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 ,uM, respectively. For

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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.

African trypanosomes are ancient eukaryotes that cause lethal disease in humans and cattle. Available drugs are inadequate and the need for new therapeutic targets is great. Trypanosoma brucei and related pathogens differ strikingly from higher eukaryotes in many aspects of nucleic acid structure and metabolism. We find yet another example of this in their unusual DNA topoisomerase IB. Type IB topoisomerases relieve the supercoils that accumulate during DNA and RNA synthesis, and are of considerable importance as the target for antitumor camptothecins. Dozens of type IB topoisomerases sequenced from eukaryotes, bacteria, and pox viruses are all encoded by a single gene that predictably contains a highly conserved DNA binding domain and C-terminal catalytic domain, linked by a nonconserved hydrophilic region. We find that topoisomerase IB in T. brucei is encoded by two genes: one for the DNA-binding domain and a second for the C-terminal catalytic domain. In keeping with this, highly purified fractions of native T. brucei topoisomerase IB catalytic activity contain two proteins, of 90 and 36 kDa. The native enzyme is conventional in its Mg 2؉ -independence, ability to relax positive and negative supercoils, and inhibition by camptothecin. Camptothecin promotes the formation of a covalent complex between 32 P-labeled substrate DNA and the small subunit. This unusual structural organization may provide a missing link in the evolution of type IB enzymes, which are thought to have arisen over time from the fusion of two independent domains. It also provides another basis for the design of selectively toxic drug candidates.T he African trypanosome, Trypanosoma brucei, is a flagellated protozoan that causes sleeping sickness in humans. This tsetse fly-transmitted meningoencephalitis is of increasing incidence and is fatal if not treated (1). Closely related organisms cause Chagas' disease and leishmaniasis. Available therapies for sleeping sickness are widely acknowledged to be inadequate: they require multiple parenteral doses for cure, are expensive, toxic, and are losing efficacy against drug-resistant parasites. Trypanosomes and leishmania are ancient eukaryotes whose distinctive features include structurally and metabolically unusual DNAs. In African trypanosomes, the nuclear genome is comprised of 11 chromosome pairs, several additional chromosomes of unknown ploidy, and Ϸ100 minichromosomes, each containing a single gene that encodes a variable surface protein (2). Surface protein genes can be activated by recombinationbased transfer from minichromosomes to transcriptionally active sites in larger chromosomes. Large polycistronic transcripts are trans-spliced to form mRNAs with a 5Ј-leader sequence and 3Ј poly(A) tail. Even more unusual is their mitochondrial DNA, termed kinetoplast or kDNA, which is in the form of a single gigantic network of interlocked relaxed DNA circles (3, 4). Mature mitochondrial messages are created by an editing process that involves systematic insertion or removal of U residues.DNA top...