ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2005, p. 1326–1330 0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.4.1326–1330.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 4
Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris, Candida albicans, and Candida dubliniensis Matthew D. Disney,1,2 Ruth Stephenson,3 Terry W. Wright,3,4 Constantine G. Haidaris,4 Douglas H. Turner,1,2,3 and Francis Gigliotti3,4* Department of Chemistry1 and Center for Human Genetics and Molecular Pediatric Disease,2 Department of Pediatrics,3 and Department of Microbiology,4 School of Medicine and Dentistry, University of Rochester, Rochester, New York Received 29 September 2004/Returned for modification 22 October 2004/Accepted 22 December 2004
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 (IC50)of 30 M in 2 or 4 mM Mg2ⴙ, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC50s 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. Hoechst 33258, however, indicating that the group I intron is not the sole target affecting growth. Similar to the effects seen for C. albicans, experiments on purified rRNA precursor from P. carinii demonstrate that Hoechst 33258 inhibits splicing of the P. carinii group I intron. Overall, these results demonstrate that compounds such as Hoechst 33258 have potential as antifungal compounds if their toxicity allows for an acceptable therapeutic index.
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
MATERIALS AND METHODS Buffers. HXMg buffer is 50 mM HEPES (25 mM Na HEPES), 135 mM KCl, and X mM MgCl2 at pH 7.5. TBE buffer is 100 mM Tris, 90 mM boric acid, and 1 mM EDTA at pH 8.4. Stop buffer is 12 M urea, 12 mM Na2-EDTA, and 0.1⫻ TBE buffer. Instruments and general protocols. All radioactivity was quantified on a Molecular Dynamics PhosphorImager with ImageQuaNT version 4.1 software. The P. carinii precursor was transcribed and purified as described previously (9, 36). Growth inhibition of Candida species. C. albicans isolates were obtained from clinical samples from the University of Rochester Medical Center, N.Y. (11). C. dubliniensis isolates were obtained from Centraalbureau voor Schimmelcultures (CBS) in The Netherlands. Candida strains were grown overnight in YPD (yeast extract peptone dextrose) media; all media were prepared as described previously (34). Cells were harvested by centrifugation, washed with an equal volume of sterile water, and placed into YNB (yeast nitrogen base) medium at pH 4.5 to give an optical density at 540 nm (OD540) of 0.01. Serial dilutions of inhibitors were added to cultures, and they were incubated at 37°C with vigorous aeration for 8 to 12 h until the OD540 in the absence of inhibitor reached about 0.6 (range, 0.6 to 0.9). The OD540 of all cultures was measured, and 50% inhibitory concentrations (IC50s) for Candida spp. were determined from plots of the ratio of OD540 with and without inhibitor versus inhibitor concentration. The plots were fit with the SIGMAPLOT 2001s Logistic four-parameter curve fit program. Splicing assays. Splicing assays on purified P. carinii-truncated precursor were completed as described previously (10). In a typical experiment, 2 nM precursor was refolded in buffer by incubation at 50°C for 3 min. The sample was placed at 37°C for at least 2 min to allow the temperature to equilibrate, and then 3 l of this solution was added to 3 l of a solution containing inhibitor and 2 mM pG
* Corresponding author. Mailing address: Department of Pediatrics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642. Phone: (585) 275-0588. Fax: (585) 273-1104. E-mail:
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FIG. 1. Chemical structures of pentamidine (top) and Hoechst 33258 (bottom). R represents OH for Hoechst 33258.
