DNA topoisomerases: a new twist for antiparasitic chemotherapy?

The parasitic protozoa are notorious for their bizarre cellular structures and metabolic pathways, a characteristic also true for their nucleic acids. Despite these florid differences from mammalian cells, however, it has proven surprisingly difficult to devise novel chemotherapy against these pathogens. In recent years, the DNA topoisomerases from parasites have been the focus of considerable study, not only because they are intrinsically interesting, but also because they may provide a target for much-needed new antiparasitic chemotherapy.

Effects of camptothecin, a topoisomerase I inhibitor, on Plasmodium falciparum.

Currently, the treatment of falciparum malaria is seriously compromised by spreading drug resistance. We studied the effects of camptothecin, a potent and specific topoisomerase I inhibitor, on erythrocytic malaria parasites in vitro. In Plasmodium falciparum, camptothecin trapped protein-DNA complexes, inhibited nucleic acid biosynthesis, and was cytotoxic. These results provide proof for the concept that topoisomerase I is a vulnerable target for new antimalarial drug development.

Topoisomerases in kinetoplastids.

Topoisomerases are enzymes that mediate topological changes in DNA that are essential for nucleic acid biosynthesis and for cell survival. The kinetoplastid protozoa, which include pathogenic trypanosomes and Leishmania, have yielded an interesting variety of purified topoisomerase activities as well as several topoisomerase genes. In these parasites, topoisomerases are involved in the metabolism of both nuclear and mitochondrial (kinetoplast) DNA. In this review, Christian Burri, Armette Bodley and Theresa Shapiro summarize what is known about topoisomerases in kinetoplastids, and consider the intriguing possibility that these enzymes may act as valuable antiparasite drug targets.

Drug cytotoxicity assay for African trypanosomes and Leishmania species.

The trypanosomes and Leishmania species are parasitic protozoa that afflict millions of people throughout the world. If not treated, African trypanosomiasis and visceral leishmaniasis are fatal. The available drugs are severely limited by toxicity, marginal efficacy, the requirement for parenteral administration, and spreading drug resistance. In this study, a spectrophotometric assay was developed and validated for measuring the cytotoxicity of test compounds against axenically cultured bloodstream-form Trypanosoma brucei (African trypanosomes) and promastigotes of Leishmania donovani. Enzymatic hydrolysis of p-nitrophenyl phosphate, monitored by a microtiter plate reader, is a reliable surrogate for parasite cell counts. The assay is simple, inexpensive, and highly reproducible. The coefficient of variation for EC50 values is < 10% for determinations obtained over several months. This method permits the rapid screening of candidates for much-needed new drugs against these parasites.

Antitrypanosomal activity of camptothecin analogs. Structure-activity correlations.

African trypanosomes (Trypanosoma brucei species) are parasitic protozoa that cause lethal diseases in humans and cattle. Previous studies showed that camptothecin, a potent and specific inhibitor of DNA topoisomerase I, is cytotoxic to African trypanosomes and related pathogenic hemoflagellates (Bodley AL and Shapiro TA, Proc Natl Acad Sci USA 92: 3726-3730, 1995). In this study, a series of camptothecin analogs was tested against axenically cultured, bloodstream form, T. brucei. Modifications to the pentacyclic nucleus of camptothecin ablated antiparasitic activity. In contrast, activity could be increased by substituents added to the parent ring system (e.g. 10,11-methylenedioxy or ethylenedioxy groups; alkyl additions to carbon 7; or 9-amino or 9-chloro substituents). Cytotoxicity was correlated with the level of cleavable complexes in trypanosomes, implicating topoisomerase I as the intracellular target for these compounds. To obtain some indication of selective toxicity, ten compounds were also tested against L1210 mouse leukemia cells. The 9-substituted-10,11-methylenedioxy analogs caused a disproportionate increase in antiparasitic activity, compared with mammalian cell toxicity. These findings provide a basis for designing further structural modifications and for selecting camptothecin analogs to test in animal models of trypanosomiasis.

Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania.

Parasites pose a threat to the health and lives of many millions of human beings. Among the pathogenic protozoa, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani are hemoflagellates that cause particularly serious diseases (sleeping sickness, Chagas disease, and leishmaniasis, respectively). The drugs currently available to treat these infections are limited by marginal efficacy, severe toxicity, and spreading drug resistance. Camptothecin is an established antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I. When trypanosomes or leishmania are treated with camptothecin and then lysed with SDS, both nuclear and mitochondrial DNA are cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin also inhibits the incorporation of [3H]thymidine in these parasites. These molecular effects are cytotoxic to cells in vitro, with EC50 values for T. brucei, T. cruzi, and L. donovani, of 1.5, 1.6, and 3.2 microM, respectively. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity.

Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide).

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.

A new mammalian DNA topoisomerase I poison Hoechst 33342: cytotoxicity and drug resistance in human cell cultures.

Hoechst dye 33342 (Ho33342), like many other DNA minor groove binding ligands and its parent compound Hoechst dye 33258 (Ho33258), nonspecifically inhibits the catalytic activities of many DNA enzymes. However, both Ho33258 and Ho33342 also specifically interrupt the breakage/reunion reaction of mammalian DNA topoisomerase I by trapping reversible topoisomerase I cleavable complexes. The enhanced membrane permeability of Ho33342 over its parent compound Ho33258 has allowed studies of the cellular action of Ho33342. Our results suggest that Ho33342 also traps topoisomerase I but not topoisomerase II into reversible cleavable complexes in human KB cells. Although Ho33342 shares a similar mechanism of action with camptothecin, a prototypic topoisomerase I poison, in trapping topoisomerase I cleavable complexes, Ho33342 differs from camptothecin in its effect on drug-resistant cells. Different from camptothecin, Ho33342 was shown to be about 200-fold less cytotoxic in MDR1-overexpressing human KB V1 cells relative to parental human KB 3-1 cells. Ho33342 is only 5-fold less cytotoxic for camptothecin-resistant CPT-K5 cells, which expresses a highly camptothecin-resistant from of topoisomerase I, than for the wild type human lymphoblast RPMI 8402 cells. Our studies suggest a potential use of Hoechst 33342 as a new topoisomerase I poison in antitumor chemotherapy.

DNA topoisomerase II-mediated interaction of doxorubicin and daunorubicin congeners with DNA.

Three groups of doxorubicin and daunorubicin analogues, differing by their substituents on the chromophore and sugar moieties, were used in this study. The 3'-N-unsubstituted (Group 1), 3'-N-acyl (Group 2), and 3'-N-alkyl (Group 3) analogues were tested for: (a) in vivo antitumor activity and in vitro cytotoxicity; (b) cellular or tissue uptake and metabolic conversion; (c) strength of DNA intercalation; and (d) interaction with DNA topoisomerase II (topo-II). Compounds of Group 1 were cytotoxic, were strongly intercalative, and, except for those with C-14 side chain substitution, induced the formation of topo-II-DNA cleavable complexes. As shown previously, esterolysis of C-14-acyl substituents was required to yield a metabolite which can interact with topo-II in the purified system. The C-14-substituted compounds of Group 2 and their C-14-unsubstituted metabolites were cytotoxic. These drugs were weak intercalators, and the C-14-unsubstituted cogeners induced cleavable complex formation in the purified system, but with reduced potency relative to doxorubicin. The type of the 3'-N-position substituent determined whether Group 3 analogues were cytotoxic and strong intercalators, or less active and nonintercalating. Although C-14-unsubstituted intercalators of Group 3 did not form cleavable complexes in the purified system, they were cytotoxic. The study shows that DNA intercalation is required but not sufficient for the activity by topo-II-targeted anthracyclines. In addition to the planar chromophore which is involved in intercalation, two other domains of the anthracycline molecule are important for the interaction with topo-II: (a) substitution of the C-14 position totally inhibits drug activity in the purified system, but enhances cytotoxicity by aiding drug uptake and presumably acting on other cellular targets; and (b) substitutions on the 3'-N position of the sugar ring can, depending on the nature of the substituent, inhibit intercalation and/or topo-II-targeting activity. These findings may provide guidance for the synthesis and development of new active analogues.

