Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdask, 11/12 Narutowicza St, 80-952 Gda
sk, Poland1
Author for correspondence: Edward Borowski. Tel: +48 58 347 25 23. Fax: +48 58 347 26 94. e-mail: borowski{at}altis.chem.pg.gda.pl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: glucosamine-6-phosphate synthase, glutamine analogues, pro-drugs, diffusion, antifungal compounds
Abbreviations: FMDP, N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid; GlcN-6-P synthase, glucosamine-6-phosphate synthase; TNBS, 2,4,6-trinitrobenzenesulfonate
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our research programme, aimed at the development of novel antifungal agents, we have focused on a glutamine analogue, N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), a potent and selective inhibitor of glucosamine-6-phosphate synthase (GlcN-6-P synthase) EC 2 . 6 . 1 . 16 from C. albicans (Andruszkiewicz et al., 1986 , 1993
; Milewski et al., 1985
). This enzyme, which plays a key role in the biosynthesis of glucosamine-containing microbial cell wall macromolecules, chitin, mannoprotein and peptidoglycan, is considered to be a target for potential antimicrobial drugs (Chmara et al., 1986
; Milewski et al., 1986
; Borowski, 2000
).
In our previous studies it has been found that FMDP, an amino acid analogue, exhibits only moderate antifungal activity and the presence of some amino acids, especially glutamine and glutamic acid, strongly decreases its antifungal efficacy (Cybulska et al., 1997 ). In order to overcome this problem, we have performed chemical modifications of the FMDP molecule aimed at the construction of latent lipophilic derivatives, which could be transported into cells by diffusion. Following uptake, the modifying group could be removed intracellularly. Very commonly, the development of pro-drugs, in particular in the penicillin group, involves the formation of esters or other easily hydrolysable derivatives of the active agent in order to increase penetration of compounds into the cell (Daehne et al., 1970
). Recently, we synthesized and examined the FMDP derivative acetoxymethyl ester. The structure of this compound is shown in Fig. 1
. This novel derivative inhibits the activity of isolated pure GlcN-6-P synthase and its enzyme inhibitory potency is only a few-fold lower than that of the unsubstituted FMDP (IC50=3·2 µM for FMDP and 11·5 µM for the FMDP ester; Zgódka et al., 1999
). In this paper, we present evidence that the FMDP acetoxymethyl ester penetrates into the fungal cells by free diffusion and, once inside, generates free FMDP upon enzymic cleavage. As a consequence, the compound inhibits growth of C. albicans cells. Our results show that the pro-drug approach can be successfully applied to the design of derivatives of GlcN-6-P synthase inhibitors which bypass the amino acid carrier system and exhibit good antifungal activity.
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Organisms.
Candida albicans ATCC 26278 cells were stored on Sabouraud Dextrose Agar slants (1% Bacto peptone, 1% yeast extract, 2% glucose, 2% agar) at 4 °C and transferred monthly.
Growth conditions.
Minimal medium (YNBG) contained 1·7 g YNB (Yeast Nitrogen Base; Difco), 20 mg L-Trp, 20 mg L-Met, 10 mg L-His and 10 g glucose in 1000 ml water. Overnight cultures in Sabouraud Dextrose medium were diluted in fresh medium to a concentration of 106 cells ml-1 and incubated for about 2 h at 30 °C with shaking (200 r.p.m.) to obtain exponential-phase culture cells.
Growth experiments
(a) Antifungal susceptibility tests.
To determine the 50% inhibitory concentrations (IC50) and MICs of FMDP and its ester, C. albicans cells grown overnight in Sabouraud Dextrose medium at 30 °C were inoculated at a cell density of 104 ml-1 in YNBG medium containing the test compounds at concentrations of 0400 µM. Cultures were incubated for 48 h at 30 °C, and cell growth was quantified by measuring the OD660 to determine the IC50 values. The MIC was defined as the lowest drug concentration preventing visible growth.
(b) Determination of kinetics of growth inhibition.
YNBG liquid medium was inoculated with 106 yeast cells ml-1 from the overnight culture in Sabouraud broth. After a 3 h preincubation at 30 °C with shaking (200 r.p.m.), the compounds were added to give a final concentration in the range of 0160 µM. Incubation was continued for 8 h under the same conditions. Growth was determined spectrophotometrically at 660 nm at hourly intervals.
Uptake studies.
