HWY-289, a novel semi-synthetic protoberberine derivative with multiple target sites in Candida albicans

Kang-Sik Parka, Kap-Chul Kanga, Ki-Young Kima, Pan-Young Jeonga, Jai-Hyun Kima, David J. Adamsb, Jung-Ho Kimc and Young-Ki Paika,*

a Department of Biochemistry, Bioproducts Research Center and Yonsei Proteome Research Center, Yonsei University, 134 Shinchon-dong, Sudaemoon-ku, Seoul, Korea; b Department of Microbiology, University of Leeds, Leeds, UK; c Hanwha Chemical Research and Development Center, Taejeon 305-345, Korea


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antifungal properties of 515 synthetic and semi-synthetic protoberberines were investigated. HWY-289 was chosen for further study because it exhibited the most significant anti-Candida activity (MICs were 1.56 mg/L for Candida albicans and Candida krusei; 6.25 mg/L for Candida guilliermondii) but did not demonstrate toxicity in rats. HWY-289 inhibited the incorporation of L-[methyl-14C]methionine into the C-24 of ergosterol in whole cells of C. albicans (IC50 20 µM). However, HWY-289 (100 µM) had no effect on mammalian cholesterol biosynthesis in rat microsomes while miconazole (100 µM) was a potent inhibitor of cholesterol biosynthesis under identical assay conditions. A second major target site for HWY-289 was identified that involves cell wall biosynthesis in C. albicans. HWY-289 was a potent inhibitor of the chitin synthase isozymes CaCHS1 and CaCHS2, with IC50 values of 22 µM for each enzyme. The effect was highly specific in that HWY-289 had no significant effect on C. albicans CaCHS3 (IC50 > 200 µM). Thus, HWY-289 compared favourably with well-established antifungal agents as an inhibitor of the growth of Candida species in vitro, and may have considerable potential as a new class of antifungal agent that lacks toxic side effects in the human host.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increasing prevalence of life-threatening fungal diseases means that it is necessary to develop new, and more effective antifungal agents. Although many research groups have carried out intensive research in attempts to develop new antifungal drugs and some have now entered clinical trials, amphotericin B and the azoles remain the mainstay of systemic therapy.1 Moreover, side effects are frequently noted for currently available antimycotics at the therapeutic dosage and reports of the isolation of strains of important fungal pathogens that are resistant to antifungals are increasing in frequency.27 The success of the development of a new drug will depend upon the identification of new antifungal targets that have no mammalian host counterpart.1,8,9 In addition, new antifungal drugs should be active against a broad spectrum of pathogenic fungi.

We have demonstrated previously that the protoberberines, berberine and palmatine, which are alkaloids isolated from Korean and Chinese medicinal plants, significantly inhibit growth of a wide range of Candida species.10 The protoberberines inhibit enzymes that are found only in fungi and that are involved in the synthesis of membrane sterols or cell wall chitin.10 This work led us to design and synthesize derivatives of protoberberines in an attempt to enhance antifungal activity. Here, we describe the effects of one such derivative, HWY-289 (Figure 1Go) on (i) the growth of Candida species; and (ii) Candida albicans chitin synthase (CaCHS) and sterol {triangleup}24-methyltransferase (24-SMT), two essential enzymes in fungi that are absent from humans. Our results indicate that semi-synthetic protoberberines such as HWY-289 may have considerable potential as novel antifungal agents that lack host toxicity.



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Figure 1. Structure of HWY-289.

 

    Materials and methods
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 Introduction
 Materials and methods
 Results
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 References
 
Chemicals and reagents

Berberine iodide, miconazole and amphotericin B were obtained from Sigma (St Louis, MO, USA) and fluconazole was obtained from Yuhan Pharmaceut. Co. (Seoul, Korea). Nikkomycin Z was purchased from Calbiochem (San Diego, CA, USA). Calcofluor White, cofactors and other biochemicals were purchased from Sigma, and were of the highest grade available. The following isotopes (specific activity) were purchased from Amersham (Buckinghamshire, UK); [14C]acetate (56 mCi/mmol), UDP-[U-14C]GlcNAc (271 mCi/mmol), [methyl-14C]S-adenosylmethionine (SAM) (58 mCi/mmol), [14C]mevalonic acid (60 mCi/mmol), 1a, 2a(n)-[3H]1-cholesterol (specific activity 46 Ci/mmol) and L-[methyl-14C]methionine (1 mCi/mmol). Silica gel plates (Kiesel gel 60F254), toluene, diethyl ether and chloroform were purchased from Merck Co. (Darmstadt, Germany). Yeast nitrogen base (YNB), neopeptone, dextrose and bacto-agar were obtained from Difco Co. (Detroit, MI, USA).

Organisms

The fungal strains used were C. albicans ATCC10231, ATCC28838, ATCC64550, ATCC44373, ATCC200955, ATCC64124, ATCC38248 and ATCC62342, and clinical isolates of C. albicans (YH-019 and YH-003; Yuhan Pharmaceut. Co., Seoul, Korea), Candida krusei, Candida glabrata and Candida guilliermondii (the culture collection of the Department of Microbiology, University of Leeds, UK).10 Cells were maintained on slants of Sabouraud's dextrose agar at 4°C. Three different mutants of C. albicans were used for the assay of each CHS isozyme; they were chs2{Delta} and chs3{Delta} for the assay of CaCHS1p, chs3{Delta} for the assay of CaCHS2p and chs2{Delta} for the assay of CaCHS3p.11 For culture of the yeast growth form, cells were inoculated and grown at 30°C for 18 h in Sabouraud's dextrose broth, pH 5.6.12 Late exponential phase cells (18 h) were harvested and washed by centrifugation, and used for the preparation of microsomes.

Broth microdilution susceptibility test

Tests were based on that described previously by Park et al.10 and the NCCLS.13 HWY-289, amphotericin B, fluconazole and miconazole were dissolved in dimethyl sulphoxide (DMSO). Stock solutions were diluted with RPMI 1640 (RPMI tissue culture medium supplemented with glutamine) (Sigma) with 2% glucose, buffered to pH 7.0 with 0.156 M 3-N-morpholinopropane-sulphonic acid (MOPS; Sigma). The final concentrations of the antifungal agents were 0.0975–100 mg/L. The inoculum size was 104 cells/mL. Tests were performed in 96-well microtitre plates and were incubated at 35°C for 48 and 72 h. The MICs of fluconazole and miconzaole were read as the lowest concentration of the agent that inhibited growth by 80%. For HWY-289 and amphotericin B the MIC was the lowest concentration of drug that completely inhibited growth.

Sterol biosynthesis assay

Cells of C. albicans (ATCC 10231) were incubated with radiolabelled substrates as described previously.10,14,15 Briefly, washed cells were incubated in medium A [YNB without amino acid and 1% (w/v) ammonium sulphate, pH 6.5] and harvested after 18 h at 30°C. Washed cells were resuspended in medium B [0.1 mM potassium phosphate buffer and 1% (w/v) glucose]. Test compounds dissolved in DMSO were added to the cell suspensions (980 µL, 2 x 109 cells/mL), which were then pre-incubated with drug for 10 min. The reaction was initiated by addition of 10 µL of [14C]acetate (1 µCi, specific activity 56 mCi/mmol) or L-[methyl-14C]methionine (1 µCi, specific activity 1 mCi/ mmol) and the cells were incubated at 30°C for 3 h. The reaction was stopped by addition of 1 mL 15% (w/v) KOH, 90% ethanol, and the samples were saponified at 80°C for 1 h. The non-saponifiable lipids were then extracted with petroleum ether and the samples evaporated to dryness. Quantification of sterol biosynthesis was as described previously.10 For the preparation of rat liver homogenates, Sprague–Dawley rats (Samyuk Experimental Animal Laboratory, Seoul, Korea) were maintained as described.16 The rate of sterol biosynthesis was measured using S10 fraction (10 000g supernatant of liver homogenates) as described previously.17

Preparation of microsomes and assay of 24-SMT

The preparation of microsomes of C. albicans was carried out by a method described previously.18 Protein concentration was determined by the method of Bradford19 with bovine serum albumin as a standard. Assays for 24-SMT activity were conducted as described previously.10 The assay mixture contained 0.1 M Tris–HCl (pH 7.5), 0.4 mg microsomal protein, 30 mM KHCO3, 5 mM MgCl2, 200 µM desmosterol and 50 µM SAM (0.25 µCi) in a volume of 500 µL in an assay vial and was incubated at 30°C in a shaking water bath for 25 min to determine the initial velocity of the reaction. The reactions were terminated by the addition of 500 µL 15% (w/v) KOH, 90% (v/v) ethanol plus {1a, 2a(n)-[3H]1-cholesterol (0.015 µCi, specific activity 46 Ci/mmol)}. Extraction and quantification of the reaction products were carried out as described above.

Assays of CaCHS

The standard assays for CaCHS were carried out with 1 mM UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) containing 9 nCi UDP-[U-14C]GlcNAc (271 mCi/mmol) in a 50 µL reaction volume by the method of Causier et al. described previously.10,20 Nikkomycin Z was used as a positive control for CaCHS inhibition.21,22 For the assay of CaCHS1p activity, microsomes (40 µg) prepared from chs2{Delta}/chs3{Delta} were incubated with 1.1 mM UDP-[U-14C]- GlcNac (400 dpm/nmol) as described.10,11 For the assay of CaCHS2p activity, microsomes (40 µg) prepared from chs3{Delta} were preincubated in the presence of 2.5% digitonin (w/w) and 10 mM Mg(OAc)2 at 30°C for 10 min and processed as described.23 The substrate {unlabelled UDPGlcNAc (32 mM) plus UDP-[U-14C]GlcNAc, 400 dpm/ nmol} was added to the reaction mixture and incubated at 30°C for 30 min. For the assay of CaCHS3p activity, the microsomes (40 µg) prepared from chs2{Delta} were incubated in the presence of 7 mM Mg(OAc)2, 10 mM Ni(OAc)2, 2 mM Co(OAc)2 and 5 µL of UDP-[U-14C]GlcNAc (400 dpm/nmol) at 30°C for 10 min. Data presented here were obtained from the initial velocity of each reaction.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of HWY-289 on the growth of Candida spp.

Of the 515 synthetic and semi-synthetic derivatives of the protoberberines examined, HWY-289 caused most significant growth inhibition of C. albicans in the primary screening procedures. The anti-Candida activity of HWY-289 for Candida spp. was investigated, and as summarized in Table IGo, HWY-289 showed a wide range of anti-Candida activities with MIC values ranging from 1.56 to 6.25 mg/L for most strains tested. In particular, HWY-289 showed almost equivalent or better potency when compared with miconazole against most standard Candida strains as well as clinical isolates (e.g. C. krusei and C. albicans YH-003), but was less potent than amphotericin B.


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Table I. Effects of HWY-289 on growth of Candida spp.a
 
Effect of HWY-289 on sterol biosynthesis

To determine the basis for growth inhibition by HWY-289 and its target site, the relative rates of total cellular sterol synthesis in whole cell homogenates of C. albicans ATCC 10231 were measured in the presence and absence of HWY-289 using either [14C]acetate or L-[methyl-14C]- methionine. This approach was based on our earlier observation that the berberine precursor of HWY-289 is a potent inhibitor of 24-SMT activity.10 In this experiment, L-[methyl-14C]methionine was added to C. albicans that had been cultured previously in a medium devoid of amino acids, in phosphate buffer containing glucose (1%, w/v). As shown in Figure 2Go(a), incorporation of either L-[methyl-14C]methionine (lane 1 versus lane 3) or [14C]acetate (lane 2 versus lane 4) into the C-24 of ergosterol was specifically inhibited in the presence of 100 µM HWY-289. The decrease in the incorporation of L-[methyl-14C]methionine into ergosterol appeared to be a consequence of inhibition of reactions involving 24-SMT, which is the only enzyme that catalyses the transmethylation of sterols from this precursor. The results shown in Figure 2Go(b) indicate a dose-dependent inhibition of the rate of L-[methyl-14C]- methionine incorporation into ergosterol by HWY-289 (IC50 20 µM). However, surprisingly, HWY-289 did not exhibit any direct inhibitory effects on the enzyme activity in vitro, suggesting that HWY-289 has some form of indirect effect on 24-SMT in vivo.




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Figure 2. Inhibition of ergosterol biosynthesis in C. albicans. (a) Autoradiograph of TLC showing the separation of labelled non-saponifiable lipids from C. albicans cells that were incubated with either L-[methyl-14C]methionine or [14C]acetate in the presence of HWY-289. Cells that had been pre-incubated in YNBG media without amino acids were incubated with L-[methyl-14C]methionine or [14C]acetate in 0.1 M potassium phosphate buffer (pH 6.5) containing 1% (w/v) glucose at 30°C for 3 h. Lane 1, methionine substrate (control); lane 2, acetate substrate (control); lane 3, methionine substrate with HWY-289 at 100 µM; lane 4, acetate substrate with HWY-289 at 100 µM. Abbreviations: S, squalene; L, 4,4-dimethyl sterols (lanosterol); M, 4{alpha}-methylsterols; E, ergosterol; O, origin. (b) Effects of HWY-289 on sterol biosynthesis (whole cells) in C. albicans ({circ}) and 24-SMT activity (•). Whole cells were incubated with L-[methyl-14C]methionine as described for (a). The data are expressed as mean ± S.D. for three separate experiments.

 
It is well known that mammals and fungi share many reactions in their pathways of sterol biosynthesis, so we investigated whether protoberberine derivatives, like the azole class of compounds, might inhibit mammalian cholesterol biosynthesis. As shown in Figure 3Go, HWY-289 (100 µM) exhibited virtually no inhibition of rat hepatic cholesterol biosynthesis while miconazole at the same concentration almost completely blocked cholesterol biosynthesis by causing the accumulation of lanosterol, which otherwise would have been demethylated by 14{alpha}-demethylase.24 We also tested other sterol biosynthesis enzymes in vitro: sterol 14-reductase,16 sterol 8-isomerase,25 sterol 24-reductase26 and 7-dehydrocholesterol reductase27 (data not shown). The decrease in activity observed in most cases was <10% at 100 µM HWY-289.



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Figure 3. Effects of sterol biosynthesis inhibitors on the incorporation of [14C]mevalonic acid into cell-free extracts of rat liver. Autoradiograph of TLC showing the separation of labelled non-saponifiable lipids from rat liver extract incubated with [14C]mevalonic acid in the presence of HWY-289 and miconazole. Lane 1, control; lane 2, HWY-289 (100 µM); lane 3, miconazole (100 µM). Abbreviations: S, squalene; L, 4,4-dimethyl sterols (lanosterol); C, desmethyl sterols (cholesterol); O, origin.

 
Effect of HWY-289 on CaCHS activity in C. albicans

In an attempt to identify other target sites, if any, for the action of HWY-289 in C. albicans, the effect of this compound on CaCHS activity in vitro was determined. Previously, palmatine, a precursor of HWY-289, was shown to cause a moderate dose-dependent inhibition of CaCHS isolated from yeast or mycelial growth forms.10 Therefore, an assay was carried out using the crude extracts of C. albicans in the presence of HWY-289. From this pilot experiment, the IC50 (µM) of HWY-289 against total CaCHS of crude extracts of C. albicans (ATCC 10231) was found to be 16.2 µM while nikkomycin Z had an IC50 of 0.63 µM (data not shown). Because it has been established that there are at least three isozymes of CaCHS,11,28 it was of interest to determine which isoform(s) was most susceptible to the action of HWY-289. Enzyme assays were carried out using the microsomes obtained from three different mutants of C. albicans: chs2{Delta} chs3{Delta} for the CaCHS1p assay, chs3{Delta} for the CaCHS2p assay in the presence of digitonin and chs2{Delta} for the assay of CaCHS3p in the presence of Ni2+ and Co2+.11,23,29 Having established optimal assay conditions for the detection of the activity of each isozyme, the effects of HWY-289 were determined. As summarized in Table IIGo, HWY-289 inhibited CaCHS1 and CaCHS2 to the same extent (IC50 for both enzymes = 22.0 µM). However, HWY-289 did not show any significant inhibitory activity against CaCHS3p (IC50 > 200 µM). These results suggest that HWY-289 preferentially inhibits CaCHS1p and CaCHS2p, which are known to be involved in the synthesis of the chitin plate in the primary septum and the majority of chitin, respectively.11,29


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Table II. Effect of HWY-289 on chitin synthase isozymes
 

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 Introduction
 Materials and methods
 Results
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 References
 
Identification of a novel antifungal drug with unique modes of action is desirable, since fungi resistant to available antifungal agents would be unlikely to be cross-resistant to these newer drugs.1,2 Based on our previous results that described the anti-Candida activities of protoberberines, we set out to find a new class of drugs derived from protoberberines that may be targeted against major enzymes involved in fungal growth. Our results indicate that chemical modifications of the natural product berberine,10 such as addition of a t-benzyl group, can lead to significant improvement in inhibitory activity against the growth of C. albicans. This modification is likely to have made the compound more lipophilic in nature, which in turn may have made it a more effective inhibitor of enzymes involved in lipid and cell wall synthesis. However, this modification appears to have changed its inhibitory behaviour against 24-SMT in vitro.10 Thus, although HWY-289 effectively blocked the 24-SMT reaction in vivo, it failed to directly inhibit this enzyme activity in vitro. One explanation for this result may be that HWY-289 is metabolized by the fungus to an inhibitor of 24-SMT activity. Alternatively, HWY-289 may have had an, as yet unidentified, indirect effect on the 24-SMT reaction in vivo.

It is tempting to seek an explanation for the marked inhibition of the yeast-to-mycelium transition of C. albicans by HWY-289 in the differential effects of the protoberberine derivative on the chitin synthase isoforms. Recent evidence indicates that CaCHS1 is expressed equally in yeast and hyphal cells while CaCHS2 and CaCHS3 are co-expressed and exhibit a transient peak of expression 1–2 h after induction of the hyphal growth phase.30 However, both {Delta}chs2 and {Delta}chs3 null mutants formed hyphae efficiently, indicating that these genes are not required for hypha formation.30 Therefore, there is no obvious relationship between potent inhibition of CaCHS1p and CaCHS2p by HWY-289 and inhibition of C. albicans morphogenesis by this compound. It was also noted that HWY-289 differentially inhibits the chitin synthase of Saccharomyces cerevisiae (ScCHS): IC50 values for ScCHS1p and ScCHS2p were found to be 30 and 170 µM, respectively. Both CaCHSp and ScCHSp appear to be regulated at the level of transcription.3032

The fact that HWY-289 may have the capacity to simultaneously target CaCHS1p, CaCHS2p and 24-SMT activities, which are absent from mammalian cells,9,11 may mean that this compound or its derivatives will represent a new class of antifungal compound with low host toxicity. These compounds may provide a safer alternative to amphotericin B, one of the most effective drugs currently used for the treatment of systemic fungal infections, which is highly toxic to human cells. For example, HWY-289 showed no sign of any side effects when subjected to genotoxicity and acute toxicity tests in laboratory animals. The acute toxicity (LD50) of HWY-289 was 3742 (male) and 2760 (female) mg/kg body weight for rats when orally administered (Y.-K. Paik & J.-H. Kim, unpublished data). Therefore, our results suggest that HWY-289 has promising selective antifungal activity and is deserving of further in vitro and in vivo investigation


    Acknowledgments
 
We would like to thank Dr M. Sudoh at Nippon Roche for his generosity in supplying chitin synthase mutant strains of C. albicans and Professor W.-J. Choi at Ehwa Women's University, Jong Chul Park at Yonsei Medical School and Professor D. M. Han for their frequent help throughout this project. This work was supported by a grant from Health Technology Planning and Evaluation Board (to Y.-K.P.) and Korea Science and Engineering Foundation through the Bioproducts Research Center at Yonsei University (to Y.K.P., 9514-0401-00-12-3), Seoul, Korea.


    Notes
 
* Correspondence address. Department of Biochemistry, Yonsei University, 134 Shinchon-dong, Sudaemun-ku, Seoul 120-749, Korea. Tel: +82-2-2123-4242/2702; Fax: +82-2-393-6589/362-9897; E-mail: paikyk{at}yonsei.ac.kr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 25 September 2000; returned 14 December 2000; revised 8 January 2001; accepted 29 January 2001