The lack of target specificity of small molecule anticancer kinase inhibitors is correlated with their ability to damage myocytes in vitro.

Many new targeted small molecule anticancer kinase inhibitors are actively being developed. However, the clinical use of some kinase inhibitors has been shown to result in cardiotoxicity. In most cases the mechanisms by which they exert their cardiotoxicity are not well understood. We have used large scale profiling data on 8 FDA-approved tyrosine kinase inhibitors and 10 other kinase inhibitors to a panel of 317 kinases in order to correlate binding constants and kinase inhibitor binding selectivity scores with kinase inhibitor-induced damage to neonatal rat cardiac myocytes. The 18 kinase inhibitors that were the subject of this study were: canertinib, dasatinib, dovitinib, erlotinib, flavopiridol, gefitinib, imatinib, lapatinib, midostaurin, motesanib, pazopanib, sorafenib, staurosporine, sunitinib, tandutinib, tozasertib, vandetanib and vatalanib. The combined tyrosine kinase and serine-threonine kinase selectivity scores were highly correlated with the myocyte-damaging effects of the kinase inhibitors. This result suggests that myocyte damage was due to a lack of target selectivity to binding of both tyrosine kinases and serine-threonine kinases, and was not due to binding to either group specifically. Finally, the strength of kinase inhibitor binding for 290 kinases was examined for correlations with myocyte damage. Kinase inhibitor binding was significantly correlated with myocyte damage for 12 kinases. Thus, myocyte damage may be multifactorial in nature with the inhibition of a number of kinases involved in producing kinase inhibitor-induced myocyte damage.

A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II.

Etoposide is a widely used anticancer drug that targets topoisomerase II, an essential nuclear enzyme. However, despite the fact that it has been in use and studied for more than 30years the specific site on the enzyme to which it binds is unknown. In order to identify the etoposide binding site(s) on topoisomerase II, a diazirine-based photoaffinity etoposide analog probe has been synthesized and its photoreactivity and biological activities have been characterized. Upon UV irradiation, the diazirine probe rapidly produced a highly reactive carbene species that formed covalent adducts containing stable carbon-based bonds indicating that it should also be able to form stable covalent adducts with amino acid residues on topoisomerase II. The human leukemia K562 cell growth and topoisomerase II inhibitory properties of the diazirine probe suggest that it targets topoisomerase II in a manner similar to etoposide. The diazirine probe was also shown to act as a topoisomerase II poison through its ability to cause topoisomerase IIalpha-mediated double-strand cleavage of DNA. Additionally, the diazirine probe significantly increased protein-DNA covalent complex formation upon photoirradiation of diazirine probe-treated K562 cells, as compared to etoposide-treated cells. This result suggests that the photoactivated probe forms a covalent adduct with topoisomerase IIalpha. In conclusion, the present characterization of the chemical, biochemical, and biological properties of the newly synthesized diazirine-based photoaffinity etoposide analog indicates that use of a proteomics mass spectrometry approach will be a tractable strategy for future identification of the etoposide binding site(s) on topoisomerase II through covalent labeling of amino acid residues.

Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents.

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIalpha-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIalpha decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIalpha covalent complex indicating that they acted as topoisomerase IIalpha poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.

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 the topoisomerase II-inactive bisdioxopiperazine ICRF-161 as a protectant against doxorubicin-induced cardiomyopathy.

Anthracycline-induced cardiomyopathy is a major problem in anti-cancer therapy. The only approved agent for alleviating this serious dose limiting side effect is ICRF-187 (dexrazoxane). The current thinking is that the ring-opened hydrolysis product of this agent, ADR-925, which is formed inside cardiomyocytes, removes iron from its complexes with anthracyclines, hereby reducing the concentration of highly toxic iron-anthracycline complexes that damage cardiomyocytes by semiquinone redox recycling and the production of free radicals. However, the 2 carbon linker ICRF-187 is also is a catalytic inhibitor of topoisomerase II, resulting in the risk of additional myelosuppression in patients receiving ICRF-187 as a cardioprotectant in combination with doxorubicin. The development of a topoisomerase II-inactive iron chelating compound thus appeared attractive. In the present paper we evaluate the topoisomerase II-inactive 3 carbon linker bisdioxopiperazine analog ICRF-161 as a cardioprotectant. We demonstrate that this compound does chelate iron and protects against doxorubicin-induced LDH release from primary rat cardiomyocytes in vitro, similarly to ICRF-187. The compound does not target topoisomerase II in vitro or in cells, it is well tolerated and shows similar exposure to ICRF-187 in rodents, and it does not induce myelosuppression when given at high doses to mice as opposed to ICRF-187. However, when tested in a model of chronic anthracycline-induced cardiomyopathy in spontaneously hypertensive rats, ICRF-161 was not capable of protecting against the cardiotoxic effects of doxorubicin. Modulation of the activity of the beta isoform of the topoisomerase II enzyme by ICRF-187 has recently been proposed as the mechanism behind its cardioprotection. This concept is thus supported by the present study in that iron chelation alone does not appear to be sufficient for protection against anthracycline-induced cardiomyopathy.

The structure-based design, synthesis, and biological evaluation of DNA-binding amide linked bisintercalating bisanthrapyrazole anticancer compounds.

A series of amide-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed using molecular modeling and docking and synthesized in order to develop an anticancer drug that formed a strongly binding bisintercalation complex with DNA. Concentration dependency for the increase in the DNA melting temperature was used to determine the DNA binding strength and whether bisintercalation occurred for the newly synthesized analogs. The ability of the compounds to inhibit the growth of the human erythroleukemic K562 cell line and inhibit the decatenation activity of DNA topoisomerase IIalpha was also measured. Finally, the compounds were evaluated for their ability to act as topoisomerase II poisons by measuring the topoisomerase IIalpha-mediated double strand cleavage of DNA. All of the bisanthrapyrazoles inhibited K562 cell growth and topoisomerase IIalpha in the low micromolar range. Compounds with either two or three methylene linkers formed bisintercalation complexes with DNA and bound as strongly as, or more strongly than, doxorubicin. In conclusion, a novel group of amide-coupled bisintercalating anthrapyrazole compounds were designed, synthesized, and evaluated for their physico-chemical and biologic properties as potential anticancer agents.

A three-dimensional quantitative structure-activity analysis of a new class of bisphenol topoisomerase IIalpha inhibitors.

After the identification of a new lead bisphenol compound that had good topoisomerase IIalpha (EC 5.99.1.3) inhibitory activity, a series of bisphenol analogs were synthesized and tested to identify the structural features that were responsible for their activity. The bisphenols represent a new structural class of topoisomerase II inhibitor that potently inhibited the growth of Chinese hamster ovary and K562 leukemia cells in the low micromolar range. The fact that cell growth inhibition was significantly correlated with topoisomerase IIalpha inhibition suggests that the catalytic inhibition of topoisomerase IIalpha probably contributed to their growth inhibitory activity. Only one of the bisphenols (O3OH) tested significantly induced topoisomerase IIalpha-mediated cleavage of DNA. Most of the bisphenols displayed only low-fold cross-resistance to a K562 subline containing reduced levels of topoisomerase IIalpha Thus, it is likely that most of the bisphenols inhibited cell growth, not by acting as topoisomerase II poisons, but rather by acting as catalytic inhibitors of topoisomerase IIalpha. Three-dimensional quantitative structure-activity analysis (3D-QSAR) was carried out on the bisphenols using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) to determine the structural features responsible for their activity. The CoMSIA analysis of the topoisomerase IIalpha inhibitory activity yielded a statistically significant model upon partial least-squares analyses. The 3D-QSAR CoMSIA analysis showed that polar meta hydrogen bond acceptor substituents on the phenyl rings favored inhibition of topoisomerase IIalpha. For the hydrogen bond donor field, para- and meta-substituted hydroxyl groups favored inhibition. Hydrophobic substituents on the bridge atoms disfavored inhibition.

Mechanisms of myocyte cytotoxicity induced by the multiple receptor tyrosine kinase inhibitor sunitinib.

The anticancer tyrosine kinase inhibitor sunitinib has been shown recently to be cardiotoxic. Using a neonatal rat myocyte model, we investigated various mechanisms that might be responsible for its cardiotoxicity. Sunitinib potently inhibited the enzyme activity of both AMP-activated protein kinase (AMPK) and the ribosomal S6 kinase RSK1 at therapeutically relevant concentrations. Heart tissue with its high energy needs might be particularly sensitive to inhibition of AMPK because of its role as an energy sensor regulating ATP levels. As measured by lactate dehydrogenase release, sunitinib treatment of myocytes caused dose-dependent damage at therapeutic levels. Sunitinib treatment also caused a dose-dependent reduction in myocyte protein levels of the phosphorylated alpha and beta isoforms of the AMPK phosphorylation target acetyl-Coenzyme A carboxylase. However, myocytes were not protected from sunitinib treatment by pretreating them with the AMPK-activating antidiabetic drug metformin. Sunitinib treatment of myocytes also did not affect cellular ATP levels. Together, these last two results do not suggest a major role for inhibition of AMPK in sunitinib-induced myocyte damage. Dexrazoxane, which is a clinically approved doxorubicin cardioprotective agent, also did not protect myocytes from damage, which suggests that sunitinib did not induce oxidative damage. In conclusion, even though sunitinib potently inhibits AMPK and RSK1, given the extreme lack of kinase selectivity that sunitinib exhibits, it is likely that inhibition of other kinases or combinations of kinases are responsible for the cardiotoxic effects of sunitinib.

The dihydroorotase inhibitor 5-aminoorotic acid inhibits the metabolism in the rat of the cardioprotective drug dexrazoxane and its one-ring open metabolites.

Dexrazoxane (ICRF-187) is clinically used as a doxorubicin cardioprotective agent and to prevent anthracycline extravasation injury. It may act by preventing iron-based oxygen free radical damage through the iron-chelating ability of its metabolite N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[(N-(2-amino-2-oxoethyl)]glycine (ADR-925). Dexrazoxane undergoes an initial metabolism to its two one-ring open intermediates [N-(2-amino-2-oxoethyl)-N-[(1S)-2-(3,5-dioxo-1-piperazinyl)-1-methylethyl]glycine (B) and N-(2-amino-2-oxoethyl)-N-[(2S)-2-(3,5-dioxo-1-piperazinyl)propyl]glycine (C)] and is then further metabolized to its presumably active metal-chelating form ADR-925. We previously showed that the first ring opening reaction is catalyzed by dihydropyrimidinase and the second by dihydroorotase (DHOase), but not vice versa. To determine whether DHOase was important in the metabolism of dexrazoxane, its metabolism and that of B and C to ADR-925 were measured in rats that were pretreated with the DHOase inhibitor 5-aminoorotic acid. In rats pretreated with 5-aminoorotic acid the area-under-the-curve concentration of ADR-925 was reduced 5.3-fold. In rats treated with a mixture of B and C, the maximum concentration of ADR-925 in the plasma was significantly decreased in rats pretreated with 5-aminoorotic acid, which indicates that DHOase directly metabolized B and C. Both heart and liver tissue levels of ADR-925 in rats were also greatly reduced by pretreatment with 5-aminoorotic acid. Together these results indicate that the metabolism of dexrazoxane and of B and C is mediated by DHOase. These results provide a mechanistic basis for the antioxidant cardioprotective activity of dexrazoxane.