Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris , Candida albicans , and Candida dubliniensis

Abstract

Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against Pneumocystis carinii f. sp. muris in a mouse model for P. carinii pneumonia and against Candida albicans and Candida dubliniensis in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with P. carinii reduced by about 100-fold the number of P. carinii organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of P. carinii by about fourfold. In vitro inhibition of P. carinii group I intron splicing was observed with a 50% inhibitory concentration (IC 50 )of 30 M in 2 or 4 mM Mg 2؉ , suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC 50 s ranged from 1 to 9 M for strains with group I introns and were 12 and 32 M for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for Pneumocystis and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.Compounds targeting nucleic acids are a promising class for new therapeutics because of the large number of potential targets in cells. For example, the mode of action for many antibiotics is disruption of normal ribosome function. Several of these drugs bind to rRNA and inhibit protein synthesis (7,17,18,22,27). Other compounds, such as distamycin, bind DNA and have antifungal activity (42).Hoechst 33258 is a compound that binds both DNA (2, 30, 33) and RNA (1,6,8). Its chemical structure (Fig. 1) is relatively simple, so derivatives are readily synthesized (21,31,32,37). Hoechst 33258 is an effective inhibitor (13) of in vitro self splicing of the group I intron isolated from the human pathogen Candida albicans (25). The group I self-splicing intron in the large-subunit (LSU) rRNA precursor is a potential drug target, because self splicing is necessary for the maturation of ribosomes (29). Group I introns are also found in the fungal pathogens Pneumocystis and Aspergillus (28, 35) but have not been found in mammalian genomes. Compounds such as pentamidine and fluorocytosine (25, 26), which have antimicrobial activity against Pneumocystis carinii and Candida spp., have also been shown to interfere with the self splicing of group I introns (23, 26). These results suggest that Hoechst 33258 may also inhibit growth of C. albicans and C. dubliniensis in vitro and P. carinii in vivo.Here, we show that Hoechst 33258 inhibits the growth of Candida in vitro and Pneumocystis in vivo. Growth of Candida isolates with and without a group I intron are both inhibited by Hoechst 33258, however, indicating that the group I intron is not the sole target affe...

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