For uptake studies, exponential-phase C. albicans cells grown in Sabouraud Dextrose medium were washed with 50 mM potassium phosphate buffer, pH 7·0, containing 1% (w/v) glucose and suspended in the same buffer to a cell density of 107 cells ml-1 (or 106 cells ml-1 to determine the effects of cell density on FMDP ester uptake). Cell suspensions were preincubated for 15 min at 30 °C and uptake was started by adding FMDP or its ester at final concentrations from 0·05 to 4 mM. At that moment, and at 5 min intervals thereafter, 2 ml samples of the cell suspensions were withdrawn, immediately collected on filter disks (GF/A Whatman filters, pore size 0·22 µm), and the filtrates were used to determine the concentration of the antifungal agents. Then, 1 ml samples of the filtrates were taken and combined with 1·25 ml of a solution containing 4% Na2B4O7 . 10H2O and 0·8 mg TNBS ml-1. Reactions were carried out at 37 °C for 30 min. The A420 was measured and concentrations of FMDP or its ester were read from standard curves. Data were plotted as nmol amino acid (or amino acid ester) taken up by 1 mg (dry wt) cells versus time. The initial uptake velocities were determined from the slopes of the linear part of the curves in the 010 min region.
In some experiments, cells were preincubated with the inhibitor or ionophore for the indicated times at 30 °C, before the determination of uptake by the antifungal agents.
Analysis of FMDP ester metabolism.
Cell-free extracts from C. albicans were prepared by the method described previously (Milewski et al., 1991 ). FMDP ester was added to the extract, to give a final concentration of 100 µM. The mixture was incubated at 30 °C, and 1 ml samples were collected at 1 min intervals and deproteinized by addition of 1 ml ethanol. Precipitate was removed by filtration and the supernatant was analysed by TLC. TLC analyses were performed on Kieselgel 60 F 254 (Merck) and cellulose plates (Merck) in the following solvent systems: A: n-propanol:NH3:CHCl3 (12:8:1, by vol.); B: n-propanol:H2O (7:3, v/v). The filtrates obtained after cell harvesting in uptake studies were also subjected to TLC analysis in the same systems.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The TLC analysis of the spent medium filtrates collected during uptake determination did not reveal any ninhydrin-positive substances, except the FMDP ester, for 25 min. After that time, the appearance of another ninhydrin-positive but UV-negative substance was observed. Its RF value amounted to 0·03 and was lower than that of the diaminopropanoic acid (RF=0·37) and FMDP (RF=0·93), but we were not able to identify this compound. We suppose that this unidentified substance could be a product of FMDP intracellular metabolism, extruded by C. albicans cells.
Effects of metabolic inhibitors on FMDP and FMDP acetoxymethyl ester uptake
The influence of several metabolic inhibitors and ionophores on the uptake of these compounds is shown in Table 1. Metabolic inhibitors such as sodium azide and sodium arsenate reduced the initial velocity of FMDP uptake to 2 and 12% of the control value, respectively, when the cells were preincubated with the appropriate inhibitor for 10 min. These inhibitors and the proton ionophore carbonyl cyanide m-chlorophenylhydrazone did not affect the rate of FMDP acetoxymethyl ester uptake into C. albicans. This indicates that uptake of this compound is neither energy-dependent nor proton-linked. N-Ethylmaleimide, an agent known to covalently modify cysteine side chains, did not reduce FMDP ester uptake.
|
Metabolism of the FMDP ester
FMDP acetoxymethyl ester was added to a crude cell-free extract from C. albicans, and the mixture was incubated at 30 °C. TLC analysis of deproteinized samples taken from this mixture at 1 min intervals revealed the immediate formation of FMDP. Traces of the FMDP ester were detected for 5 min, but totally absent from samples taken later. When proteins present in the cell-free extract were denatured by heating for 3 min at 100 °C prior to the addition of the FMDP ester, decomposition of this compound was not observed. The ester was also stable in 50 mM potassium phosphate buffer (pH 7·0) as well as in the chromatographic solvent system used in TLC analysis. We can therefore conclude that the FMDP acetoxymethyl ester is rapidly hydrolysed to FMDP by unidentified enzymes present in the cytoplasm of C. albicans cells. It is very likely that this process also takes place inside intact fungal cells treated with the agent. Intracellular fast hydrolysis of the FMDP ester must lower the intracellular concentration of this agent to nearly negligible amounts, thus permitting its continuous influx. Moreover, the generated free FMDP is expected to be eliminated from the cytoplasmic environment due to the binding to its target, GlcN-6-P synthase. If the hydrolysis did not occur, the FMDP ester could be accumulated by free diffusion only until its intracellular concentration equilibrates with the extracellular one. According to Ziegelbauer et al. (1998) , the intracellular volume of 107 C. albicans cells is roughly 5 µl. Therefore, such an amount of fungal cells, present in 1 ml of suspension used in our uptake studies, would have been able to accumulate only 0·5% of the antifungal agent present in the medium. Our results clearly show that the accumulation rate is much higher. One may therefore assume that the intracellular hydrolysis of the FMDP ester keeps the concentration gradient of this compound as a driving force for continuous free diffusion.