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum.

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.

Regulation of DNA topoisomerases during cellular differentiation.

DNA topoisomerase II has been shown to be a nuclear marker for cell proliferation and a therapeutic target in cancer chemotherapy. In order to study the regulation of DNA topoisomerases during cellular differentiation, MELC were induced to differentiate by treatment with 5 mM HMBA. At day five, approximately 95% of MELC had reproducibly undergone differentiation. The level of topoisomerase II, as measured by immunoblotting with anti-topoisomerase II antisera, showed a parallel decrease to approximately 5-10% of the control level by day five. Activity measurements showed a similar decrease during the time course of MELC differentiation. Immunofluorescence studies at day five showed that only about 5% of the MELC had strong nuclear immunofluorescence. These results indicate that the level and activity of DNA topoisomerase II are significantly lower in differentiated versus undifferentiated cells. We also observed that the level and activity of topoisomerase II dropped twofold as cells grew to high cell densities in the absence of HMBA. In contrast, the topoisomerase I levels in MELC remained relatively constant throughout growth and differentiation.

Involvement of intracellular ATP in cytotoxicity of topoisomerase II-targetting antitumor drugs.

The effect of the ATP pool on the cytotoxic action of teniposide (VM-26) has been studied in mouse leukemia cells (L1210). L1210 cells in tissue culture were treated with VM-26 (10 microM) in the presence of DNP, an uncoupler of oxidative phosphorylation. The simultaneous treatment of DNP (1 mM) increased cell survival 100-200 fold. Pre- or post-treatment with DNP had little effect on cell survival. Other uncouplers and inhibitors of ATP synthesis had effects similar to DNP. The interference of DNP with the cytotoxic action of VM-26 was also seen with another topoisomerase II-targetting drug, m-AMSA, but not with the topoisomerase I-targetting drug camptothecin. Studies using either purified topoisomerase II or cultured mammalian cells had shown that DNP had little effect on the amount of cleavable complexes induced by VM-26. We propose that an ATP requiring process(es) which occurs subsequent to the formation of the cleavable complexes is involved in the cytotoxic action of topoisomerase II-targetting drugs.

Metabolic activation of N-acylanthracyclines precedes their interaction with DNA topoisomerase II.

The N-acylanthracyclines AD32 (N-trifluoroacetyladriamycin-14-valerate) and AD143 (N-trifluoroacetyladriamycin-14-O-hemiadipate) are analogs of Adriamycin (ADR) undergoing clinical or advanced pre-clinical screening. Their principal metabolites, following the cleavage of the 14-acyl side-chain, are N-trifluoroacetyladriamycin (AD41) and its reduced form N-trifluoroacetyladriamycinol (AD92). Both these compounds are biologically active and detectable in treated patients, laboratory animals, and in tissue culture cells. Unlike ADR, AD32, as well as AD143 and metabolites, show no detectable binding to double-strand DNA. Their effects on DNA have been previously investigated in vivo and in vitro using the alkaline filter-elution assay. It has been shown that all of the compounds cause approximately equivalent amounts of protein-associated DNA breaks (PAB) and DNA-protein crosslinks in a mouse lymphoma and in tissue-culture leukemia cells. In order to establish whether the induction of PAB by the drugs requires DNA topoisomerase II mediation, cleavage mapping analysis was done with tested compounds using purified human topoisomerase II. DNA fragmentation was significantly enhanced in the presence of the enzyme and either AD41 or AD92. In contrast, no fragmentation enhancement was observed in the presence of the parental drugs AD32 or AD143. The results strongly suggest that metabolic activation of N-acylanthracyclines by nonspecific esterases is a prerequisite for their interaction with DNA topoisomerase II and for stabilization of the cleavable complex.