Inhibition of H+-ATPase-mediated proton pumping in Cryptococcus neoformans by a novel conjugated styryl ketone

Elias K. Manavathua,*, Jonathan R. Dimmockb, Sarvesh C. Vashishthab and P. H. Chandrasekara

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We investigated the in vitro susceptibility of clinical isolates of Cryptococcus neoformans to the novel conjugated styryl ketone NC1175 by broth microdilution. The MIC90 and the MFC of NC1175 for C. neoformans were 1 and 2 mg/L, respectively. NC1175 at low concentrations (1–4 mg/L) completely inhibited the glucose-induced acidification of the external medium caused by the extrusion of intracellular protons mediated by the plasma membrane located H+-ATPase. These data suggest that NC1175 is a fungicidal agent for C. neoformans and its possible cellular target(s) include the H+-ATPase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cryptococcus neoformans is an encapsulated, neurotropic pathogenic yeast that causes severe and often fatal infections in immunocompromised and at times in immunocompetent patients.1 Amphotericin B and fluconazole are the mainstay of treatment for cryptococcal infections. Adverse side effects at higher doses of amphotericin B and emergence of clinical isolates of C. neoformans with reduced susceptibility to amphotericin B and azoles have compromised their effectiveness against cryptococcal infection.2,3 To improve the efficacy of currently available antifungal agents against cryptococcal infections refractory to treatment, new approaches such as the introduction of lipid formulations of amphotericin B and the use of combinations of antifungal agents have been sought, with some success.4,5 In vitro susceptibility assays performed to evaluate the feasibility of combination therapy with new and existing drugs produced promising results.6 In addition, efforts are underway for the discovery and development of new antifungal agents directed at novel cellular targets.

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Test organisms

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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Susceptibility studies

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 TableGo, 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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Table. In vitro susceptibility of C. neoformans to various antifungal agents
 
Inhibition of external medium acidification

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 FigureGo, 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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Figure. Effect of NC1175 on the proton pumping (as measured by the acidification of external medium) of C. neoformans. Each value represents the mean of two experiments, and the standard deviation was ±10% of the mean. The pH of the external medium in the presence of various concentrations of NC1175 was plotted against time by linear regression (95% confidence level) third-order curve fitting (SigmaPlot 3.0; Jandel Scientific Software, San Rafael, CA, USA). The concentrations of NC1175 used were 0 (•), 1 ({blacktriangledown}), 2 ({diamondsuit}), 4 ({blacksquare}) and 8 ({blacktriangleup}) mg/L.

 
Amphotericin B and fluconazole are the mainstay of treatment against cryptococcal infection in immunocompromised patients. The efficacy of these agents against cryptococcal infection is limited by increased toxicity at higher doses of amphotericin B and the emergence of resistance to fluconazole among clinical isolates. So, one of the objectives of this investigation was to examine the in vitro susceptibility of C. neoformans species to the novel conjugated styryl ketone NC1175 directed to new cellular target(s) and compare the results with those obtained for amphotericin B and various azoles, including the investigational triazoles voriconazole and posaconazole. All compounds tested showed excellent in vitro acitivity against C. neoformans isolates, and in all cases the MIC90 values were <=1 mg/L. The MIC values of NC1175 were three- to four-fold higher than those obtained for other antifungal agents. However, the activity of NC1175 against C. neoformans was approximately six-fold higher than that obtained against Candida and Aspergillus species in a previous study9 indicating that C. neoformans is more susceptible to this novel conjugated styryl ketone.

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.


    Acknowledgments
 
The authors wish to thank Drs William J. Brown (Microbiology Laboratory, Detroit Medical Center, Detroit, MI, USA) and Mahmoud Ghannoum (Mycology Reference Laboratory, Cleveland Medical Center, Cleveland, OH, USA) for kindly providing various C. neoformans isolates used in this study. The authors also thank Jessica L. Cutright for excellent technical assistance.


    Notes
 
* Corresponding author. Tel: +1-313-577-1931; Fax: +1-313-993-0302; E-mail: aa1388{at}wayne.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Mitchell, T. G. & Perfect, J. R. (1995). Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clinical Microbiology Reviews 8, 515–48.[Abstract]

2 . Lozano-Chiu, M., Paetznick, V. L., Ghannoum, M. A. & Rex, J. H. (1998). Detection of resistance to amphotericin B among Cryptococcus neoformans clinical isolates: performances of three different media assessed by using E-test and National Committee for Clinical Laboratory Standards M27-A methodologies. Journal of Clinical Microbiology 36, 2817–22.[Abstract/Free Full Text]

3 . Nguyen, M. H. & Yu, C. Y. (1998). In vitro comparative efficacy of voriconazole and itraconazole against fluconazole-susceptible and -resistant Cryptococcus neoformans isolates. Antimicrobial Agents and Chemotherapy 42, 471–2.[Abstract/Free Full Text]

4 . Schurmann, D., de Matos Marqués, B., Grunewald, T., Ponte, H. D., Hahn, H. & Ruf, B. (1991). Safety and efficacy of liposomal amphotericin B in treating AIDS-asssociated disseminated cryptococcosis. Journal of Infectious Diseases 164, 620–2.[ISI][Medline]

5 . Bennett, J. E., Dismukes, W. E., Duma, R. J., Medoff, G., Sande, M. A., Gallis, H. et al. (1979). A comparison of amphotericin B alone and combined with flucytosine in the treatment of cryptococcal meningitis. New England Journal of Medicine 301, 126–31.[Abstract]

6 . Franzot, S. P. & Casadevall, A. (1997). Pneumocandin L-743,872 enhances the activities of amphotericin B and fluconazole against Cryptococcus neoformans in vitro. Antimicrobial Agents and Chemotherapy 41, 331–6.[Abstract]

7 . Mutus, B., Wagner, J. D., Talpas, C. J., Dimmock, J. R., Phillips, O. A. & Reid, R. S. (1989). 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one hydrobromide, a sulphydryl-specific compound which reacts irreversibly with protein thiols but reversibly with small molecular weight thiols. Analytical Biochemistry 177, 237–43.[ISI][Medline]

8 . Manavathu, E. K., Vashishtha, S. C., Alangaden, G. J. & Dimmock, J. R. (1998). In vitro antifungal activity of some Mannich bases of conjugated styryl ketones. Canadian Journal of Microbiology 44, 74–9.[ISI][Medline]

9 . 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, 2950–9.[Abstract/Free Full Text]

10 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard M27-A. NCCLS, Wayne, PA.

Received 22 May 2000; returned 9 August 2000; revised 16 November 2000; accepted 2 January 2001





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