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 Mg2+ 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.

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Consequence of Hoechst 33342-mediated Leishmania DNA topoisomerase-I inhibition on parasite replication

Parasitology ◽

10.1017/s0031182002002524 ◽

2003 ◽

Vol 126 (1) ◽

pp. 21-30 ◽

Cited By ~ 10

Author(s):

J.-F. MARQUIS ◽

M. DROLET ◽

M. OLIVIER

Keyword(s):

Topoisomerase I ◽

Parasite Growth ◽

Dependent Manner ◽

Minor Groove Binding ◽

Groove Binding ◽

Hoechst 33342 ◽

Dna Topoisomerase ◽

Dna Breakage ◽

Pbr322 Dna ◽

Parasite Replication

This study reports that inhibition of Leishmania Topo-I with the minor groove-binding ligands (MGBLs) Hoechst 33342 (Ho342) blocks parasite growth in culture by mechanisms involving DNA breakage. While Ho342 inhibited the replication of several species of Leishmania in a dose- and time-dependent manner, Ho258 was not effective. Cytofluorometric analysis suggested that superior effectiveness of Ho342 over Ho258 was attributed to Leishmania parasites being more permeable toward Ho342. This observation was supported by the ability of both Ho342 and Ho258 to block the relaxation of supercoiled pBR322 DNA by Leishmania Topo-I. The Ho342 specificity toward L. donovani Topo-I was reinforced by the observation that increased Topo-I gene expression and Topo-I activity in Leishmania was paralleled by augmented resistance for this compound. Furthermore, the capacity of NaCl treatment to reverse MGBL-mediated DNA break suggests that Ho342 targetted Topo-I. Moreover, we observed that Ho342-inducible arrest of Leishmania growth was accompanied by G1 arrest and induction of cell death that closely resembles apoptosis. Taken together, our results suggest that MGBL compounds show promise as Topo-I inhibitors against Leishmania infection.

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