in buffer at 37°C. Samples were incubated at 37°C for 1 h, and a 2/3 volume of stop buffer was added to quench the reactions. Products were separated on a denaturing 5% polyacrylamide gel and were quantified with a PhosphorImager (10). The IC50s were determined as described previously (5). Mouse modeling of P. carinii pneumonia. A mouse model of P. carinii pneumonia (Pcp) was used to test the activity of Hoechst 33258 against P. carinii. For this model, 20-g CB-17 SCID mice (Jackson Laboratories, Bar Harbor, Maine) were inoculated intranasally with 80 l of physiologic saline containing 5 ⫻ 105 P. carinii cysts that had been partially purified from freshly isolated infected mouse lungs. Twenty-four and 48 h after inoculation, the mice were given a 100-g intraperitoneal (i.p.) dose of gentamicin (Gibco, Rockville, Md.) to prevent bacterial pneumonias which sometimes occur when SCID mice are infected by inoculation rather than by cohousing. Two weeks after inoculation, when infection was well established, the mice were treated with either Hoechst 33258, trimethoprim-sulfamethoxazole (TMP-SMX) (Gensiasicor Pharmaceuticals, Irvine, Calif.), or physiologic saline. Hoechst 33258 was diluted in saline and given daily at a dose of approximately 7.5, 22.5, or 37.5 mg/kg of body weight by i.p. injection. Dosages were calculated based on the average body weight of the experimental mice. TMP-SMX was also diluted in saline and given at a single daily dose of 15 to 20 mg of the TMP component/kg. Control mice received saline alone. These treatments were continued for 14 days, at which time the mice were sacrificed and their lungs removed for quantitation of P. carinii by real-time PCR using primers specific for the single-copy kex1 gene of P. carinii sp. f. muris (38). The statistical significance in the number of P. carinii in the various treatment groups was determined by two-tailed t test. Results were graphed with Sigma Plot.
RESULTS Growth of Candida strains in the presence of small molecules. Growth inhibition of Candida spp. was tested for Hoechst 33258 and compared with pentamidine and miconazole as positive controls. Strains tested included clinical iso-
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lates that either harbor or do not harbor a group I intron in their LSU rRNA precursor. The presence of the intron was determined by PCR as described previously (10). The results are shown in Table 1. Miconazole was tested because its therapeutic target is cell membrane biosynthesis, not RNA (20). The IC50s ranged from 10 to 190 nM for the strains tested (Table 1). There is no correlation between growth inhibition and the presence of a group I intron. Pentamidine was tested for inhibition of growth because part of its antifungal activity is due to inhibition of group I intron self splicing (26). For example, pentamidine is usually more effective at slowing growth of C. albicans strains that harbor a group I intron than ones that do not. For strains tested here, intron-containing strains have IC50s of 0.6 and 6 M and the intronless strains have IC50s of 0.9 and 100 M (Table 1). Hoechst 33258 slows growth of all C. albicans strains tested (Table 1). The two strains that do not harbor a group I intron had IC50s of 32 and 12 M. The two strains that contain a group I intron had IC50s for Hoechst 33258 of 9 and 7 M. Anti-Candida compounds were also tested for inhibition of C. dubliniensis growth (Table 1). All reported isolates of C. dubliniensis have a group I intron in their LSU rRNA precursor (3). As shown in Table 1, the three C. dubliniensis strains tested had IC50s of ⬃1.5 M for Hoechst 33258, which is lower than the IC50 for any C. albicans strain. Antimicrobial activity of Hoechst 33258 on in vivo infection with P. carinii. Preliminary toxicity studies were done in normal BALB/c mice given 5 and 25 mg of Hoechst 33258 i.p./kg once daily for 10 days. Mice displayed decreased movement about the cages after the first two doses, but thereafter they displayed normal activity and appearance over the remainder of the 10-day trial. Lung tissue was obtained from mice after 2 days of treatment with Hoechst 33258 and examined by fluorescent microscopy. Cell nuclei were fluorescent, indicating that the compound was reaching the lung. Based on preliminary experience, four groups of five SCID mice each were treated once daily with saline (Group A), TMP-SMX (Group B), 7.5 mg Hoechst 33258/kg (Group C), or 37.5 mg Hoechst 33258/kg (Group D), beginning 14 days after the mice were inoculated with P. carinii. Treatments were given for 14 days, and the mice were sacrificed one day after the last dose and assayed for the presence of P. carinii. As shown in Fig. 2A, there was an almost 2 log10 drop in P. carinii
TABLE 1. Inhibition of growth of C. albicans and C. dubliniensis by small molecules Strain
IC50 (M)a
Intron present Hoechst 33258
Pentamidine
Miconazole
C. albicans RO102 RO99 RO20 RO109
No No Yes Yes
32 ⫾ 8 (13.6 ⫾ 3.4) 12 ⫾ 3 (5.1 ⫾ 1.3) 9 ⫾ 2 (3.8 ⫾ 0.8) 7 ⫾ 0.4 (3.0 ⫾ 0.2)
100 ⫾ 20 (34.2 ⫾ 6.8) 0.9 ⫾ 0.3 (0.3 ⫾ 0.1) 6 ⫾ 2 (2.1 ⫾ 0.7) 0.6 ⫾ 0.2 (0.2 ⫾ 0.1)
0.01 ⫾ 0.006 (0.004 ⫾ 0.002) 0.06 ⫾ 0.02 (0.02 ⫾ 0.01) 0.05 ⫾ 0.01 (0.02 ⫾ 0.004) 0.05 ⫾ 0.01 (0.02 ⫾ 0.004)
C. dubliniensis CBS8500 CBS7987 CBS7988
Yes Yes Yes
1.8 ⫾ 0.2 (.76 ⫾ 0.08) 1.2 ⫾ 0.5 (.51 ⫾ 0.21) 1.0 ⫾ 0.5 (.42 ⫾ 0.21)
1.2 ⫾ 0.5 (0.4 ⫾ 0.2) 6 ⫾ 3 (2.1 ⫾ 1.0) 0.4 ⫾ 0.2 (0.1 ⫾ 0.1)
0.05 ⫾ 0.03 (0.02 ⫾ 0.01) 0.13 ⫾ 0.11 (0.05 ⫾ 0.05) 0.19 ⫾ 0.75 (0.08 ⫾ 0.31)
a
Numbers in parentheses are calculated IC50 in micrograms per milliliter.
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FIG. 2. Copies of P. carinii as determined by real-time PCR from harvested mouse lung after treatment. Low, intermediate (Mid), and high doses of Hoechst 33258 are 7.5, 22.5, and 37.5 mg/kg, respectively. TMP-SMX dose is 15 to 20 mg of TMP/kg and 75 to 100 mg of the SMX component/kg.
in mice receiving 37.5 mg of Hoechst 33258/kg compared to saline-treated animals as determined by PCR. (Group A, 3.9 ⫻ 106 ⫾ 2.7 ⫻ 106 kex1 copies/ml; Group D, 5.9 ⫻ 104 ⫾ 9.6 ⫻ 104 kex1 copies/ml; P ⫽ 0.01). Mice receiving the low dose of Hoechst 33258 had organism counts similar to those of the saline-treated mice. PCR results were cross-checked by silver staining the lung homogenates to identify P. carinii cysts. Cyst counts were 2.4 ⫻ 106 in Group A, 8.9 ⫻ 104 in Group B, 2.0 ⫻ 106 in group C, and undetectable in Group D. To test reproducibility, the experiment was repeated with the addition of a group of five SCID mice that received an intermediate dose of Hoechst 33258. For this experiment, mice received saline (Group A), TMP-SMX (Group B), 7.5 mg of Hoechst 33258/kg (Group C), 22.5 mg of Hoechst 33258/kg (Group D), or 37.5 mg of Hoechst 33258/kg (Group E). Mice receiving the highest dose of Hoechst 33258 again had a 2 log10 reduction in organism numbers (Fig. 2B) (Group A, 4.6 ⫻ 106 ⫾ 2.0 ⫻ 106 kex1 copies/ml; Group E, 4.0 ⫻ 104 ⫾ 2.8 ⫻ 104 kex1 copies/ml; P ⫽ 0.002). Mice receiving the 22.5-mg/kg dose of Hoechst 33258 had a smaller but still statistically significant drop in P. carinii counts (Group A, 4.6 ⫻ 106 ⫾ 2.0 ⫻ 106 kex1 copies/ml; Group, D 1.1 ⫻ 106 ⫾ 3.0 ⫻ 105; P ⫽ 0.005). The lowest dose again had no effect on organism clearance. To test whether Hoechst 33258 might affect group I intron splicing in P. carinii, its IC50 was measured (Fig. 3) for inhibi-
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. Autoradiogram of a gel showing inhibition of self splicing of the P. carinii group I intron at 2 mM Mg2⫹ by Hoechst 33258. From top to bottom, the bands are 5⬘ exon-intron-3⬘ exon (i.e., truncated precursor), 5⬘ exon-intron, intron-3⬘ exon intron, ligated exons, and 5⬘ exon. The plot shows percentages of intron versus the concentration of Hoechst 33258 (in micromolars) on a logarithmic scale.
tion of self splicing of the purified truncated precursor rRNA developed by Testa et al. (36). As shown in Table 2, the IC50 is about 30 M at 2 and 4 mM Mg2⫹, which is similar to expected Mg2⫹ concentrations in vivo (14, 19, 39). The IC50 at 4 mM Mg2⫹ is not very sensitive to addition of up to 29 mM nucle-
TABLE 2. IC50 (M) for inhibition of truncated precursor self splicing by Hoechst 33258 [Torula yeast bulk RNA] in nucleotides
[Mg2⫹] (mM)
0
2.9 mM
9.5 mM
29 mM
P. carinii
2 4 10
31 ⫾ 17 27 ⫾ 10 85 ⫾ 20
NDa 18 ⫾ 2 ND
ND 40 ⫾ 10 ND
ND 77 ⫾ 10 ND
C. albicansb
2 4 10
17 ⫾ 3 41 ⫾ 5 80 ⫾ 11
16 ⫾ 3 39 ⫾ 5 85 ⫾ 20
27 ⫾ 4 54 ⫾ 4 160 ⫾ 50
90 ⫾ 20 125 ⫾ 20 200 ⫾ 40
Species
a
ND, not determined. This truncated precursor was cloned from C. albicans strain RO109. Data are from reference 13. b
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otides of Torula yeast bulk RNA, suggesting some specificity in binding to the group I intron (Table 2). Similar results have been reported (13) for the truncated precursor rRNA of C. albicans (Table 2). DISCUSSION Hoechst 33258 binds both DNA (2, 30, 33) and RNA (1, 6, 8). It is known (13) to inhibit the function of one class of catalytic RNA, group I self-splicing introns (41), that is a potential drug target (Table 2). To determine whether Hoechst 33258 has antifungal activity, we tested it against P. carinii in vivo and Candida spp. in vitro. In vivo the activity of Hoechst against P. carinii exceeded that of a daily dose of TMP-SMX. It also demonstrated in vitro activity against C. albicans and C. dubliniensis. While antifungal activity was observed for all three organisms, the activity was not completely due to inhibition of self splicing. In vivo, Hoechst 33258 at a single daily dose of 37.5 mg/kg given for 14 days to SCID mice infected with P. carinii resulted in a 99% decrease in organisms compared to levels for salinetreated control mice. A lower dose of 22.5 mg/kg still resulted in a statistically significant drop in organisms. The activity of Hoechst 33258 in this model of Pcp was as effective, or more so, over a 14-day period than was TMP-SMX, which was given at a similar dosing range to validate the ability of this model to demonstrate an anti-pneumocystis effect for a given compound. Because no pharmacokinetic studies were employed, however, direct comparison of the potency of Hoechst 33258 to TMP-SMX is not appropriate. However, Yasuoka et al. showed that a single daily i.p. injection of a similar dose of TMP-SMX for 21 days was effective treatment for Pcp in a nude mouse model (40). Thus, we can conclude from our experimental design that Hoechst 33258 brought about a more rapid drop in P. carinii than did TMP-SMX. Biochemical experiments show that Hoechst 33258 inhibits self splicing of the group I intron from C. albicans (13) and P. carinii (Fig. 3), suggesting that this could be an antifungal target for Hoechst 33258. In cell culture, however, C. albicans strains that harbor a group I intron, as well as those that do not, were both inhibited by Hoechst 33258, suggesting that the compound has targets in addition to the group I intron demonstrated in vitro (13). One possible mechanism for this activity is through binding to DNA topoisomerase I (4). Uptake and toxicity have been measured for Hoechst derivatives in mammalian cells. For example, Hoechst 33258 is internalized in mammalian cells at 10-fold lower concentrations than Hoechst 33342 (15). The only structural difference between these compounds is the substitution of a hydroxyl group for an ethoxy group. While toxicity issues are observed with Hoechst 33342 at concentrations greater than 5 and 10 M with HeLa cells (16) and myeloid cells (24), respectively, Hoechst 33258 will likely be less toxic. The effects of intravenous administration of Hoechst 33342 in mice have also been studied (24). High doses (0.3 M) of Hoechst 33342 were administered in these studies and resulted in morphological changes to the spleen and lungs. The IC50 data here suggest that much lower concentrations of Hoechst 33258 will be required to slow fungal growth. These considerations, and the fact that Candida infections often involve skin or mucous mem-
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branes that allow drug to be applied topically, may reduce toxicity. Likewise, Pcp can potentially be treated with aerosolized drug as a means to reduce toxicity. Such an approach has been utilized to reduce systemic hematologic toxicity from ribavirin in the treatment of respiratory syncytial virus pneumonia. Finally, the relatively simple structure of Hoechst dyes allows the synthesis of many derivatives that can be tested for efficacy and toxicity (21, 31, 32, 37). For example, oligonucleotides can be attached covalently, which might allow rational optimization toward RNA and DNA targets (12, 31, 37). In summary, Hoechst 33258 has clearly demonstrable antimicrobial activity against P. carinii in vivo and against C. albicans and C. dubliniensis in vitro. The effect of Hoechst 33258 against these fungi may be due to interaction with DNA, RNA, or both. Determination of the cellular target should allow derivatization of Hoechst 33258 to enhance its effectiveness and specificity. ACKNOWLEDGMENTS We thank Sherry Spinelli for suggesting and obtaining C. dubliniensis strains. This work was supported by NIH grants GM22939 (D.H.T.) and HL071659 (F.G.). REFERENCES 1. Bailly, C., P. Colson, C. Houssier, and F. Hamy. 1996. The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism. Nucleic Acids Res. 24:1460–1464. 2. Bontemps, J., C. Houssier, and E. Fredericq. 1975. Physico-chemical study of the complexes of ‘33258 Hoechst’ with DNA and nucleohistone. Nucleic Acids Res. 2:971–984. 3. Boucher, H., S. Mercure, S. Montplaisir, and G. Lemay. 1996. A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron. Gene 180:189–196. 4. Chen, A. Y., C. Yu, B. Gatto, and L. F. Liu. 1993. DNA minor groove-binding ligands: a different class of mammalian DNA topoisomerase I inhibitors. Proc. Natl. Acad. Sci. USA 90:8131–8135. 5. Childs, J. L., M. D. Disney, and D. H. Turner. 2002. Oligonucleotide directed misfolding of RNA inhibits Candida albicans group I intron splicing. Proc. Natl. Acad. Sci. USA 99:11091–11096. 6. Cho, J., and R. R. Rando. 2000. Specific binding of Hoechst 33258 to site 1 thymidylate synthase mRNA. Nucleic Acids Res. 28:2158–2163. 7. Cundliffe, E. 1990. Recognition sites for antibiotics within rRNA, p. 479–490. In W. Hill, A. Dahlberg, R. Garrett, P. Moore, D. Schlessinger, and J. Warner (ed.), The ribosome: structure, function and evolution. American Society for Microbiology, Washington, D.C. 8. Dassonneville, L., F. Hamy, P. Colson, C. Houssier, and C. Bailly. 1997. Binding of Hoechst 33258 to the TAR RNA of HIV-1. Recognition of pyrimidine bulge-dependent structure. Nucleic Acids Res. 25:4487–4492. 9. Disney, M. D., S. M. Testa, and D. H. Turner. 2000. Targeting a Pneumocystis carinii group I intron with methylphosphonate oligonucleotides: backbone charge is not required for binding or reactivity. Biochemistry 39:6991–7000. 10. Disney, M. D., C. G. Haidaris, and D. H. Turner. 2001. Recognition elements for 5⬘ exon substrate binding to the Candida albicans group I intron. Biochemistry 40:6507–6519. 11. Disney, M. D., C. G. Haidaris, and D. H. Turner. 2003. Uptake and antifungal activity of oligonucleotides in Candida albicans. Proc. Natl. Acad. Sci. USA 100:1530–1534. 12. Disney, M. D., J. L. Childs, and D. H. Turner. 2004. New approaches to targeting RNA with oligonucleotides: inhibition of group I intron self-splicing. Biopolymers 73:151–161. 13. Disney, M. D., J. L. Childs, and D. H. Turner. 2004. Hoechst 33258 selectively inhibits group I self-splicing by affecting RNA folding. ChemBioChem 5:1–6. 14. Donahue, C. P., R. S. Yadara, S. M. Nesbitt, and M. J. Fedor. 2000. The kinetic mechanism of the hairpin ribozyme in vivo: influence of RNA helix stability on intracellular cleavage kinetics. J. Mol. Biol. 295:693–707. 15. Elstein, K. H., and R. M. Zucker. 1994. Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations. Exp. Cell Res. 211:322–331. 16. Filatov, M. V., and E. Y. Varfolomeeva. 1995. Active dissociation of Hoechst 33342 from DNA in living mammalian cells. Mutat. Res. 327:209–215. 17. Fourmy, D., M. I. Recht, S. C. Blanchard, and J. D. Puglisi. 1996. Structure
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