Molecular Characterization of Recombinant Pneumocystis carinii Topoisomerase I: Differential Interactions with Human Topoisomerase I Poisons and Pentamidine

Abstract

The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The P. carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg 2؉ requirement. Cleavage assays reveal that both the human and P. carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4,6-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that P. carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme, P. carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of P. carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with P. carinii topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.Pneumocystis carinii pneumonia (PCP) is an opportunistic infection common in immune compromised individuals (35, 48). The incidence of this infection has decreased dramatically in individuals infected with human immunodeficiency virus due to the use of highly active antiretroviral therapy. However, in human immunodeficiency virus-infected individuals and cancer patients with CD4 cell counts lower than 200/l, long-term prophylaxis with the standard PCP drug combination sulfamethoxazole-trimethoprim (co-trimoxazole) is recommended (2). Continued use of co-trimoxazole in the treatment of Plasmodium falciparum, Escherichia coli, and Streptococcus pneumoniae infections is associated with the generation of point mutations in the dihydropteroate synthase gene, which confers resistance to sulfonamides. Such resistance has been seen in P. carinii-infected individuals (31,36,37,42). Therefore, novel antipneumocystis agents must be developed to address these concerns.Topoisomerases from a variety of organisms have been effective targets for cytotoxic drug development. These nuclear enzymes modulate the topology of DNA during processes such as transcription, replication, and recombination. Type I topoisomerases cleave a single DNA strand and allow "controlled rotation" of the strand to relieve torsional stress one linking number at a time (9,30,56). The type II enzymes cleave both DNA strands and change the linking number by two by passing intact, double-stranded DNA through the cut (reviewed in references 10 and 14). In the presence of certain drugs, topoisomerases are converted into cytotoxic molecules. Camptothecin (CPT) reversibly traps ...

Highly Active Anti- Pneumocystis carinii Compounds in a Library of Novel Piperazine-Linked Bisbenzamidines and Related Compounds

“…Because pentamidine and related compounds were shown to bind to the minor groove of the DNA molecule, it was initially thought that the anti-P. carinii properties were reliant upon inhibition of the organism's topoisomerase I (13,28). This was not found to be the case, and later studies with recombinant P. carinii topoisomerase I as a reagent rather than semipurified extracts provided further evidence that the cytotoxic mechanisms of pentamidine were not targeted to this enzyme (35). Topoisomerase II (3) or inhibition of group I intron splicing (22,41) were suggested as other potential targets.…”

Combination Treatment of Invasive Fungal Infections

“…The aromatic diamidine pentamidine (PN) displays multiples effects against protozoa, bacteria and fungi (17, 53, 58, 132, 137, 217a, 237). Among the fungi, its effect has been mostly studied against Pneumocystis carinii (95,228), C. neoformans (10), C. albicans (150,162), and A. fumigatus (3). The in vitro effect of PN-plus-ITRA and PN-plus-KETO combinations on 11 C. albicans strains (including one azole-resistant isolate) has also been studied (217a).…”

Benzo[c]phenanthrene Adducts and Nogalamycin Inhibit DNA Transesterification by Vaccinia Topoisomerase

“…Thus, the outcome of nogalamycin intercalation with respect to the mammalian topoisomerase will depend upon the sequence context and the position of the intercalated drug relative to the cleavage site. From a pharmacological perspective, the topo I poison mechanism dominates cytotoxicity, which allows for drug efficacy even if the topoisomerase is nonessential for cell growth (55) and even if only a fraction of the potential topoisomerase-DNA complexes can be trapped by the drug.…”