a Division of Infectious Diseases, Department of Internal Medicine, 427 Lande Building, Wayne State University, 550 E. Canfield, Detroit, MI 48201, USA; b College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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Abstract |
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Introduction |
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We showed previously that Mannich bases of certain conjugated styryl ketones, which react preferentially with low molecular weight and protein-associated thiols,7 had activity against a wide spectrum of pathogenic fungi.8 NC1175 {3-[3-(4-chlorophenyl)-2-propenoyl]-4-[2-(4-chlorophenyl)vinylene]-1-ethyl-4-piperidinol hydrochloride} is a conjugated styryl ketone, which inhibits H+-ATPasemediated proton pumping in Candida and Aspergillus species.9 Moreover, vanadate-sensitive ATP hydrolysis by membrane preparations of Candida albicans is completely inhibited by low concentrations of NC1175, suggesting that the proton translocating H+-ATPase is a possible cellular target of this compound.9 To investigate the H+-ATPase targeted antifungal activity of NC1175 further in other clinically important fungal species, we examined the in vitro susceptibility of C. neoformans to NC1175 and compared the results with those obtained for other antifungal agents. We report here that the activity of NC1175 against C. neoformans is at least partly due to the inhibition of plasma membrane located H+-ATPase-mediated extrusion of intracellular protons.
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Materials and methods |
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Clinical isolates of C. neoformans (n = 15) used in our study were obtained from the Microbiology Laboratory, Detroit Medical Center, Detroit, MI, USA and the Mycology Reference Laboratory, Cleveland Medical Center, Cleveland, OH, USA. Long-term stock cultures were maintained in 25% glycerol at 80°C. Working cultures were stored at 4°C on Sabouraud dextrose agar (SDA). The liquid cultures were routinely grown in peptone yeast extract glucose (PYG: peptone 2 g, yeast extract 2 g, glucose 5 g, per litre of water) broth at 30°C on a gyratory shaker.
Antifungal agents
Amphotericin B (batch no. 20-914-29670), itraconazole (R51 211, batch no. STAN-9304-005-1), voriconazole (batch no. 25381-57-8) and posaconazole (batch no. 97-56592-X-208) were obtained as pure powders from Squibb Institute for Medical Research (Princeton, NJ, USA), Janssen Pharmaceutica (Beerse, Belgium), Pfizer Pharmaceuticals (New York, NY, USA) and Schering-Plough Research Institute (Kenilworth, NJ, USA), respectively. NC1175 was obtained from the College of Pharmacy and Nutrition, University of Saskatchewan. All antifungals were dissolved in dimethylsulphoxide (DMSO) at a concentration of 1 g/L and stored as 0.25 mL aliquots at 20°C. The frozen stock was thawed at room temperature and gently vortexed several times to ensure that any remaining crystals were dissolved completely before use. Comparable concentrations of DMSO were used to examine its effect on the growth of C. neoformans.
MIC and minimum fungicidal concentration (MFC) determinations
The MICs of various antifungal agents for C. neoformans were determined by the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards,10 but using PYG broth as growth medium. We substituted PYG broth for RPMI 1640 for the MIC studies since the latter medium failed to support the growth of C. neoformans species adequately for an MIC determination within 48 h. The MIC was defined as the lowest concentration of the drug that inhibited growth by 80% for the azoles and 100% for amphotericin B and NC1175 compared with the drug-free control after 48 h of incubation at 35°C. The MIC determination for each isolate was repeated once, and the data were either identical or within plus or minus one dilution.
The MFCs of NC1175 for various C. neoformans isolates were determined by subculturing 0.1 mL aliquots in duplicate from all MIC wells showing no visible growth, on to SDA plates. The plates were incubated at 35°C for 48 h and the MFC was defined as the lowest concentration of the antifungal agent that produced 100% killing.
Measurement of acidification of external medium
The proton pumping activity of C. neoformans was determined by monitoring glucose-induced acidification of the external medium by measuring the pH by means of an electrode as we described previously for C. albicans.9 Control experiments were performed in the presence of DMSO to measure acidification of the external medium without the drug.
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Results and discussion |
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We examined the in vitro susceptibilities of C. neoformans to the conjugated styryl ketone NC1175 by a previously published method and the results were compared with those obtained for amphotericin B and three members of the azole family of antifungal agents.9,10 As shown in the Table, all compounds tested were effective against C. neoformans. The MICs of various compounds obtained for C. neoformans ranged from 0.0625 to 2 mg/L. A pair-wise comparison of the MIC50 and MIC90 values showed that posaconazole (0.125 and 0.25 mg/L) and the styryl ketone NC1175 (1 and 2 mg/L) had the lowest and the highest MIC values, respectively. The MFCs of NC1175 for various C. neoformans isolates tested were either the same or two-fold higher than the MIC values.
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The glucose-induced acidification of the external medium is an indirect measurement of H+-ATPase activity in vivo. The conjugated styryl ketone NC1175 inhibits the H+-ATPase-mediated acidification of the external medium by Saccharomyces, Candida and Aspergillus species, suggesting that this key enzyme is a possible cellular target for the activity of NC1175.9 As shown in the Figure, NC1175 inhibited the glucose-induced acidification of the external medium by C. neoformans in a time- and concentration-dependent manner. For instance, at 4 mg/L the medium acidification was completely inhibited within 30 min, suggesting that the H+-ATPase of C. neoformans is a potential cellular target of action for NC1175.
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Our previous studies revealed that NC1175 is fungicidal against Candida and Aspergillus species. Similarly, this novel conjugated styryl ketone killed C. neoformans cells rapidly in a time- and concentration-dependent manner (data not shown). This finding is not surprising considering the possible mode of action of this compound. Concentrations of the compound at its MIC and at several times its MIC inhibited the H+-ATPase-dependent extrusion of protons, which prevented the acidification of the external medium. Thus, it appears that the conjugated styryl ketones are a new group of antifungal agents whose cellular target(s) include the H+-ATPase.
Although the measurement of the external medium acidification was undertaken in the same manner, the proton pumping ability of C. neoformans was lower than that of C. albicans (data not shown). An identical number of cells (1 x 108 cells/mL) produced a significantly lower degree of acidification of the external medium. Moreover, incubation of KCl-washed C. neoformans cells overnight in 50 mM KCl for the depletion of carbon reserve resulted in the complete loss of proton pumping in the presence of glucose. Thus, in our experiments we incubated the cells at 25°C for 1 h for the depletion of carbon reserve, which provided the best results. Future studies are planned in which the in vitro activity of NC1175 on the H+-ATPase activity of microsomal fractions prepared from C. neoformans will be assessed.
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Acknowledgments |
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Notes |
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References |
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Manavathu, E. K., Dimmock, J. R., Vashishtha, S. C. & Chandrasekar, P. H. (1999). Proton pumping ATPase targeted antifungal activity of a novel conjugated styryl ketone. Antimicrobial Agents and Chemotherapy 43, 29509.
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Received 22 May 2000; returned 9 August 2000; revised 16 November 2000; accepted 2 January 2001