Our earlier studies on physico-chemical properties of the acetoxymethyl ester of FMDP showed that the apparent lipophilicity of this compound is higher than that of FMDP (Zgódka et al., 1999 ). Results of the studies presented in this paper suggest that the formation of acetoxymethyl esters can be a way of obtaining amino acid analogues lipophilic enough to diffuse through the fungal cell membrane. The application of the lipophilic pro-drug approach for the design of derivatives of GlcN-6-P synthase inhibitors was shown to be a possible way of obtaining novel compounds exhibiting better antifungal properties than the parent compound.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andruszkiewicz, R., Chmara, H., Milewski, S., Kasprzak, L. & Borowski, E. (1993). Structural determinants of inhibitory activity of N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid towards glucosamine-6-phosphate synthase. Pol J Chem 67, 673-683.
Aoki, Y., Kondoh, M., Nakamura, M., Fujii, T., Yamazaki, T., Shimada, H. & Arisawa, M. (1994). A new methionine antagonist that has antifungal activity: mode of action. J Antibiot 47, 909-916.[Medline]
Borowski, E. (2000). Novel approaches in the rational design of antifungal agents of low toxicity. Farmaco 55, 206-208.[Medline]
Capobianco, J., Zakula, D. Z., Coen, M. L. & Goldman, R. C. (1993). Anti-Candida activity of cispentacin: the active transport by amino acid permeases and possible mechanism of action. Biochem Biophys Res Commun 190, 1037-1044.[Medline]
Chmara, H., Andruszkiewicz, R. & Borowski, E. (1986). Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs. Biochim Biophys Acta 870, 357-366.[Medline]
Cybulska, B., Milewski, S. & Andruszkiewicz, R. (1997). Antifungal effect of FMDP. In Abstracts of the 6th International Symposium on Molecular Aspects of Chemotherapy, Gdask 912 July 1997, Poland, p. 148.
Daehne, W., Frederiksen, E., Gundersen, E., Lund, F. & Morch, P. (1970). Acyloxymethyl esters of ampicillin. J Med Chem 13, 607-612.[Medline]
Ghannoum, M. A. (1997). Future of antimycotic chemotherapy. Dermatol Ther 3, 104-111.
Horak, J. (1986). Amino acid transport in eucaryotic microorganisms. Biochim Biophys Acta 864, 223-256.[Medline]
Konishi, M., Nishio, M., Saitoh, K., Miyaki, T., Oki, T. & Kawaguchi, K. (1989). Cispentacin, a new antifungal antibiotic. I. Production, isolation, physico-chemical properties and structure. J Antibiot 42, 1749-1755.[Medline]
Milewski, S., Chmara, H., Andruszkiewicz, R. & Borowski, E. (1985). Synthetic derivatives of N3-fumaroyl-L-2,3-diaminopropanoic acid inactivate glucosamine synthetase from Candida albicans. Biochim Biophys Acta 828, 247-254.[Medline]
Milewski, S., Chmara, H. & Borowski, E. (1986). Anticapsin: an active site directed inhibitor of glucosamine-6-phosphate synthase from Candida albicans. Drugs Exp Clin Res 12, 577-583.[Medline]
Milewski, S., Andruszkiewicz, R., Kasprzak, L., Mazerski, J., Mignini, F. & Borowski, E. (1991). Mechanism of action of anticandidal dipeptides containing inhibitors of glucosamine-6-phosphate synthase. Antimicrob Agents Chemother 35, 36-43.[Medline]
Polak, A. & Hartman, P. G. (1991). Antifungal chemotherapy are we winning? Prog Drug Res 37, 181-269.[Medline]
Prasad, R. (1987). Nutrient transport in Candida albicans, a pathogenic yeast. Yeast 3, 209-221.[Medline]
Yamaki, H., Yamaguchi, M., Nishimura, T., Shinoda, T. & Yamaguchi, H. (1988). Unique mechanism of action of an antifungal antibiotic RI-331. Drugs Exp Clin Res 14, 467-472.[Medline]
Zgódka, D., Cybulska, B., Milewski, S. & Borowski, E. (1999). Acyloxymethyl esters of N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (pro-drugs). In Abstracts of The Multidisciplinary Conference on Drug Research, Muszyna, 35 March 1999, Poland, p. P-76.
Ziegelbauer, K., Babczinski, P. & Schonfeld, W. (1998). Molecular mode of action of the antifungal amino acid BAY 10-8888. Antimicrob Agents Chemother 42, 2197-2205.
Received 21 July 2000;
revised 23 February 2001;
accepted 19 March 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |